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Journal Cover
Journal of Molecular Catalysis B: Enzymatic
Journal Prestige (SJR): 0.522
Citation Impact (citeScore): 2
Number of Followers: 1  
 
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 1381-1177
Published by Elsevier Homepage  [3161 journals]
  • One-pot synthesis of nitrocyclopropane: α-Amylase-catalyzed Michael
           addition initiated ring-closure sequence reactions
    • Authors: Xue-Dong Zhang; Jian Song; Na Gao; Zhi Guan; Yan-Hong He
      Pages: 1 - 8
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xue-Dong Zhang, Jian Song, Na Gao, Zhi Guan, Yan-Hong He
      This article presents a one-pot synthesis of nitrocyclopropanes via Michael addition initiated ring-closure sequence reactions of bromonitroalkane to α,β-unsaturated enones. Moderate to favorable yields (55–93%) and certain enantioselectivities were obtained with α-amylase from hog pancreas as the catalyst. This strategy utilizes the unnatural ability of enzymes to provide a convenient and biocatalytic method for green organic synthesis.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.006
      Issue No: Vol. 134 (2017)
       
  • Biocatalytic epoxidation of α-pinene to oxy-derivatives over
           cross-linked lipase aggregates
    • Authors: Madalina Tudorache; Andreea Gheorghe; Ana S. Viana; Vasile I. Parvulescu
      Pages: 9 - 15
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Madalina Tudorache, Andreea Gheorghe, Ana S. Viana, Vasile I. Parvulescu
      Lipase-based cross-linked aggregates were investigated for a non-specific reaction, i.e. the epoxidation of α-pinene to its oxygenated derivatives. The activity of the biocatalysts has been evaluated in a green context, i.e. ethyl acetate as both acetate-supplier and organic solvent with H2O2/UHP/TBHP as oxidant. Screening of the lipase sources indicated Aspergillus niger lipase as the most efficient biocatalyst for this reaction. Different immobilization protocols ((i) cross-linked enzyme aggregates (CLEA), (ii) cross-linked enzyme aggregates onto magnetic particles (CLEMPA) and (iii) covalent immobilized enzyme (CIE) onto magnetic particles (MP)) were evaluated considering the activity as main parameter. Thus, CLEA and CLEMPA afforded better epoxidation yields of α-pinene towards CIE. The investigated biocatalytic systems allowed to transform α-pinene into oxigenated derivatives with industrial and commercial applications (e.g. α-pinene oxide, camphene, pinanediol and camphonelic aldehyde). FTIR investigations on the biocatalysts revealed the effects of the immobilization protocol on the enzyme secondary-structure. Additionally, textural characterizations were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) analysis.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.009
      Issue No: Vol. 134 (2017)
       
  • Investigation of structural stability and enzymatic activity of glucose
           oxidase and its subunits
    • Authors: Fatemeh Janati-Fard; Mohammad Reza Housaindokht; Hassan Monhemi
      Pages: 16 - 24
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Fatemeh Janati-Fard, Mohammad Reza Housaindokht, Hassan Monhemi
      Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of β-d glucose utilizing molecular oxygen as an electron acceptor to produce d-glucono-1,5-lactoneand hydrogen peroxide, which has applications in food, biotechnology and medical industries. It was known that dimer form was considered to be active and monomer form has inactive conformation. However, there are no evidences at the molecular levels for Glucose oxidase (GOx) inactivation through dissociation. Here, using molecular dynamic simulation, it has been investigated for the first time that why dimer form of the enzyme is active. We have performed a series of molecular dynamics simulations at different forms of GOx (monomer and dimer with and without FAD cofactor). The analysis of tertiary structure showed that monomer is more unstable and has more deviation from the crystal structure. In contrast, dimer has a stable conformation during simulation. These results are in good agreement with experimental data about enzyme inactivation by dissociation. It was also found that when FAD is removed from monomer, it became more unstable in comparison with monomer containing cofactor. This shows essential role of FAD in both activity and stability of the enzyme. According to the MD simulation, enzyme inactivation is associated with changing in secondary structure at the interface. Interestingly, it was found that some secondary structures are destructed while some structures are formed in monomer upon dissociation. The analysis of active site structure during simulation revealed that both dissociation and release of the FAD influence on inactivation of GOx. This study provided novel insight to understand the mechanism of enzyme inactivation upon dissociation, which would be useful for rational enzyme design.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.008
      Issue No: Vol. 134 (2017)
       
  • A highly efficient immobilized MAS1 lipase for the glycerolysis reaction
           of n-3 PUFA-rich ethyl esters
    • Authors: Xiumei Wang; Daoming Li; Weifei Wang; Bo Yang; Yonghua Wang
      Pages: 25 - 31
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xiumei Wang, Daoming Li, Weifei Wang, Bo Yang, Yonghua Wang
      This study reported that immobilized MAS1 lipase showed high catalytic efficiency in the production of triacylglycerols (TAG) highly enriched with n-3 polyunsaturated fatty acids (PUFA) by glycerolysis of ethyl esters (EE). Immobilized MAS1 lipase was found to have no regiospecificity and be a more suitable catalyst for the glycerolysis of n-3 PUFA-rich EE compared with other enzymes. Higher TAG content (73.9%) and EE conversion (82%) were obtained by immobilized MAS1 lipase than those by Novozym 435 (29.6% and 54.8%, respectively) and Lipozyme RM IM (10% and 49%, respectively). Besides, the effects of temperature, enzyme loading and n-3 PUFA-rich EE/glycerol molar ratio on TAG content were evaluated using response surface methodology. The results showed that temperature, enzyme loading and n-3 PUFA-rich EE/glycerol molar ratio had significant effects on TAG content. The maximum TAG content (76.5%) was achieved under the optimal conditions (enzyme loading of 163.8U/g substrate, n-3 PUFA-rich EE/glycerol molar ratio of 4.13:1 at 65°C). Subsequently, the glycerolysis reaction mixtures were further purified by molecular distillation and highly pure n-3 PUFA-rich TAG (96.2%) with similar fatty acids composition to the substrate (EE) was obtained in the final products. In addition, the obtained final products had low acid value and peroxide value (0.03mg KOH/g and 3.2meq/kg, respectively). These results indicated that immobilized MAS1 lipase is a promising catalyst for the synthesis of TAG in industrial application.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.012
      Issue No: Vol. 134 (2017)
       
  • Differential effect of a chemical denaturant on activity and stability of
           a serine protease in nonaqueous media
    • Authors: Shivcharan Prasad; Villendra S. Negi; Joydev K. Laha; Ipsita Roy
      Pages: 32 - 36
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Shivcharan Prasad, Villendra S. Negi, Joydev K. Laha, Ipsita Roy
      With reference to industrial application, reusability of the biocatalyst is an important criterion which determines the cost of the final product. Urea-induced structural perturbation of proteases has led to higher enzymatic activity, especially in nonaqueous media. The mechanism behind this phenomenon has not been investigated in detail. Using the transesterification activity of subtilisin Carlsberg in nonaqueous media as an illustration, we report that the higher activity is due to simultaneous decrease in Michaelis constant and increase in turnover number of the enzyme. However, we show that this perturbed architecture is unable to retain the high activity-conformation for further rounds of catalysis. Thus, we conclude that the use of an enzyme for commercial applications is dependent upon a compromise between activity and (operational) stability.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.011
      Issue No: Vol. 134 (2017)
       
  • The synergism of hot water pretreatment and enzymatic hydrolysis in
           depolymerization of lignocellulosic content of palm kernel cake
    • Authors: Shuofu Mi; Hongqiang Li; Shuying Li; Yejun Han
      Pages: 37 - 42
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Shuofu Mi, Hongqiang Li, Shuying Li, Yejun Han
      Palm kernel cake (PKC), mainly composed of mannan, lignin and protein, is abundant renewable resource with commercial value. To develop clean and efficient way for PKC refinery, the method based on the synergism of hot water pretreatment (HWP), steam pretreatment (SP) and enzymatic hydrolysis were developed. HWP of 180°C, 20min and SP of 121°C, 20min showed similar performance for sugar release from PKC. The main saccharides produced from PKC by HWP and SP were mannose and manno-oligosaccharides, while no furfural formed. The surface structure analyzed by SEM showed that HWP enhanced the microporosity of PKC, and the accessibility of which was increased thereafter. When HWP pretreated PKC was further hydrolyzed with enzyme cocktail (cellulase, xylanase, endo-mannanase), 45% of PKC was solubilized compared with the control. The manno-oligosaccharides produced by HWP and SP were converted to mannose and mannobiose by endo-mannanase. The results suggested that both HWP and SP promote enzymatic hydrolysis of PKC by releasing oligosaccharides and enhancing microporosity, and the synergism of which was effective for PKC decomposition.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.004
      Issue No: Vol. 134 (2017)
       
  • Enantioselective acetylation of (R,S)-atenolol: The use of Candida rugosa
           lipases immobilized onto magnetic chitosan nanoparticles in
           enzyme-catalyzed biotransformation
    • Authors: Adam Sikora; Dorota Chełminiak-Dudkiewicz; Tomasz Siódmiak; Agata Tarczykowska; Wiktor Dariusz Sroka; Marta Ziegler-Borowska; Michał Piotr Marszałł
      Pages: 43 - 50
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Adam Sikora, Dorota Chełminiak-Dudkiewicz, Tomasz Siódmiak, Agata Tarczykowska, Wiktor Dariusz Sroka, Marta Ziegler-Borowska, Michał Piotr Marszałł
      This paper describes the enzyme immobilization protocol as well as the enzymatic method for the direct resolution of (R,S)-atenolol. The used magnetic enzyme carriers possess on their surface new-synthetized chitosan derivatives with free amine groups distanced by ethyl or butyl chain. Additionally the catalytic activity of two types of commercially available lipases from Candida rugosa immobilized onto two different magnetic nanoparticles were compared. The highest values of enantioselectivity (E=66.9), enantiomeric excess of product (eep =94.1%) and conversion (c=41.84%) were obtained by using lipase from Candida rugosa OF immobilized onto Fe3O4-CS-EtNH2. The study confirmed that even after 5 reaction cycles the immobilized lipase maintain its high catalytic activity.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.017
      Issue No: Vol. 134 (2017)
       
  • Efficient production of ethyl (R)-4-chloro-3-hydroxybutanoate by a novel
           alcohol dehydrogenase from Lactobacillus curieae S1L19
    • Authors: Yiping Zhang; Hualei Wang; Lifeng Chen; Kai Wu; Jingli Xie; Dongzhi Wei
      Pages: 51 - 60
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Yiping Zhang, Hualei Wang, Lifeng Chen, Kai Wu, Jingli Xie, Dongzhi Wei
      Ethyl (R)-4-chloro-3-hydroxybutanoate ester [(R)-CHBE] is an important chiral intermediate for the synthesis of chiral drugs. In this study, a novel short-chain, NADH-dependent dehydrogenase (LCRIII) from Lactobacillus curieae S1L19 was discovered to exhibit high activity and enantioselectivity in the production of (R)-CHBE by reduction of ethyl 4-chloroacetoacetate (COBE). LCRIII was heterologously overexpressed in Escherichia coli and the protein was purified to homogeneity. Characterization of LCRIII showed broad substrate specificity towards a variety of ketones. In addition, an efficient cofactor regeneration system was constructed by co-expressing LCRIII and glucose dehydrogenase (GDH) in E. coli cells. Up to 1.5M (246.8g/L) COBE could be completely reduced to (R)-CHBE with excellent enantiomeric excess (>99% ee) in a monophasic aqueous system. Moreover, the process could be performed even without external addition of cofactors. These results demonstrate the great potential of this process in industrial applications.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.010
      Issue No: Vol. 134 (2017)
       
  • A simple approach to a vastly improved acetylcholinesterase activity and
           stability at elevated temperatures using magnetic microbeads and
           poly(N-(3-aminopropyl methacrylamide)) hydrogel supports
    • Authors: Lisa C. Shriver-Lake; Paul T. Charles; Andre A. Adams; Jake Fontana; Brett D. Martin
      Pages: 61 - 69
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Lisa C. Shriver-Lake, Paul T. Charles, Andre A. Adams, Jake Fontana, Brett D. Martin
      The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme (“EM Conjugate”) are incubated overnight in a dry state at 63°C (140°F), the catalytic efficiency (kcat/Km) of the latter is higher than the former by six orders of magnitude (a factor of 2.16×106). This effect arises mostly through a ∼29,700-fold decrease in Km experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63°C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34×104) and that of the EM Conjugate alone (a factor of 1.78×104) after all are incubated overnight at 63°C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38×104 relative to that of the EM Conjugate, reaching a value of 2.18×10−3 M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.007
      Issue No: Vol. 134 (2017)
       
  • Screening of fungi from the genus Penicillium for production of β-
           fructofuranosidase and enzymatic synthesis of fructooligosaccharides
    • Authors: A.K.C. Nascimento; C. Nobre; M.T.H. Cavalcanti; J.A. Teixeira; A.L.F. Porto
      Pages: 70 - 78
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): A.K.C. Nascimento, C. Nobre, M.T.H. Cavalcanti, J.A. Teixeira, A.L.F. Porto
      Eight new isolated fungi of the genus Penicillium were evaluated for β- fructofuranosidase (FFase) production. From these, Penicillium citreonigrum was selected for FFase and fructooligosaccharides (FOS) production. The influence of temperature, yeast extract concentration, pH and fermentation time on the FFase activity when using the whole microorganism was evaluated by 24 and 23 designs. The pH was set at 6.5 and no yeast extract was used in the optimization experiments since both shown low significant effects on FFase activity. After optimization, temperature and fermentation time, were set to 25.5°C and 67.8h. Under these conditions, the model predicted a FFase production of 301.84U/mL. The scaled-up process in a 2L bioreactor enhanced the enzyme productivity up to 1.5 times (6.11U/mLh). A concentration of 58.7g/L of FOS was obtained, where kestose was the main product. Assays performed for enzyme characterization showed that 50°C and a pH 5.0 are the optimal conditions for FFase activity. FFase showed to be stable at temperatures between 25 and 30°C and pH 4.0–10.0 and its activity increased in the presence of ions, especially Cu4+. Results obtained in this primary report are a clear indication on the interest of using P. citreonigrum as a source of FFase for further FOS production.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.005
      Issue No: Vol. 134 (2017)
       
  • In vivo cytotoxicity, molecular docking and study of yeast alcohol
           dehydrogenase on polycarbazole-titanium dioxide nanocomposite
    • Authors: Mohammad Shakir; Mohd. Shoeb Khan; Umair Baig; Md. Fazle Alam; Hina Younus; Mahboob Alam
      Pages: 79 - 88
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Mohammad Shakir, Mohd. Shoeb Khan, Umair Baig, Md. Fazle Alam, Hina Younus, Mahboob Alam
      The present work deals with the synthesis of an electrically conductive polycarbazole-titanium dioxide (PCz/TiO2-6%) nanocomposite employing facile in-situ oxidative polymerization of carbazole monomer. In order to immobilize the yeast alcohol dehydrogenase (YADH) enzyme, the polymerization reaction was done in the presence of TiO2 (titanium dioxide). The pristine PCz and PCz/TiO2-6% nanocomposites were fully characterized using Fourier transform infra-red spectroscopy, Scanning electron microscopy, Transmission electron microscopy, Thermogravimetric analysis and Differential thermal analysis. The studies revealed that the TiO2 and YADH loading changes nanocomposite morphology in comparison to pristine PCz. YADH immobilization was efficient and successfully carried out on PCz and PCz/TiO2-6% nanocomposite with a loading efficiency of 67.4% and 88.2% respectively. Immobilized YADH on the PCz/TiO2-6% nanocomposite enhanced YADH stability, recycling efficiency, and residual activity, which makes it ideally suited for industrial applications. A total of four 3D molecular field descriptors or field points were used to characterize and define the necessary properties required for a molecule to bind into a specified active site, in a characteristic fashion. 3D molecular dynamics and a molecular docking simulation were employed to predict the modes of interactions of YADH with either PCz or PCz/TiO2-6%. The in vivo cytotoxicity profiles of PCz and PCz/TiO2-6% nanocomposite were obtained by lethality bioassay against brine shrimp nauplii.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.018
      Issue No: Vol. 134 (2017)
       
  • Purification and characterization of an alkaline chloride-tolerant laccase
           from a halotolerant bacterium, Bacillus sp. strain WT
    • Authors: Maryam Siroosi; Mohammad Ali Amoozegar; Khosro Khajeh
      Pages: 89 - 97
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Maryam Siroosi, Mohammad Ali Amoozegar, Khosro Khajeh
      Laccases are multicopper oxidases with various biotechnological applications that oxidize different aromatic or inorganic substrates. In present work, different bacterial strains isolated from Urmia lake, a hypersaline lake in northwest of Iran, were screened to find laccase-producing ones. Spore and an extracellular enzyme from a halotolerant spore-forming bacterium, Bacillus sp. strain WT, showed laccase activity toward typical laccase substrates: syringaldazine and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate). The extracellular laccase (0.01UmL−1) decolorized sulphonyl green BLE up to 97% at pH 7.0 after two h incubation at 35°C, without any addition of mediators. This enzyme with apparent molecular mass of 180kDa was purified using ammonium sulfate precipitation method and anion exchange chromatography. The optimum laccase activity toward 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine was at 55°C and pH values of 5.0 and 8.0, respectively. One mM of metal ions, Na+ and Ni2+, increased the enzyme activity by 12%. The enzyme from Bacillus sp. strain WT could be able to tolerate up to 600–800mM NaCl (a very strong laccase inhibitor) and showed halotolerant nature with maximum activity at 100mM NaCl. One mM NaN3 (another potent laccase inhibitor) almost had no effect on the laccase activity; however, 1mM l-Cys reduced 87% of its original activity. K M values for the purified enzyme on 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine were determined to be 132.7 and 3.7μM, with corresponding k cat values of 309 and 51s−1, respectively. The present study is among the first studies on laccase activity of a halotolerant bacterial strain isolated from a hypersaline lake.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.001
      Issue No: Vol. 134 (2017)
       
  • Purification and characterization of a fibrinolytic enzyme from the
           food-grade fungus, Neurospora sitophila
    • Authors: Xiao lan Liu; Narasimha kumar Kopparapu; Hong chen Zheng; Priti Katrolia; Yong ping Deng; Xi qun Zheng
      Pages: 98 - 104
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xiao lan Liu, Narasimha kumar Kopparapu, Hong chen Zheng, Priti Katrolia, Yong ping Deng, Xi qun Zheng
      A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, Neurospora sitophila. The enzyme displayed a molecular mass of 34kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3±0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41°C and displayed remarkable stability in a wide pH range (4–11) and up to 52°C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.006
      Issue No: Vol. 134 (2017)
       
  • Overexpression and characterization of CCD4 from Osmanthus fragrans and
           β-ionone biosynthesis from β-carotene in vitro
    • Authors: Xuesong Zhang; Jianjun Pei; Linguo Zhao; Feng Tang; Xianying Fang; Jingcong Xie
      Pages: 105 - 114
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xuesong Zhang, Jianjun Pei, Linguo Zhao, Feng Tang, Xianying Fang, Jingcong Xie
      In this study, a recombinant carotenoid cleavage dioxygenase 4 was produced from Osmanthus fragrans by E. coli under different bacterial growth conditions and used to develop a biotechnological method for preparation of natural β-ionone from β-carotene. β-ionone was analyzed by HPLC and OfCCD4 activity was measured based on concentration change of β-ionone. At pH 8.0 and 35°C, the greatest activity of the purified recombinant protein was 14.3U/mg and the maximum concentration of β-ionone was 71.186mg/L within 1h. Both the enzyme activity and the concentration of β-ionone could increase by nearly 6 times with addition of 9% Triton X-100 and 2% liquid paraffin.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.003
      Issue No: Vol. 134 (2017)
       
  • Efficient production of 5-aminovalerate from l-lysine by engineered
           Escherichia coli whole-cell biocatalysts
    • Authors: Xin Wang; Peipei Cai; Kequan Chen; Pingkai Ouyang
      Pages: 115 - 121
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xin Wang, Peipei Cai, Kequan Chen, Pingkai Ouyang
      In this study, we developed a whole-cell biocatalysis process for high-level conversion of l-lysine into 5-aminovalerate. To obtain the highly efficient whole-cell biocatalyst, five expression plasmids were constructed to optimize the expression of 5-aminovaleramide amidohydrolase and l-lysine 2-monooxygenase in Escherichia coli. The engineered strain BL-22A-RB-YB harboring plasmid pET22b-davA, pRSFDuet-davB and pACYCDuet-davB was correspondingly obtained. Subsequently, the effects of induction conditions, reaction temperature, metal ion additives, and cell permeability on the whole-cell biocatalyst system were evaluated to improve biocatalytic efficiency. Under optimized reaction conditions, 95.3g/L 5-aminovalerate was synthesized from 120g/L l-lysine with a yield of 99.1%, and 103.1g/L 5-aminovalerate was produced from 150g/L l-lysine with a molar yield of 85.7%. The 5-aminovalerate production was then further improved using a l-lysine fed-batch strategy, and a hyper 5-aminovalerate production of 240.7g/L was achieved within 28h with a yield of 86.8%. The whole-cell biocatalytic system described here demonstrated an environmentally friendly strategy for industrial production of 5-aminovalerate.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.008
      Issue No: Vol. 134 (2017)
       
  • Characterization of a thermostable mannitol dehydrogenase from
           hyperthermophilic Thermotoga neapolitana DSM 4359 with potential
           application in mannitol production
    • Authors: Marwa Yagoub Farag Koko; Hinawi Abdo Mustafa Hassanin; Rebaone Letsididi; Tao Zhang; Wanmeng Mu
      Pages: 122 - 128
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Marwa Yagoub Farag Koko, Hinawi Abdo Mustafa Hassanin, Rebaone Letsididi, Tao Zhang, Wanmeng Mu
      Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135kDa. K m and V max values for reduction of D-fructose to D-mannitol were 20mM and 200U mg-1 respectively. k cat for reduction direction was 180s−1 and k cat/K m were 9mM−1 s−1. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90°C with 100% relative activity. The purified enzyme was quite stable at 75°C and had half of initial activity after 1h of incubation at 90°C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP+ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.010
      Issue No: Vol. 134 (2017)
       
  • Immobilization of lipase on mesoporous silica nanoparticles with
           hierarchical fibrous pore
    • Authors: Zafar Ali; Lei Tian; Panpan Zhao; Baoliang Zhang; Nisar Ali; Muhammad Khan; Qiuyu Zhang
      Pages: 129 - 135
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Zafar Ali, Lei Tian, Panpan Zhao, Baoliang Zhang, Nisar Ali, Muhammad Khan, Qiuyu Zhang
      Lipase from Candida Ragusa (CRL) was successfully covalently immobilized on fibrous silica nanoparticles KCC-1, and the properties of immobilized enzyme were investigated. Mesoporous fibrous silica nanoparticles (MSNPs) were synthesized with particles size 200nm pore size 15–30nm; followed by amino-functionalization. Scanning Electron Microscopy (SEM), Transmittance Electronic microscopy (TEM), Fourier Transform Infrared Spectroscopy (FT-IR) and N2 adsorption were used for the characterization of nanoparticles. Further SiO2@NH2 nanoparticles were activated by glutaraldehyde as a bifunctional cross linker, and were used for lipase immobilization. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to better resistance to temperature and pH inactivation in comparison to the free lipase, and hence widened the reaction pH and temperature regions, with the optimum pH and temperature of 7.5 and 40°C, respectively. The immobilized Lipase Candida Ragusa (ICRL) maintained above 81% of the initial activity after 28days and 80% activity after 8 repeated cycles. Thus ICRL showed improved storage stability reusability and 700U/g of protein as immobilization efficiency.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.011
      Issue No: Vol. 134 (2017)
       
  • RHA-P: Isolation, expression and characterization of a bacterial
           α-l-rhamnosidase from Novosphingobium sp. PP1Y
    • Authors: Federica De Lise; Francesca Mensitieri; Vincenzo Tarallo; Nicola Ventimiglia; Roberto Vinciguerra; Annabella Tramice; Roberta Marchetti; Elio Pizzo; Eugenio Notomista; Valeria Cafaro; Antonio Molinaro; Leila Birolo; Alberto Di Donato; Viviana Izzo
      Pages: 136 - 147
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Federica De Lise, Francesca Mensitieri, Vincenzo Tarallo, Nicola Ventimiglia, Roberto Vinciguerra, Annabella Tramice, Roberta Marchetti, Elio Pizzo, Eugenio Notomista, Valeria Cafaro, Antonio Molinaro, Leila Birolo, Alberto Di Donato, Viviana Izzo
      α-l-Rhamnosidases (α-RHAs) are a group of glycosyl hydrolases of biotechnological potential in industrial processes, which catalyze the hydrolysis of α-l-rhamnose terminal residues from several natural compounds. A novel α–RHA activity was identified in the crude extract of Novosphingobium sp. PP1Y, a marine bacterium able to grow on a wide range of aromatic polycyclic compounds. In this work, this α-RHA activity was isolated from the native microorganism and the corresponding orf was identified in the completely sequenced and annotated genome of strain PP1Y. The coding gene was expressed in Escherichia coli, strain BL21(DE3), and the recombinant protein, rRHA-P, was purified and characterized as an inverting monomeric glycosidase of ca. 120kDa belonging to the GH106 family. A biochemical characterization of this enzyme using pNPR as substrate was performed, which showed that rRHA-P had a moderate tolerance to organic solvents, a significant thermal stability up to 45°C and a catalytic efficiency, at pH 6.9, significantly higher than other bacterial α-RHAs described in literature. Moreover, rRHA-P was able to hydrolyze natural glycosylated flavonoids (naringin, rutin, neohesperidin dihydrochalcone) containing α-l-rhamnose bound to β-d-glucose with either α-1,2 or α-1,6 glycosidic linkages. Data presented in this manuscript strongly support the potential use of RHA-P as a biocatalyst for diverse biotechnological applications.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.002
      Issue No: Vol. 134 (2017)
       
  • Nearly aqueous-like activity of lipoprotein lipase treated with
           glucose-headed surfactant in organic solvent
    • Authors: Yeonock Oh; Yoon Kyung Choi; Inyeol Yun; Eungyeong Lee; Kyungwoo Kim; Mahn-Joo Kim
      Pages: 148 - 153
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Yeonock Oh, Yoon Kyung Choi, Inyeol Yun, Eungyeong Lee, Kyungwoo Kim, Mahn-Joo Kim
      In this work, we explored the activation of a lipoprotein lipase from Burkholderia species by glucose-headed surfactants (GHSs) for enhancing its catalytic activity in organic solvent. Three GHSs were prepared and then tested as the additives for inducing the activation of lipoprotein lipase. The kinetic parameters of GHS-treated lipoprotein lipase were determined for the hydrolysis or alcoholysis of p-nitrophenyl acetate. It was found that GHS-treated lipoprotein lipase was 4 to 5 orders of magnitude more active than its native counterpart in organic solvent. Such a dramatic activity enhancement was largely the result of a huge increase in the turnover frequency k cat. Surprisingly, the k cat values in organic solvent were one order of magnitude greater than their aqueous counterparts. As a result, the k cat/K m of GHS-treated lipoprotein lipase in organic solvent became comparable to the aqueous level within one order of magnitude. We thus have demonstrated for the first time that a lipase can display nearly aqueous-like activity in organic solvent. As an illustrative application of GHS-treated lipoprotein lipase, we performed the dynamic kinetic resolution of two secondary alcohols, which provided the products of high enantiopurity (98–99%ee) with high yields (90–91%).
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.009
      Issue No: Vol. 134 (2017)
       
  • Thermostability enhancement of xylanase Aspergillus fumigatus RT-1
    • Authors: Mohd Khairul Hakimi bin Abdul Wahab; Mohd Anuar bin Jonet; Rosli Md Illias
      Pages: 154 - 163
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Mohd Khairul Hakimi bin Abdul Wahab, Mohd Anuar bin Jonet, Rosli Md Illias
      This study aimed to improve the thermostability of endo-1,4-β-xylanase (afxynG1) from Aspergillus fumigatus RT-1 using error-prone PCR. Since the wild type enzyme has an optimum temperature stability at 50°C, the improvement of its stability will widen its application in industries with operating processes at higher temperatures. A library containing approximately 5000 afxynG1 mutants was generated and thermally screened at 60°C for 30min. Four mutants (T16A/T39I/L176Q, S68R, A60D and Q47P/S159R) were selected for enzymatic characterization because of their higher catalytic activity compared to the wild type. Among these mutants, the mutant T16A/T39I/L176Q showed highest stability at 70°C and retained 45.9% of its activity after 60min of incubation while the wild type had lost its activity completely after 50min of incubation. The other mutants, A60D, S68R and Q47P/S159R also showed improvement in thermostability by retaining 33.2%, 25.8% and 23.8% of their activity respectively. The optimum temperature for mutants also significantly increased. The optimum temperature for T16A/T39I/L176Q increased up to 70°C, followed by A60D increased up to 60°C while the rest remained the same, similar to the wild type enzyme. The mutant T16A/T39I/L176Q had the highest half-life time (t1/2) of 42min at 70°C, which is a 3.5-fold increase compared to the wild type enzyme which only showed a t1/2 of 12min at 70°C. This is followed by mutant A60D, t1/2 of 31min (2.7-fold), S68R, t1/2 of 29min (2.4-fold) and Q47P/S159R, t1/2 of 27min (2.25-fold). Based on homology modelling conducted to analyze the mutants’ structures, it showed that hydrophobicity and hydrogen bonds were the driving forces that lead to the improvement of the thermal stability of these xylanase mutants.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.020
      Issue No: Vol. 134 (2017)
       
  • Magnetic cellulose nanocrystals: Synthesis by electrostatic self-assembly
           approach and efficient use for immobilization of papain
    • Authors: Feng Zhang; Ruonan Wang; Cheng Zhen; Bin Li
      Pages: 164 - 171
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Feng Zhang, Ruonan Wang, Cheng Zhen, Bin Li
      Novel magnetic cellulose nanocrystals (MCNCs) prepared via electrostatic self-assembly approach were used as magnetic carriers for efficient immobilization of papain and facilitated recovery of this immobilized enzyme. Zeta potential measurements, Fourier transform infrared spectroscopy and Scanning electron microscope were applied to evaluate the forming mechanism and surface structure of MCNCs. Cellulose nanocrystals (CNCs) were successfully combined with cationic polyethyleneimine (PEI) modified Fe3O4 nanoparticles (NPs), and the electrostatic interaction between them was a key driving force. The prepared MCNCs were successfully used for the immobilization and separation of papain from the reaction system. When enzyme concentration and pH value of enzyme solution were 0.4mgmL−1 and 6, respectively, the resultant immobilized enzyme exhibited the highest enzymatic activity about 227μgmin−1 g−1. Better pH and thermo stabilities than those of the free papain were also achieved after immobilizing the enzyme on MCNCs. Furthermore, the immobilized papain manifested enhanced tolerability to three different solvents, namely n-butyl alcohol, n-hexane and [Cnpy][NTf2], respectively. The prepared MCNCs as the efficient carrier materials have a strong application potential for enzyme immobilization.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.017
      Issue No: Vol. 134 (2017)
       
  • Recycling Rhizopus oryzae resting cells as biocatalyst to prepare near
           eutectic palmitic-stearic acid mixtures from non-edible fat
    • Authors: Pau Gallart-Sirvent; Edinson Yara; Gemma Villorbina; Mercè Balcells; Núria Sala; Ramon Canela-Garayoa
      Pages: 172 - 177
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Pau Gallart-Sirvent, Edinson Yara, Gemma Villorbina, Mercè Balcells, Núria Sala, Ramon Canela-Garayoa
      Here we studied non-edible fat waste as a starting material to prepare eutectic mixtures of biomaterials. Initially, the fat was hydrolyzed using water and R. oryzae resting cells. The hydrolysis was performed in organic solvent-free media and the degree of hydrolysis at 1h was 42% while hydrolytic values of 86% and 98% were achieved at 12 and 48h, respectively. To recover the resting cells, they were extracted in consecutive cycles with solvents or supercritical CO2. Compared with solvents, supercritical CO2 allowed the highest reuse. Hence, R. oryzae was used for 336h (7 reaction cycles), yielding 56.5g of free fatty acid/g biocatalyst. Crude glycerol was recovered, showing a purity of 66.0% and an ash and water content of 2.3% and 1.8%, respectively. The hydrolyzed fat was crystallized with several solvents to yield palmitic and stearic acid mixtures with melting point characteristics of eutectic mixtures. We recovered 76% to 90% of the palmitic and stearic acids present in the initial hydrolyzed animal fat, depending on the solvent. The palmitic:stearic acid ratios determined by GC-FID were similar to those reported for eutectic mixtures whit phase change materials properties, as were the melting points, which ranged from 51.5°C to 54.8°C.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.015
      Issue No: Vol. 134 (2017)
       
  • Conversion of a Monascus ruber esterase into a lipase by disrupting a salt
           bridge
    • Authors: Zi-Tong Meng; Chen Hu; Yan Zhang; Hai-Lun Guo; Mu Li
      Pages: 178 - 185
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Zi-Tong Meng, Chen Hu, Yan Zhang, Hai-Lun Guo, Mu Li
      Cold-active lipases have emerged as an important class of biocatalysts for chemical and food industries due to their high efficiency at low temperature and long-chain substrate preference. In an effort to explore the feasibility of converting a cold-active esterase from Monascus ruber (Lip10) into a cold-active lipase, an Y264F variant in which the salt bridge between K243 and Y264 was disrupted has been constructed and characterized. The interfacial kinetic parameter, Km app for pNP-laurate (C12) and pNP-palmitate (C16), of Lip10 esterase was 4.2 and 5.7 times higher than those of the Y264F variant, respectively. Substrate specificity of the Y264F variant changed from shot-chain length substrate to medium- and long-chain length substrates, indicating that the Y264F variant turned into a lipase. Meanwhile, the Y264F variant displayed 48.6% maximum activity at 4°C and 3.2kcal/mol activation energy in the range of 5–30°C, suggesting that it was still cold-active. Based on analysis of the structure-function relationships, it suggests that the shape of substrate channel controlled by the conserved salt bridge was very important for the substrate specificity. This study provides a way to alter the substrate preference of the Lip10 esterase as well as new insight into the structural basis of esterase substrate specificity.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.012
      Issue No: Vol. 134 (2017)
       
  • Isoquercitrin production from rutin catalyzed by naringinase under
           ultrasound irradiation
    • Authors: Dan Zhu; An Gong; Yan Xu; D’assise Kinfack Tsabing; Fuan Wu; Jun Wang
      Pages: 186 - 195
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Dan Zhu, An Gong, Yan Xu, D’assise Kinfack Tsabing, Fuan Wu, Jun Wang
      Isoquercitrin, a rare flavonol glycoside with wide biological activities and key synthetic intermediate for the production of enzymatically modified isoquercitrin (EMIQ), was conducted by naringinase-catalyzed conversion of rutin under ultrasound irradiation. The maximum yields were obtained to 98.35±3.13% and 95.20±2.52% under conventional heating and ultrasound irradiation, respectively. The optimal results under ultrasound irradiation were obtained under the following conditions: rutin concentration 0.8g/L, naringinase concentration 3000U/L, reaction temperature 40°C for 20min, which was more economical than that with conventional heating. The reaction time was reduced from 60min to 20min, and the apparent kinetic parameter (V m/K m) was increased 3.72-fold. The lower activity energy E a under ultrasonic irradiation was 0.7-fold of that in an incubator reactor, which could easily initiate the enzymatic reaction. The association saturation constant K a was 1.98-fold higher than that with conventional heating, showed a better affinity between rutin and naringinase detected by surface plasmon resonance (SPR) analysis. These results suggest that ultrasound irradiation can accelerate the enzymatic synthesis of isoquercitrin from rutin.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.011
      Issue No: Vol. 134 (2017)
       
  • Detoxification of furanic and phenolic lignocellulose derived inhibitors
           of yeast using laccase immobilized on bacterial cellulosic nanofibers
    • Authors: Thiyagarajan Saravanakumar; Han-Sung Park; Ae-Young Mo; Myoung-Suk Choi; Dae-Hyuk Kim; Seung-Moon Park
      Pages: 196 - 205
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Thiyagarajan Saravanakumar, Han-Sung Park, Ae-Young Mo, Myoung-Suk Choi, Dae-Hyuk Kim, Seung-Moon Park
      Biotransformation of lignocellulose by microbial fermentation is usually preceded by thermo-chemical pretreatments followed by enzymatic hydrolysis of cellulose. Derivatives formed during the pretreatment of the lignocellulosic biomass inhibit enzymatic hydrolysis as well as microbial fermentation. Pretreated lignocellulose hydrolysate contains many derivatives of either furanic or phenolic inhibitory derivatives. In the present study, laccase was used to detoxify three different types of lignocellulosic derivatives that are highly toxic to microbial fermentation due to their low hydrophilic nature, namely furfural, acetosyringone, and coniferyl aldehyde. A minimal inhibitory concentration (MIC) test was carried out with Saccharomyces cerevisiae. The MIC of furfural, acetosyringone, and coniferyl aldehyde was 12mM, 24mM, and 1.5mM, respectively. Laccase was immobilized on to cellulose nanofiber produced by Gluconacetobacter xylinus. Immobilized laccase showed a better pH and thermal stability than free laccase. Reuse of immobilized laccase retains 85% of its enzyme activity after 16 recycles. Immobilized laccase completely degraded the three lignocellulose inhibitory derivatives after 36h of incubation at 40°C. Finally, the degradation was confirmed by ultraviolet visible spectroscopy (UV–VIS spectrum), high performance liquid chromatography and liquid chromatography mass spectrometry. Interestingly, it was found that the effect of enzymatic degradation depends on the structural variation of the lignocellulosic derivatives as laccase alone detoxified the furfural and coniferyl aldehyde, whereas a redox mediator HOBt was needed for the detoxification of ketone based lignin derivative acetosyringone.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.006
      Issue No: Vol. 134 (2017)
       
  • Wickerhamomyces subpelliculosus as whole-cell biocatalyst for
           stereoselective bioreduction of ketones
    • Authors: Viktória Bódai; László Nagy-Győr; Róbert Örkényi; Zsófia Molnár; Szabolcs Kohári; Balázs Erdélyi; Zsuzsanna Nagymáté; Csaba Romsics; Csaba Paizs; László Poppe; Gábor Hornyánszky
      Pages: 206 - 214
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Viktória Bódai, László Nagy-Győr, Róbert Örkényi, Zsófia Molnár, Szabolcs Kohári, Balázs Erdélyi, Zsuzsanna Nagymáté, Csaba Romsics, Csaba Paizs, László Poppe, Gábor Hornyánszky
      Newly isolated strains of Wickerhamomyces subpelliculosus were recognized as excellent whole-cell biocatalyst for bioreduction of various ketones. The biocatalytic properties of the new strains were demonstrated in this study by stereoselective bioreduction of acetophenone 1a, 2-heptanone 1b, phenylacetone 1c, 3,4-dimethoxyphenylacetone 1d and 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e. Our study is the first report on application of W. subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of prochiral ketones. In these processes, both the freshly harvested cell paste and the lyophilized cell powder were tested as biocatalyst using glucose or 2-propanol at various concentrations as cosubstrates for cofactor regeneration. The newly isolated strains of W. subpelliculosus showed diverse characteristics, including optimal pH, temperature and organic solvent tolerance. Bioreductions of phenylacetone 1c applying glucose as cosubstrate under various mild conditions resulted (S)-1-phenylpropanol [(S)-2c] in good to excellent conversion (c =63.4%–99.9%) with excellent enantiomeric excess [ee (S)-2c =98.7%–99.8%].
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.003
      Issue No: Vol. 134 (2017)
       
  • A new member of family 8 polysaccharide lyase chondroitin AC lyase
           (PsPL8A) from Pedobacter saltans displays endo- and exo-lytic catalysis
    • Authors: Aruna Rani; Arun Goyal
      Pages: 215 - 224
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Aruna Rani, Arun Goyal
      Chondroitin lyases are therapeutically important enzymes. The functional aspects of a chondroitin AC lyase (PsPL8A) from Pedobacter saltans DSM12145 were investigated. PsPL8A was cloned in to pET28a(+) vector, expressed in E. coli BL-21(DE3) cells exhibited a molecular size of approximately, 77kDa. PsPL8A displayed maximum activity with chondroitin 4-sulphate, C4S (489Umg−1) followed by chondroitin 6-sulphate, C6S (214Umg−1) and hyaluronic acid (43.2Umg−1). PsPL8A was maximally active at 39°C and pH 7.2. 100mM Na+ and 20mM Ca2+ ions enhanced the activity of PsPL8A by 2-fold. The time dependent TLC analysis of PsPL8A degraded products of C4S revealed the presence of higher degree of polymerization (DP) chondroitin sulphate (CS) oligosaccharides at initial stage, but after 1h, only ΔC4S disaccharide was produced as the major product. This result displayed that PsPL8A follows initially a concomitant endo- and exo-lytic mode which finally shifted to exolytic mode of catalysis. The oligosaccharides released were identified as di-, hexa-, octa- and dodeca-saccharide by ESI–MS analysis. The ΔC4S disaccharide showed a peak at m/z 458 (ESI–MS) while in MS/MS mode it gave the peak at m/z 300. ESI–MS/MS, 1H- and 13C- NMR analyses confirmed the structure of ΔC4S disaccharide product obtained after 24h reaction of C4S with PsPL8A. The enzyme reported in present study can be used for cancer mitigation, spinal cord injury treatment and CS oligosaccharides production which act as anti-inflammatory agents. This is the first study reporting the cloning and expression of chondroitin AC lyase from Pedobacter saltans DSM 12145.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.001
      Issue No: Vol. 134 (2017)
       
  • Genetic modification and optimization of endo-inulinase for the enzymatic
           production of oligofructose from inulin
    • Authors: Jiang-Ke Yang; Ji-Wen Zhang; Lin Mao; Xun You; Guang-Jun Chen
      Pages: 225 - 232
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Jiang-Ke Yang, Ji-Wen Zhang, Lin Mao, Xun You, Guang-Jun Chen
      The enzymatic hydrolyzation of inulin by endo-inulinase to produce oligofructoses, a new type of food additive and health product, is a promising, “green”, and environmentally friendly technique. To identify novel genetic sources of endo-inulinase genes and facilitate their industrial application for oligofructose production, we cloned an endo-inulinase gene from a Fusarium oxysporum strain and achieved high-level expression in the genetically modified Pichia pastoris strain in a pilot-scale bioreactor by using strategies such as C-terminal truncation and mutagenesis of protease-sensitive sites. We then optimized the parameters of the inulinase reaction and the amount of enzyme used to inulin hydrolysis and oligofructose production. The results of this study should facilitate the bulk production of inulinase and provide a reference for the industrial production of oligofructose from inulin.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.020
      Issue No: Vol. 134 (2017)
       
  • A Green approach towards the synthesis of chiral alcohols using
           functionalized alginate immobilized Saccharomyces cerevisiae cells
    • Authors: Narmada Muthineni; Manikanta Swamy Arnipally; Sridhar Bojja; Harshadas Mitaram Meshram; Ajay Kumar Srivastava; Bhaskar Rao Adari
      Pages: 233 - 237
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Narmada Muthineni, Manikanta Swamy Arnipally, Sridhar Bojja, Harshadas Mitaram Meshram, Ajay Kumar Srivastava, Bhaskar Rao Adari
      The stereochemistry of the drug molecule is gaining greater therapeutic importance and thus much attention was drawn in synthesis of chiral compounds by the pharmaceutical industry. In this study Saccharomyces cerevisiae cells immobilized on functionalized alginate beads, catalyze the bio-reduction of prochiral ketones 1a–12a to their corresponding chiral alcohols 1b–12b in higher yields of 60–99% and.excellent optical purity 75–97%. The synthesized chiral azido alcohols 10b-12b were further subjected to hydrogenation using Palladium(Pd) nanoparticles (≤5nm), to obtain chiral amino alcohols 10c–12c of therapeutic importance. Thus, a simple, green and inexpensive continuous chemo-enzymatic process has been developed in the synthesis of chiral alcohols/amino alcohols to enhance the scope of the methodology towards industrial application.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.016
      Issue No: Vol. 134 (2017)
       
  • Exploring the catalase activity of unspecific peroxygenases and the
           mechanism of peroxide-dependent heme destruction
    • Authors: Alexander Karich; Katrin Scheibner; René Ullrich; Martin Hofrichter
      Pages: 238 - 246
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Alexander Karich, Katrin Scheibner, René Ullrich, Martin Hofrichter
      The catalase activity of three unspecific peroxygenases (UPOs) from the agaric basidiomycetes Agrocybe aegerita, Coprinopsis cinerea and Marasmius rotula was investigated. The study included analysis of pH dependency of the catalase reaction and H2O2 mediated enzyme inactivation as well as experiments on the influence of a second substrate on the course of catalase reaction. Apparent kinetic parameters (Km, kcat) for the catalase activity of UPOs were determined. Inactivation of UPOs by H2O2 is discussed with regard to O2 production and remaining UPO activity. Furthermore formation of biliverdin as heme destruction product was demonstrated along with the formation of UPO compound III as a possible intermediate that forces the destruction process. Radical trapping experiments with methyl benzoate gave indication for the formation of hydroxyl radicals in the presence of excess H2O2. Eventually, a plausible pathway of heme destruction has been proposed, proceeding via UPO compound III and subsequent hydroxyl radical formation, which in turn may cause heme bleaching and verdoheme and biliverdin formation.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.014
      Issue No: Vol. 134 (2017)
       
  • Investigation on the underlying mechanism: How fusion xylanase-ELPs
           self-assembles into insoluble active aggregates
    • Authors: Shuyu Wu; Yilin Wang; Tingting Shen; Junhui Wang; Guangya Zhang
      Pages: 247 - 252
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Shuyu Wu, Yilin Wang, Tingting Shen, Junhui Wang, Guangya Zhang
      We have successfully obtained a recombinant xylanase by fusing with elastin-like polypeptides (ELPs), the xylanase SoxB underwent a sharp irreversible phase transition, and self-assembled into an insoluble but more catalytically operative particle. This was analogous to the immobilized xylanase to a large extent and aroused our interest to gain new insights into the determinant factor that may cause this phenomenon. We herein listed several candidate factors including the length of ELPs, linker sequence, buffer properties, and the target protein, subsequently we evaluated their contributions to the formation of the active aggregates. The results suggested that SoxB was fused with ELPs as desired protein partners, neither ELPs length nor the linker type made crucial contribution to the formation of active aggregates. However, when Na2CO3 was chosen as the salt to trigger the phase transition, the catalytic activities detected in aggregates accounted for more than 87.7% of total activity, whereas above 83.8% of the activity remained in supernatant when using Na2SO4. Then we introduced an alkali-tolerant xylanase termed as Xyl and compared it with SoxB, and found that the activity ratio in insoluble particle dropped to 15.3% in Na2CO3 and 19.3% in Na2SO4 respectively, only a few insoluble aggregates observed during the purification. Therefore, we speculated the property of xylanase partner fused to ELPs should be the predominant factor to form the catalytically active insoluble aggregates, and this provides a promising not yet reported perspective for industrial application of “immobilized” SoxB.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.012
      Issue No: Vol. 134 (2017)
       
  • Characterization of recombinant β- galactosidase and its use in enzymatic
           synthesis of lactulose from lactose and fructose
    • Authors: Xue-Yi Liao; Qian-Wang Zheng; Qian-ling Zhou; Jun-Fang Lin; Li-Qiong Guo; Fang Yun
      Pages: 253 - 260
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A
      Author(s): Xue-Yi Liao, Qian-Wang Zheng, Qian-ling Zhou, Jun-Fang Lin, Li-Qiong Guo, Fang Yun
      The gene encoding a β- galactosidase was cloned from Lactobacillus plantarum FMNP01 and expressed in Escherichia coli BL21(DE3). The characteristics of this purified recombinant enzyme, L.pFMNP01Gal, were determined, and its transgalactosylation reaction conditions for the production of lactulose were optimized. Using ONPG as substrate, the L.pFMNP01Gal showed specific activity of 980U/g with a Km of 6.86mM and a Kcat of 22.47/s. This enzyme was most stable at 40–50°C, and exhibited optimum catalytic activity at 40°C and pH 7.0. The activity of L.pFMNP01Gal was greatly inhibited by Cu2+, while other tested metal ions had little influence on it. For the optimization of transgalactosylation reaction, high lactulose production was achieved when 60% (W/V) sugars were used as substrates with a lactose/fructose mass ratio of 2:1, and 2U/mL of L.pFMNP01Gal as catalyst. Under these optimum conditions, 18.38±2.17g/L of lactulose was synthesized in 6h at 50°C. This study provides an alternative method for enzymatic synthesis of lactulose.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.019
      Issue No: Vol. 134 (2017)
       
  • Special issue OxiZymes 2016
    • Authors: Willem van Berkel; Marco Fraaije; Frank Hollmann
      First page: 273
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Willem van Berkel, Marco Fraaije, Frank Hollmann


      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.009
      Issue No: Vol. 134 (2017)
       
  • Green routes towards industrial textile dyeing: A laccase based approach
    • Authors: Cinzia Pezzella; Simona Giacobbe; Valerio Guido Giacobelli; Lucia Guarino; Sibel Kylic; Mehmet Sener; Giovanni Sannia; Alessandra Piscitelli
      Pages: 274 - 279
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Cinzia Pezzella, Simona Giacobbe, Valerio Guido Giacobelli, Lucia Guarino, Sibel Kylic, Mehmet Sener, Giovanni Sannia, Alessandra Piscitelli
      Laccase-catalyzed synthesis of dye molecules represents a green choice to reduce the environmental footprint of conventional synthetic processes. Textile industry will benefit from this green technology since the synthesized dyes can be exploited to colour different fabrics. This work describes the application of the Pleurotus ostreatus POXA1b laccase in polymeric dye synthesis using resorcinol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA) as substrates. The potential of the resorcinol/2,5-DABSA coupling route was transferred to a chemical industry, Setaş Colour Center, by introducing a greener synthesis step within the process routinely used for textile dyeing. Dye synthesis was performed at different precursor ratios (1:1 and 1:10 2,5-DABSA: resorcinol) and their dyeing properties were compared on different fibres. The two mixtures of synthesized dyes proved to be effective on nylon and wool, with 1:10 ratio displaying the best performances in terms of dyeing efficiency and colour strength. Good and comparable end quality and “performances during use” were observed for nylon and wool coloured with both synthesized dyes.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.016
      Issue No: Vol. 134 (2017)
       
  • Chemoenzymatic Total Synthesis of (+)- &
           (−)-cis-Osmundalactone
    • Authors: Fabian Blume; Yu-Chang Liu; Daniel Thiel; Jan Deska
      Pages: 280 - 284
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Fabian Blume, Yu-Chang Liu, Daniel Thiel, Jan Deska
      Both optical antipodes of the cis-isomers of osmundalactone, a hydroxypyranone natural product and core structure of the angiopterlactones, have been synthesized from acetylfuran in only three steps through a redox cascade utilizing oxidoreductases and transition metal catalysis in a concerted fashion. The key step in this fully catalytic strategy is the enzyme-mediated Achmatowicz reaction via selective furan oxygenation to furnish the pyran core structure.
      Graphical abstract image

      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.010
      Issue No: Vol. 134 (2017)
       
  • An engineered outer membrane pore enables an efficient oxygenation of
           aromatics and terpenes
    • Authors: Anna Joëlle Ruff; Marcus Arlt; Maike van Ohlen; Tsvetan Kardashliev; Monika Konarzycka-Bessler; Marco Bocola; Alexander Dennig; Vlada B. Urlacher; Ulrich Schwaneberg
      Pages: 285 - 294
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Anna Joëlle Ruff, Marcus Arlt, Maike van Ohlen, Tsvetan Kardashliev, Monika Konarzycka-Bessler, Marco Bocola, Alexander Dennig, Vlada B. Urlacher, Ulrich Schwaneberg
      Biocatalysis with cytochrome P450 enzymes are important for the industrial production of fine chemicals, pharmaceuticals, fragrance and flavor compounds since chemoselective hydroxylation of aromatics and terpenes are chemically difficult to achieve. A few P450 based industrial processes have been developed based on whole cell catalysis. However, the outer membrane of microbial cells forms an effective barrier, which reduces the uptake of hydrophobic substrates. The coexpression of outer membrane proteins in E. coli such as the ferric hydroxamate uptake protein (FhuA) can provide alternative solutions to chemical or physical methods for increasing compound flux through the outer membrane of E. coli and thereby to boost productivities. In this study we employed an engineered FhuA Δ1-160 variant in which the “cork domain” was removed (first 160 residues are deleted); FhuA Δ1-160 has a cross-section of 39–46Å with a “free” inner diameter of about 14 Å that serves as passive diffusion channel. FhuA WT and Δ1-160 were coexpressed on a bicistronic system with two P450 BM3 variants for regiospecific hydroxylation of aromatic compounds toluene and anisole as well as for oxidation of two terpenes (α)-pinene and (R)-(+)-limonene. The presence of FhuA Δ1‐160 resulted in a doubled product concentration for toluene (35μ to 50μM), anisole (25μM to 45μM), pinene (12μM to 20μM) and limonene (12μM to 25μM) and five times higher for the coumarin derivative BCCE. In order to characterizes and compensate for expression variations a quantification method based on Chromeo546-labled StrepTactinII was developed to quantify the number of FhuA Δ1-160 in the outer E. coli membrane (∼44000 of FhuA Δ1-160 per cell). Morphology studies showed that a 6% E. coli surface coverage can be achieved with FhuA Δ1‐160 without significantly influencing the E. coli rod shape. In summary, FhuA Δ1-160 efficiently increases uptake of hydrophobic aromatics and terpenes for whole-cell biotransformations and can likely be used for other enzymes and/or substrates.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.11.007
      Issue No: Vol. 134 (2017)
       
  • Laccase-catalyzed dimerization of glycosylated lignols
    • Authors: Ivan Bassanini; Paolo Gavezzotti; Daniela Monti; Jana Krejzová; Vladimír Křen; Sergio Riva
      Pages: 295 - 301
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Ivan Bassanini, Paolo Gavezzotti, Daniela Monti, Jana Krejzová, Vladimír Křen, Sergio Riva
      Phenylpropanoid glucosides (PPGs) are naturally occurring and bioactive phenolic derivatives, largely distributed in plants. In this work different PPGs have been chemically or enzymatically synthesized from the lignols coniferyl and p-coumaryl alcohols as substrates for a laccase-catalyzed oxidative coupling. The biooxidation of these PPGs has been investigated here and novel dihydrobenzofuran-based structurally modified analogues have been isolated and characterized. Specifically, the presence of a carbohydrate moiety increased the water solubility of these compounds and reduced the number of dimeric products, as pinoresinol-like structures could not be formed. Looking for a possible sugar-promoted stereochemical enrichment of the obtained diastereomeric mixtures of dimers, different carbohydrate moieties (d-glucose, l-glucose and the disaccharide rutinose) were considered and the respective d.e. values of the dimeric products were measured by 1H NMR and HPLC. However, it was found that the sugar substituent had a minor effect on the stereochemical outcome of the radical coupling reactions, the best measured result being a d.e. value of 21%.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.019
      Issue No: Vol. 134 (2017)
       
  • Intensifying the O2-dependent heterogeneous biocatalysis: Superoxygenation
           of solid support from H2O2 by a catalase tailor-made for effective
           immobilization
    • Authors: Juan M. Bolivar; Sabine Schelch; Martin Pfeiffer; Bernd Nidetzky
      Pages: 302 - 309
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Juan M. Bolivar, Sabine Schelch, Martin Pfeiffer, Bernd Nidetzky
      Besides merely destroying H2O2, an important use of the catalase reaction, H2O2 →1/2 O2 +H2O, is to supply O2 to oxygenation reactions. Due to convenient spatiotemporal control over O2 release, oxygenation from H2O2 is useful in particular for enzymatic reactions confined to solid supports. Because commercial catalases are difficult to immobilize, we have developed a one-step procedure of purification and immobilization of Bordetella pertussis catalase, recombinantly produced in Escherichia coli. Fusion of the catalase to a positively charged binding module enabled effective immobilization of the chimeric enzyme on anionic support (Relisorb SP 400), giving a controllable activity loading of between 5000 and 100,000 units/g support. Use of the immobilized catalase in combination with H2O2 feeding provided O2 to the reaction of glucose oxidase in solution for a range of volumetric conversion rates (0.2–1.5mM/min). Using optical sensing to measure the O2 concentration in the liquid but also in the solid phase, we showed that internal superoxygenation of the support was made possible under these conditions, resulting in an inverted (that is, negative) O2 concentration gradient between the bulk and the particle and allowing the internal O2 concentration to exceed by up to 4-fold the limit of atmospheric-pressure air saturation in solution. By tailored immobilization of B. pertussis catalase, therefore, an efficient biocatalytic system for hydrogen peroxide conversion in porous solid support was developed. This could find application for bubble-free oxygenation of O2-dependent enzymes co-immobilized with the catalase whereby enhanced internal availability of O2 would contribute to biocatalytic reaction intensification.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.017
      Issue No: Vol. 134 (2017)
       
  • Chemodivergent fungal oxidation of isochroman
    • Authors: Gabriela I. Furque; Fabricio R. Bisogno; Virginia E. Sosa
      Pages: 310 - 316
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Gabriela I. Furque, Fabricio R. Bisogno, Virginia E. Sosa
      This work extends the present knowledge about the ability of filamentous fungi and Baker’s Yeast to selectively transform oxygen-containing compounds. Previously, it has been demonstrated that several species of the Aspergillus genus are able to perform selective oxidation of benzopyrans. Isochroman or 3,4-dihydro-1H-benzopyran (1) was chosen as model substrate for the biotransformation since related motifs are often found in the structure of natural products with important biological and pharmacological activities. All the tested strains showed the ability to oxidize 1. Chemodivergent reaction pathways between the employed microorganisms were observed. The use of cytochrome P450 enzyme inhibitors, and different oxygenation conditions allowed to inquire about the type of enzymes involved in the process. The results obtained were compared with chemical one-electron oxidation of compound 1 and thus, a metabolic pathway was proposed.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.015
      Issue No: Vol. 134 (2017)
       
  • Studies of carbon monoxide dehydrogenase from Oligotropha carboxidovorans
    • Authors: Stephanie Dingwall; Jarett Wilcoxen; Dimitri Niks; Russ Hille
      Pages: 317 - 322
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Stephanie Dingwall, Jarett Wilcoxen, Dimitri Niks, Russ Hille
      We have undertaken physicochemical studies of the CO dehydrogenase from the aerobe Oligotropha carboxidovorans, probing both the binuclear Mo- and Cu-containing active site where CO is oxidized to CO2 and the enzyme’s FAD, where the reducing equivalents obtained from CO are transferred to the quinone pool. Regarding the FAD site, we have characterized the semiquinone oxidation state by EPR and identified it to be of the blue neutral form with a linewidth of 20G. The signature long-wavelength absorbance of FADH is also observed in the absorption spectrum of partially reduced enzyme at low pH. The enzyme exhibits a pH-dependent absorption spectrum in the oxidized state that is lost upon covalent modification of the enzyme by the flavin-specific agent diphenyliodonium cation. The pH dependence is attributed to Tyr 193 of the FAD-containing CoxM subunit, which sits atop the re face of the isoalloxazine ring in van der Waals contact with it. Electron equilibration among the enzyme’s four redox-active centers (including two [2Fe-2S] clusters in addition to the binuclear center and FAD) is found to be pH-dependent, but too fast to be followed using a stopped-flow pH jump protocol. Electron transfer from the iron-sulfur clusters to the FAD is thus much faster than in other members of the xanthine oxidase family of molybdenum-containing enzymes to which CO dehydrogenase belongs. Finally, a complex of the binuclear center with bicarbonate has been characterized by EPR, where the absence of observed hyperfine coupling using 13C-labeled bicarbonate suggests strongly that the bicarbonate is not directly coordinated to the Mo(V) of the partially reduced binuclear center.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.007
      Issue No: Vol. 134 (2017)
       
  • Evolved alkaline fungal laccase secreted by Saccharomyces cerevisiae as
           useful tool for the synthesis of C–N heteropolymeric dye
    • Authors: Ana I. Vicente; Javier Viña-Gonzalez; Paloma Santos-Moriano; Carlos Marquez-Alvarez; Antonio O. Ballesteros; Miguel Alcalde
      Pages: 323 - 330
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Ana I. Vicente, Javier Viña-Gonzalez, Paloma Santos-Moriano, Carlos Marquez-Alvarez, Antonio O. Ballesteros, Miguel Alcalde
      Enzymatic production of C–N heteropolymeric dyes at alkaline pHs is an attractive process for the textile industry. In this work, we have designed a fungal laccase by directed evolution so that it may be used at alkaline pHs for the synthesis of C–N heteropolymeric dyes (C–N polydye) from catechol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA). Firstly, several medium- and high-redox potential fungal laccases from previous laboratory evolution campaigns were benchmarked for the synthesis of the C–N polydye at pH 8.0, choosing an alkaline laccase mutant from Myceliophthora thermophila as the departure point for further engineering. Mutant libraries were then constructed, expressed in Saccharomyces cerevisiae and screened using a high-throughput colorimetric assay for the detection of the C–N polydye. By combining directed and focused molecular evolution, a novel, strongly expressed alkaline laccase variant was identified. This laccase was secreted at 37mg/L and its catalytic efficiency for the oxidation of catechol and 2,5-DABSA at pH 8.0 was enhanced 3.5-fold relative to that of the wild-type, promoting the synthesis of the C–N polydye at basic pHs. While the improved expression was mostly the result of accumulating mutations that favor the yeast’s codon usage together with the recovery of a secretion mutation, the enhanced C–N polydye synthetic activity of the mutant laccase was dependent on the alkaline mutations it inherited. Readily secreted, this laccase mutant would appear to be a valuable platform for organic synthesis at basic pHs.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.004
      Issue No: Vol. 134 (2017)
       
  • Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase
           catalyzed oxidations
    • Authors: Gustav Rehn; Asbjørn Toftgaard Pedersen; John M. Woodley
      Pages: 331 - 339
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Gustav Rehn, Asbjørn Toftgaard Pedersen, John M. Woodley
      Biocatalytic oxidations can offer clear advantages compared to chemically catalyzed oxidations in terms of chemo, regio and stereoselectivity as well as a reduced environmental impact. One of the most industrially important reactions is the oxidation of alcohols, which can be carried out using alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables the use of molecular oxygen as a substrate, generating either water or hydrogen peroxide as a by-product. The use of these enzymes is now gaining an increased interest, and several different enzymes of both types have been applied for proof-of-concept. In this review, we give an overview of the state-of-the-art, and discuss several important issues for future implementation in a production process.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.016
      Issue No: Vol. 134 (2017)
       
  • A more polar N-terminal helix releases MBP-tagged Thermus thermophilus
           proline dehydrogenase from tetramer-polymer self-association
    • Authors: Mieke M.E. Huijbers; Willem J.H. van Berkel
      Pages: 340 - 346
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Mieke M.E. Huijbers, Willem J.H. van Berkel
      Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from Thermus thermophilus (TtProDH) is overproduced in Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.014
      Issue No: Vol. 134 (2017)
       
  • Towards preparative peroxygenase-catalyzed oxyfunctionalization reactions
           in organic media
    • Authors: Elena Fernández-Fueyo; Yan Ni; Alvaro Gomez Baraibar; Miguel Alcalde; Lukas M. van Langen; Frank Hollmann
      Pages: 347 - 352
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Elena Fernández-Fueyo, Yan Ni, Alvaro Gomez Baraibar, Miguel Alcalde, Lukas M. van Langen, Frank Hollmann
      The peroxygenase from Agrocybe aegerita (AaeUPO) has been evaluated for stereoselective oxyfunctionalization chemistry under non-aqueous reaction conditions. The stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was performed in neat substrate as reaction medium together with the immobilized biocatalyst and tertBuOOH as oxidant. Stability and activity issues still have to be addressed. Nevertheless, gram-scale production of enantiopure (R)-1-phenylethanol was achieved with respectable 90,000 turnovers of the biocatalyst.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.013
      Issue No: Vol. 134 (2017)
       
  • Enzymes in the p-hydroxyphenylacetate degradation pathway of Acinetobacter
           baumannii
    • Authors: Kittisak Thotsaporn; Ruchanok Tinikul; Somchart Maenpuen; Jittima Phonbuppha; Pratchaya Watthaisong; Pirom Chenprakhon; Pimchai Chaiyen
      Pages: 353 - 366
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Kittisak Thotsaporn, Ruchanok Tinikul, Somchart Maenpuen, Jittima Phonbuppha, Pratchaya Watthaisong, Pirom Chenprakhon, Pimchai Chaiyen
      p-Hydroxyphenylacetate (HPA) can be derived from the biodegradation of lignin or from man-made compounds. The pathway involved for HPA degradation has been characterized for several species, but little is known on the degradation of HPA in Acinetobacter sp. In this report, the HPA degradation operon in A. baumannii TH was investigated using genome walking and PCR amplification to identify the genes encoded by the operon. The results showed that there are thirteen ORFs that are involved in this process and their arrangement in the operon of A. baumannii TH is different from that in the operons of other previously reported species. ORFs 8-12 show clear variation compared to orthologous genes from other species, particularly at ORF9 which encodes for succinic semialdehyde dehydrogenase (SSADH) that is absent in other species. The ssadh gene was overexpressed and the results confirmed that this enzyme is indeed succinate semialdehyde dehydrogenase. The results suggest that the final metabolites in this pathway are pyruvate and succinate, different from other species which have pyruvate and succinic semialdehyde as final products. Functional studies of the proteins encoded by ORF 8 and 10-12 have confirmed their roles in the HPA degradation pathway as an aldolase, a transporter protein, a hydroxylase and a reductase. Analysis of the sequence similarity network of enzymes encoded by ORFs 8-12 has revealed several interesting features. The designation of enzymes homologous to the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase in the database should be reassigned, as they were mostly incorrectly assigned as acyl-CoA dehydrogenases. An understanding of the enzymatic reactions which convert aromatic compounds into pyruvate and succinate should be highly useful for future metabolic engineering for converting waste-derived aromatic compounds into useful biochemicals.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.09.003
      Issue No: Vol. 134 (2017)
       
  • Peroxidase activity enhancement of myoglobin by two cooperative distal
           histidines and a channel to the heme pocket
    • Authors: Lei-Bin Wu; Ke-Jie Du; Chang-Ming Nie; Shu-Qin Gao; Ge-Bo Wen; Xiangshi Tan; Ying-Wu Lin
      Pages: 367 - 371
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Lei-Bin Wu, Ke-Jie Du, Chang-Ming Nie, Shu-Qin Gao, Ge-Bo Wen, Xiangshi Tan, Ying-Wu Lin
      To reveal the structure-function relationship of heme proteins, and to provide clues for creating artificial heme proteins with improved functions, we here use myoglobin (Mb) as a model protein, and report that its peroxidase activity can be enhanced by construction of two distal histidines and a channel to the heme pocket. It showed that in addition to a single distal histidine with a suitable distance to the heme iron (Phe43 to His43 mutation), a second distal histidine (Leu29 to His29 mutation) can work cooperatively to increase the turnover number, mimicking the role of well-known His-Arg pair in native peroxidases. Moreover, a channel created to the heme pocket by removal of the native His64 gate (His64 to Ala64 mutation) was shown to facilitate the binding of substrate, resulting in enhanced catalytic efficiency for the triple mutant L29H/F43H/H64AMb, which is beyond the addition of both double mutants, L29H/H64A Mb and F43H/H64AMb. These results provide valuable information for elucidating the structure-function relationship of heme proteins. In addition, this study provides clues for design of artificial heme proteins, and the strategy of creating a channel to the heme active center is expected be extended to design of other artificial enzymes with improved catalytic performance.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.08.018
      Issue No: Vol. 134 (2017)
       
  • High overexpression of dye decolorizing peroxidase TfuDyP leads to the
           incorporation of heme precursor protoporphyrin IX
    • Authors: Dana I. Colpa; Marco W. Fraaije
      Pages: 372 - 377
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Dana I. Colpa, Marco W. Fraaije
      The heterologous overexpression level of the bacterial dye decolorizing peroxidase TfuDyP in Escherichia coli was increased sixty fold to approximately 200mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly overexpressed SUMO-TfuDyP was, however, almost inactive. Analysis of the enzyme by UV–vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly overexpressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.08.017
      Issue No: Vol. 134 (2017)
       
  • Identification and characterization of a FAD-dependent putrescine
           N-hydroxylase (GorA) from Gordonia rubripertincta CWB2
    • Authors: Catherine O. Esuola; Olubukola O. Babalola; Thomas Heine; Ringo Schwabe; Micheal Schlömann; Dirk Tischler
      Pages: 378 - 389
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Catherine O. Esuola, Olubukola O. Babalola, Thomas Heine, Ringo Schwabe, Micheal Schlömann, Dirk Tischler
      A putrescine N-hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine>cadaverine>hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His10-tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent K m and k cat of 361.6±0.1μM and 0.266±0.011s−1, respectively, whereas the hydroxylation assay showed GorA with an apparent Km and k cat of 737.1±0.1μM and 0.210±0.001s−1. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.
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      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.08.003
      Issue No: Vol. 134 (2017)
       
  • Expression and characterization of a thermostable organic solvent-tolerant
           laccase from Bacillus licheniformis ATCC 9945a
    • Authors: Nikola Lončar; Nataša Božić; Zoran Vujčić
      Pages: 390 - 395
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Nikola Lončar, Nataša Božić, Zoran Vujčić
      Bacterial laccases have proven advantages over fungal and plant counterparts in terms of wider pH optimum, higher stability and broader biocatalytic scope. In this work, Bacillus licheniformis ATCC 9945a laccase is produced heterologously in Escherichia coli. Produced laccase exhibits remarkably high temperature optimum at 90°C and possess significant thermostability and resistance to inactivation by organic solvents. Laccase has an apparent melting temperature of 79°C at pH 7.0 and above 70°C in range of pH 5.0–8.0, while having half-life of 50min at 70°C. Presence of 10% organic solvents such as acetonitrile, dimethylformamide, dimethylsulfoxide or methanol reduces melting temperature to 45–52°C but activity remains practically unimpaired. With 50% of acetonitrile and methanol laccase retained ∼40% of initial activity. EDTA and 300mM sodium-chloride have positive effect on activity. Enzyme is active on syringaldazine, ABTS, phenols, amines, naphthol, lignin and lignin model compounds and mediates CC bond formation via oxidative coupling after one electron oxidation of phenolic group. Successful polymerization of 2-naphthol was achieved with 77% conversion of 250mg/L 2-naphtol in only 15min which may further expand substrate scope of this enzyme towards polymer production and/or xenobiotics removal for environmental applications.
      Graphical abstract image

      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.06.005
      Issue No: Vol. 134 (2017)
       
  • Oxidative biotransformations of phenol substrates catalysed by toluene
           dioxygenase: A molecular docking study
    • Authors: Patrick Höring; Kyle Rothschild-Mancinelli; Narain D. Sharma; Derek R. Boyd; Christopher C.R. Allen
      Pages: 396 - 406
      Abstract: Publication date: December 2016
      Source:Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part B
      Author(s): Patrick Höring, Kyle Rothschild-Mancinelli, Narain D. Sharma, Derek R. Boyd, Christopher C.R. Allen
      Toluene dioxygenase-catalysed (TDO) oxidation converts substituted phenol substrates into catechols, hydroquinones, and chiral cyclohexenone cis-diol products. The ratio between the isolated products varied widely even between similar substrates, e.g. o-cresol, m-cresol and p-cresol. These differences are caused by different binding interactions within the active site of TDO. This study provides insight into the binding interactions by molecular docking using AutoDock tools. The nature of binding of phenolic substrates was of major interest, in order to explain the observed regio- and stereo-selectiviy of product formation. The ellipse-shaped binding pocket of TDO consists of a polar and a hydrophobic region, limiting the possible substrate orientations. The phenolic hydroxyl group was preferentially hydrogen bonded with Gln-215 and His-311 in the active site. In some cases, a hydrogen bond was formed with other amino acids, e.g. Asp-219 and Met-220, instead. The position and type of the substituent on the phenol ring influences the formation of transient intermediates, and thus the nature and stability of the major isolated product.
      Graphical abstract image

      PubDate: 2017-12-23T18:31:51Z
      DOI: 10.1016/j.molcatb.2016.10.013
      Issue No: Vol. 134 (2017)
       
 
 
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