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Enzyme and Microbial Technology
Journal Prestige (SJR): 0.754
Citation Impact (citeScore): 3
Number of Followers: 14  
 
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0141-0229
Published by Elsevier Homepage  [3168 journals]
  • Overexpression of the transcription factor HAC1 improves nerolidol
           production in engineered yeast
    • Abstract: Publication date: Available online 2 December 2019Source: Enzyme and Microbial TechnologyAuthor(s): Zhenzhen Qu, Lili Zhang, Shaoming Zhu, Wei Yuan, Jiawei Hang, Dong Yin, Xuechao Tang, Jianyong Zheng, Zhao Wang, Jie SunAbstractIncreasing the metabolic flux of the mevalonate pathway, reducing the metabolic flux of competing pathway and utilizing the diauxie-inducible system constructed by GAL promoters are strategies commonly used in yeast metabolic engineering for the production of terpenoids. Using these strategies, we constructed a series of yeast strains with a strengthened mevalonate pathway and finally produced 336.5 mg/L nerolidol in a shake flask. The spliced HAC1 mRNA assay indicated that the unfolded protein response (UPR) occurred in the strains that we constructed. UPR strains exhibited the low transcriptional activities of GAL1 promoter. HAC1-overexpressing strain exhibited dramatically enhanced transcriptional activity of GAL1 promoter at 72 h of fermentation in flasks. HAC1 overexpression also increased the nerolidol titer by 47.7 %, reaching 497.0 mg/L and increased cell vitality. RNA-seq showed that the genes whose transcription responded to HAC1-overexpression were involved in the regulation of monocarboxylic acid metabolic processes and cellular amino acid biosynthetic process, indicating that the metabolic regulation may be part of the reason of the improved nerolidol synthesis. Our findings enrich the knowledge of the relationship between the construction of sesquiterpene-producing cell factories and UPR regulation. This study provides an effective strategy for sesquiterpene production in yeast.
       
  • Profiles of plant core-fucosylated N-glycans of acid alpha-glucosidases
           produced in transgenic rice cell suspension cultures treated with eight
           different conditions
    • Abstract: Publication date: Available online 30 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Jihye Kim, Jonghye Do, Hong-Yeol Choi, Sun-Dal Kim, Heajin Park, Seungkwan You, Wooseok Kim, Yeonjoo Jang, Donghwi Kim, Junmyoung Lee, Jongkwan Ha, Minkyoo Ji, Dong-Il Kim, Ha Hyung KimAbstractRecombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 μM of 2-fluoro-L-fucose (2-FF), 50 μM of 2-FF, 100 μM of 2-FF, 100 μM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 μM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 μM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.
       
  • Identification of key sites determining the cofactor specificity and
           improvement of catalytic activity of a steroid 5β-reductase from Capsella
           rubella
    • Abstract: Publication date: Available online 30 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Yuanyuan Li, Hongyan Pan, Yaowen Chang, Na Dong, Lei Zou, Ping Liang, Wei Tian, Zunxue ChangAbstractProgesterone 5β-reductases (P5βRs) are involved in 5β-cardenolide formation by stereo-specific reduction of the △4,5 double bond of steroid precursors. In this study a steroid 5β-reductase was identified in Capsella rubella (CrSt5βR1) and its function in steroid 5β-reduction was validated experimentally. CrSt5βR1 is capable of enantioselectively reducing the activated C = C bond of broad substrates such as steroids and enones by using NADPH as a cofactor and therefore has the potential as a biocatalyst in organic synthesis. However, for industrial purposes the cheaper NADH is the preferred cofactor. By applying rational design based on literature and complementary mutagenesis strategies, we successfully identified two key amino acid residues determining the cofactor specificity of the enzyme. The R63 K mutation enables the enzyme to convert progesterone to 5β-pregnane-3,20-dione with NADH as cofactor, whereas the wild-type CrSt5βR1 is strictly NADPH-dependent. By further introducing the R64H mutation, the double mutant R63K_R64H of CrSt5βR1 was shown to increase enzymatic activity by13.8-fold with NADH as a cofactor and to increase the NADH/NADPH conversion ratio by 10.9-fold over the R63 K single mutant. This finding was successfully applied to change the cofactor specificity and to improve activity of other members of the same enzyme family, AtP5βR and DlP5βR. CrSt5βR1 mutants are expected to have the potential for biotechnological applications in combination with the well-established NADH regeneration systems.
       
  • A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing
           xylobiose from the non-reducing end
    • Abstract: Publication date: Available online 30 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Katarína Šuchová, Vladimír Puchart, Nikolaj Spodsberg, Kristian B.R. Mørkeberg Krogh, Peter BielyAbstractXylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear β-1,3-β-1,4-xylan) from which also an isomeric xylotriose Xyl-β-1,3-Xyl-β-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.
       
  • Novel approach to produce biomass-derived oligosaccharides simultaneously
           by recombinant endoglucanase from Trichoderma reesei
    • Abstract: Publication date: Available online 27 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Yuheng Tao, Lei Yang, Limin Yin, Chenhuan Lai, Caoxing Huang, Xin Li, Qiang YongAbstractThe recombinant endoglucanase gene (EG I) from Trichoderma reesei was successfully expressed in Pichia pastoris for the purpose of producing oligosaccharides from various biomass-derived substrates. Interestingly, the recombinant endoglucanase I (ReEG I) showed the catalytic activity towards both cellulose and xylan hydrolysis, yet it was more efficient with xylans. Among various glucans and xylans substrates (paper pulp, carboxymethylated cellulose, oat spelt xylan, birchwood xylan), birchwood xylan displayed a higher yield of xylooligosaccharides (XOS) (69.5% after optimization). Eventually, it was observed that ReEG I could simultaneously produce XOS and COS, when the alkali-extracted corncob residues were used as substrate. This is the first report on simultaneous production of XOS and COS by recombinant endoglucanase I from Trichoderma reesei expressed in Pichia pastoris, where a novel application of genetically engineered enzymes is proposed to provide an attractive application for high value utilization of biomass.
       
  • Production of isofloridoside from galactose and glycerol using
           α-galactosidase from Alicyclobacillus hesperidum
    • Abstract: Publication date: Available online 26 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Lei Wang, Liya Song, Xiaoxian He, Fei Teng, Meirong Hu, Yong TaoABSTRACTIsofloridoside (D-isofloridoside and L-isofloridoside) is the main photosynthetic product in red algae. Here, given the importance of isofloridoside, a potentially effective method to produce isofloridoside from galactose and glycerol using whole-cell biocatalysts harboring α-galactosidase was developed. α-Galactosidase-encoding genes from Alicyclobacillus hesperidum, Lactobacillus plantarum, and Bifidobacterium adolescentis were cloned and the proteins were overproduced in Escherichia coli. The α-galactosidase from A. hesperidum (AHGLA) was chosen to synthesize isofloridoside. The effects of reaction pH, temperature, and substrate concentration were investigated. In the optimum biotransformation conditions, the final isofloridoside concentration reached 0.45 M (galactose conversion 23%). The reaction mixtures were purified using activated charcoal and calcined Celite, and the purified product was identified as a mixture of D- and L-isofloridoside by liquid chromatography-mass spectrometry and nuclear magnetic resonance. This study provides a possible feasible method for the biosynthesis of isofloridoside from low-cost glycerol and galactose.
       
  • Enzymatic synthesis and biological characterization of a novel mangiferin
           glucoside
    • Abstract: Publication date: Available online 23 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Iis Septiana, Thi Thanh Hanh Nguyen, Sangyong Lim, Seonmin Lee, Byeongsu Park, Sohyung Kwak, Sunghee Park, Seong-Bo Kim, Doman KimMangiferin, a major constituent of Mangifera indica L., has attracted substantial attention due to its anti-oxidant, anti-diabetic, anti-inflammatory, and anti-microbial activities. However, its poor solubility in water limits its use in food and pharmaceutical industries. In this study, novel mangiferin-(1→6)-α-D-glucopyranoside (Mg-G1) was enzymatically synthesized from mangiferin and sucrose using glucansucrase from Leuconostoc mesenteroides B-512F/KM, and optimized using response surface methodology. The water solubility of Mg-G1 was found to be 824.7 mM, which is more than 2,300-fold higher than that of mangiferin. Mg-G1 also showed DPPH radical scavenging activity and superoxide dismutase (SOD)-like scavenging activity, which were 4.77- and 3.71-fold higher than that of mangiferin, respectively. Mg-G1 displayed inhibitory activity against human intestinal maltase and COX-2. Thus, the novel glucosylated mangiferin may be used as an ingredient in functional food and pharmaceutical application.Graphical Graphical abstract for this article
       
  • Efficient heterologous expression of nicotinate dehydrogenase in Comamonas
           testosteroni CNB-2 with transcriptional, folding enhancement strategy
    • Abstract: Publication date: Available online 22 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Zhen-Hua Lu, Li-Rong Yang, Jian-Ping WuAbstractNicotinate dehydrogenase (NDHase) from Comamonas testosteroni JA1 catalyzes the C6 hydroxylation of 3-cyanopyridine with high regional selectivity, which is a very difficult and complex reaction for chemical synthesis. However, because NDHase is a membrane protein with three subunits (ndhS, ndhL and ndhM), it is difficult to express the enzyme in a functional form using common hosts such as Escherichia coli, Bacilus subtilis or Pichia pastoris. Furthermore, the enzyme requires special electron transfer chains in the membrane system for proper catalytic activity. Thus, we investigated the expression of NDHase in non-model bacterial strains, which are evolutionarily similar to C. testosteroni JA1, using several broad-host plasmids with different copy numbers as expression vectors. We successfully expressed NDHase in soluble from using the pVLT33 vector in C. testosteroni CNB-2, and found the activity of enzyme to be 40.6 U/L. To further improve the expression of NDHase in C. testosteroni CNB-2, we trialed a T7-like MmP1 system, composed of MmP1 RNA polymerase and an MmP1 promoter, which is used for transcriptional control in non-model bacteria. This increased protein expression and enzyme activity doubled to 90.5 U/L. A molecular chaperone was co-expressed using pBBR1 MCS-5 in the same host to improve the efficiency of folding and assembly of multi-subunit structures. The maximum activity was 115 U/L using the molecular chaperone GroES-EL, far surpassing the previously reported level, although expression was almost equivalent. These results indicate that a strategy involving the construction of a T7-like system and co-expression of a molecular chaperone offers an efficient approach for heterologous expression of enzymes that are difficult to express in functional forms using conventional hosts.
       
  • Autodisplay of alpha amylase from Bacillus megaterium in E. coli for the
           bioconversion of starch into hydrogen, ethanol and succinic acid
    • Abstract: Publication date: Available online 19 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Ana K. Gutiérrez-García, Cecilia Lizeth Alvarez-Guzmán, Antonio De Leon-RodriguezAbstractIn this work, the expression of an α-amylase from Bacillus megaterium on the cell surface of Escherichia coli strains WDHA (Δ hycA and Δ ldhA) and WDHFP (Δ hycA, Δ frdD and Δ pta) by the autodisplay adhesin involved in diffuse adherence (AIDA) system was carried out with the purpose to confer the ability to E. coli strains to degrade starch and thus produce hydrogen, ethanol and succinic acid. For the characterization of the biocatalyst, the effect of temperature (30-70 °C), pH (3-6) and CaCl2 concentration (0-25 mM), as well as the thermostability of the biocatalyst (55-80 °C) at several time intervals (15-60 min) were evaluated. The results showed that the biocatalyst had a maximum activity at 55 °C and pH 4.5. Calcium was required for the activity as well for the thermal stability of the biocatalyst. The calculated Vmax and Km values were 0.24 U/cm3 and 5.8 mg/cm3, respectively. Furthermore, a set of anaerobic batch fermentations was carried out using 10 g/dm3 of starch and 1 g/dm3 of glucose as carbon sources in 120 cm3 serological bottles, using WDHA and WDHFP strains harboring the pAIDA-amyA plasmid. The hydrogen production for WDHA was 1056.06 cm3/dm3 and the succinic acid yield was 0.68 g/gstarch, whereas WDHFP strain produced 1689.68 cm3/dm3 of hydrogen and an ethanol yield of 0.28 g/gstarch. This work represents a promising strategy to improve the exploitation of starchy biomass for the production of biofuels (hydrogen and ethanol) or succinate without the need of a pre-saccharification process.
       
  • Expression, biochemical characterization, and mutation of a water forming
           NADH: FMN oxidoreductase from Lactobacillus rhamnosus
    • Abstract: Publication date: Available online 17 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Fei-Long Li, Wen Bin Su, Qing-Lan Tao, Liao-Yuan Zhang, Ye-Wang ZhangEnzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg-1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29 G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.Graphical abstractGraphical abstract for this article
       
  • Aspergillus niger production of pectinase and α-galactosidase for
           enzymatic soy processing
    • Abstract: Publication date: Available online 17 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Qian Li, Christopher S. Ray, Nicholas V. Callow, Abdullah Al Loman, S.M. Mahfuzul Islam, Lu-Kwang JuAbstractSoybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we addressed this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers.
       
  • Directed evolution of RhlI to generate new and increased quorum sensing
           signal molecule catalytic activities
    • Abstract: Publication date: Available online 16 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Biao Zhang, Lili Ren, Dayong Xu, Haonan Wang, Zhuo Chen, Bingrong Zhang, Xin Zeng, Lianhong Sun, Feng LiAbstractQuorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhlI, an enzyme in the RhlI–RhlR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HHL), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to identify RhlI mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coli. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. RhlI-4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129 N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds.
       
  • Cloning and expression of the Bacillus amyloliquefaciens transglutaminase
           gene in E. coli using a bicistronic vector construction
    • Abstract: Publication date: Available online 12 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Lovaine Silva Duarte, Laísa Quadros Barsé, Pedro Ferrari Dalberto, William Tadeu Santos da Silva, Rafael Costa Rodrigues, Pablo Machado, Luiz Augusto Basso, Cristiano Valim Bizarro, Marco Antônio Záchia AyubTransglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.Graphical abstractGraphical abstract for this article
       
  • Bio-assay of Acintobacter baumannii using DNA conjugated with gold
           nano-star: A new platform for microorganism analysis
    • Abstract: Publication date: Available online 12 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Farnaz Bahavarnia, Ahmad Mobed, Mohammad Hasanzadeh, Arezoo Saadati, Soodabeh Hassanpour, Ahad MokhtarzadehAcinetobacter baumannii is a non-motile, gram-negative member of the gamma proteobacteria. A specific and sensitive approach was established for the detection of Acintobacter baumannii via DNA based bio-assay. In this study, gold nano-star was synthesized and used for bio-conjugation with pDNA toward the detection of target sequences. Synthesized probe (5ʹ TTG TGA ACT ATT TAC GTC AGC ATG C3ʹ) of Acinetobacter baumannii was found with excellent sensitivity. After the hybridization of pDNA with cDNA, target DNA (5ʹ GCA TGC TGA CGT AAA TAG TTC ACA A 3ʹ) was easily measured. According to ultra-sensitivity of the engineered optical DNA-based bio-assay, it is potentially applied in the bacterial detection of the environmental and clinical specimens. Here, the selection of engineered biosensor in the presence of two mismatch sequences was investigated. The results indicated an acceptable choice for DNA-based assays. The low limit of quantification (LLOQ) of genosensor was obtained as 1 fM. The present study is a very important diagnostic examination to recognize Acinetobacter baumannii, which can be a best alternative to the traditional methods.Graphical abstractGraphical abstract for this article
       
  • Cutinases as Stereoselective Catalysts: Specific Activity and
           Enantioselectivity of Cutinases and Lipases for Menthol and its Analogs
    • Abstract: Publication date: Available online 12 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): An Su, Serpil Kiokekli, Mariam Naviwala, Abhijit N.Shirke, Ioannis V. Pavlidis, Richard A. GrossThe specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylhexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC’s Phe195 aromatic ring; HiC’s hydrophobic Leu 174 and Ile 169 groups; TLL’s ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.Graphical abstractGraphical abstract for this article
       
  • Modulating the properties of the lipase from Thermomyces lanuginosus
           immobilized on octyl agarose beads by altering the immobilization
           conditions
    • Abstract: Publication date: Available online 6 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Yuliya Lokha, Sara Arana-Peña, Nathalia S. Rios, Carmen Mendez-Sanchez, Luciana R.B. Gonçalves, Fernando Lopez-Gallego, Roberto Fernandez-LafuenteThe lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.Graphical abstractGraphical abstract for this article
       
  • Rational molecular design for improving digestive enzyme resistance of
           beta-glucosidase from Trichoderma viride based on inhibition of bound
           state formation
    • Abstract: Publication date: Available online 6 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Hao Wang, Xiangna Lin, Shuang Li, Jianlin Lin, Chunfang Xie, Daling Liu, Dongsheng YaoAbstractBeta-glucosidase (BGL1) is widely used in animal feed industries. However, degradation caused by digestive enzymes in the intestine hampers its application. Improving the resistance of feed enzymes against proteases is crucial in livestock farming. To improve the resistance of beta-glucosidase against pepsin and trypsin, a rational molecular design based on the inhibition of bound-state formation and secondary design was developed. The strategy includes: (1) prediction of the interaction surface of the pepsin-BGL1 complex structure, (2) prediction of key amino acids affecting the formation of the complex, (3) optimization of pepsin-resistant mutants by structural evaluation, (4) secondary molecular design based on pepsin-resistant mutants, and optimization of pepsin and trypsin-resistant mutants. Two BGL1 protein mutants (BGL1Q627C and BGL1Q627C/R543H/R646W) were constructed, and then mutated and wild-type BGL1s were expressed in Pichia pastoris. The half-life of BGL1Q627C and BGL1Q627C/R543H/R646W were 1.36 and 1.51 times that of the wild type upon pepsin exposure, respectively. For trypsin resistance, the half-life were 0.93 and 1.53 times that of the wild type, respectively. Compare to those of the wild type, most of the basic enzymatic properties of both mutants were not significantly changed except for increased Michaelis constants. The rational design method can be used as a guide for modifying other feed enzymes.
       
  • High-level production of linalool by engineered Saccharomyces cerevisiae
           harboring dual mevalonate pathways in mitochondria and cytoplasm
    • Abstract: Publication date: Available online 5 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Yaoyao Zhang, Jin Wang, Xianshuang Cao, Wei Liu, Hongwei Yu, Lidan YeAbstractLinalool, a valuable monoterpene alcohol, is widely used in cosmetics and flavoring ingredients. However, its scalable production by microbial fermentation is not yet achieved. In this work, considerable increase in linalool production was obtained in Saccharomyces cerevisiae by dual metabolic engineering of the mevalonic acid (MVA) pathway in both mitochondria and cytoplasm. A farnesyl pyrophosphate synthase mutant ERG20F96W/N127W and a linalool synthase from Cinnamomum osmophloeum (CoLIS) were introduced and meanwhile the endogenous ERG20 was down-regulated to prevent the competitive loss of precursor. In addition, overexpression of the proteins of CoLIS and ERG20F96W/N127W and another copy of the same enzymes CoLIS/ERG20F96W/N127W with mitochondrial localization signal (MLS) were carried out to further pull the flux to linalool. Finally, a maximum linalool titer of 23.45 mg/L was obtained in a batch fermentation with sucrose as carbon source. This combinatorial engineering strategy may provide hints for biosynthesis of other monoterpenes.
       
  • The properties of the linker in a mini-scaffoldin influence the catalytic
           efficiency of scaffoldin-mediated enzyme complexes
    • Abstract: Publication date: Available online 5 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Dongdong Meng, Juan Wang, Chun YouSynthetic enzyme complexes have been successfully used to accelerate the reaction rate of cascade enzyme biocatalysis. Protein scaffold-mediated enzyme complexes are often constructed by assembling cascade enzymes on the artificial protein scaffoldin to form sophisticated biomimetic architectures and enhance the catalytic efficiency of biocatalytic processes. However, the effects of the linker in scaffoldin on the performance of the enzyme complexes have not been clarified. In this study, a scaffoldin-mediated two-enzyme complex containing triosephosphate isomerase (TIM) and fructose-1,6-bisphosphate aldolase/phosphatase (FBPA) was constructed, and the initial production rate of fructose 6-phosphate (F6P) was determined with different types of fine-tuning linkers. Enzyme complexes with linker length of 25 amino acids in scaffoldin exhibited the highest initial F6P production rate compared with linker length of 0, 10, or 57 amino acids in scaffoldin. This result indicated that an appropriate interdomain spacing between functional domains was required by multienzyme complexes to facilitate effective cascade catalysis. Then, the most popular flexible linker GGGGS (unit F) and rigid linker EAAAK (unit R) were introduced into this 25 amino acid linker to investigate the effect of linker flexibility on the initial reaction rate of the TIM − FBPA enzyme complex. The synthetic enzyme complex with the semirigid linker FRRRF in scaffoldin showed the highest initial F6P production rate of 10.16 μM min-1, which indicates that the linker's amino acid composition in scaffoldin may lead to significant changes in the spatial architecture of the TIM − FBPA complex and consequently affect the initial reaction rate. Precise linker length and flexibility allow an appropriate interdomain conformation to enable efficient cascade reactions. Collectively, our results showed that fine-tuning the initial reaction rate of enzyme complexes is an integrated systematic engineering, including adjusting the multienzyme architecture, linker length, and linker flexibility, which provides rational guidance for designing effective multienzyme complexes in the future.Graphical abstractGraphical abstract for this article
       
  • CRISPR/Cas9 technology enables the development of the filamentous
           ascomycete fungus Penicillium subrubescens as a new industrial enzyme
           producer
    • Abstract: Publication date: Available online 4 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Sonia Salazar-Cerezo, Roland S. Kun, Ronald P. de Vries, Sandra GarriguesAbstractPenicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.
       
  • De novo Biosynthesis of 2-Phenylethanol in Engineered Pichia
           pastoris
    • Abstract: Publication date: Available online 1 November 2019Source: Enzyme and Microbial TechnologyAuthor(s): Sijia Kong, Hong Pan, Xiaoyun Liu, Xun Li, Daoyi GuoAbstract2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odor, which is widely used in cosmetics and food industries. Natural 2-PE is costly and cannot meet the market demand due to the relative low content of 2-PE in the plants. Thus, there is an increasing interest in the search for alternative routes for 2-PE production. Here we demonstrate the engineering of Pichia pastoris to produce 2-PE directly from simple sugars for the first time. We first demonstrated that improving downstream pathway from phenylpyruvate to 2-PE by overexpressing ARO10 and ADH6 could increase the biosynthesis of 2-PE. Then several genetic engineering strategies were developed to increase phenylpyruvate availability to improve 2-PE production. 1169 mg/L of 2-PE was accumulated in the final engineered strain. This study showed the potential of P. pastoris as a host strain to produce industrially interested 2-PE by metabolic engineering.
       
  • Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to
           enhance lipid accumulation
    • Abstract: Publication date: Available online 31 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Way-Rong Lin, I-Son NgMicroalgae biorefinery is an alternative, sustainable and promising trend to solve the problem of fossil oil depletion and carbon dioxide emission. However, considering the innate limitation of cell growth and oil content in microalgae, to accelerate metabolic balance by CRISPR/Cas9 system is attractive. At first, plasmid based from Agrobacterium tumefaciens and a fragment of mGFP was transformed into Chlorella sorokiniana and Chlorella vulgaris FSP-E by electroporation, respectively. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM), and an increase of fluorescent was observed by 67% compared with that in wild type, which proved the Agrobacterium-mediated plasmid is suitable for gene insertion in Chlorella species. Consequently, plasmid with similar structure as mentioned previously containing fragment of Cas9 with sgRNA designed on omega-3 fatty acid desaturase (fad3) gene was constructed and showed a higher accumulation of lipid content by 46% (w/w) in C. vulgaris FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in Chlorella.Graphical abstractGraphical abstract for this article
       
  • Highly sensitive in situ-synthesized cadmium sulfide (CdS) nanowire
           photosensor for chemiluminescent immunoassays
    • Abstract: Publication date: Available online 31 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Hong-Rae Kim, Byoung-Gi An, Young Wook Chang, Min-Jung Kang, Jae-Gwan Park, Jae-Chul PyunAbstractHighly sensitive in situ-synthesized cadmium sulfide (CdS) nanowires (NWs) for the detection of chemiluminescence in immunoassays with a photoresist (PR) layer to stabilize the CdS NWs before and after coating with a parylene film were developed. The thickness of the PR layer was controlled by adjusting the viscosity of the PR solution used for spin-coating. PR2005 was the optimal PR for passivation of the NW surface. After the addition of a parylene coating on the CdS NWs, the photocurrent increased by as much as 50% over a broad range of light intensities, and the additional PR layer increased the photoresponse over the whole range of light intensities. When the photoresponses of the CdS NWs with and without the parylene film were compared after the addition of a PR layer, significant differences were observed in the photocurrent behavior after the incident light was turned off. For the CdS NWs with a parylene film and PR layer, the photocurrent reached the baseline within milliseconds of the incident light being turned off. However, the CdS NWs without a parylene film but with a PR layer required>60 s to reach the baseline level. This difference was due to the capacitance arising from the contact between the NWs. The in situ-synthesized CdS NW photosensor passivated by the parylene film and a PR layer was used in a chemiluminescence-based immunoassay. Finally, the detection of human immunodeficiency virus antibodies was demonstrated via a chemiluminescent enzyme-linked immunosorbent assay based on the CdS NW photosensor in comparison with the optical-density measurement for the chromogenic reaction of TMB(3,3',5,5'-Tetramethylbenzidine).
       
  • Metabolic engineering of Ashbya gossypii for enhanced FAD production
           through promoter replacement of FMN1 gene
    • Abstract: Publication date: Available online 24 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Manan V. Patel, T.S. ChandraAbstractRiboflavin (vitamin B2), Flavin Mononucleotide (FMN), Flavin Adenine Dinucleotide (FAD) are essential biomolecules for carrying out various metabolic activities of oxidoreductases and other enzymes. Riboflavin is mainly used as food and feed supplement while the more expensive FAD has pharmacological importance. Although Ashbya gossypii has been metabolically engineered for industrial production of riboflavin, there are no reports on FAD production. In the present study, a transcriptional analysis of the time course of flavin genes expression, indicated that riboflavin to FMN conversion by riboflavin kinase enzyme encoded by FMN1 gene could be the major rate limiting step in FAD synthesis. Overexpression of FMN1gene was attempted by placing the ORF of FMN1 under control of the stronger constitutively expressed GPD (Glyceraldehyde-3-phosphate dehydrogenase) promoter replacing its native promoter. A 2.25Kb promoter replacement cassette (PRC) for FMN1 gene was synthesized from cloned pUG6-GPDp vector and used for transformation of Ashbya gossypii. Resultant recombinant strain CSAgFMN1 had 35.67-fold increase in riboflavin kinase enzyme. A 14.02-fold increase in FAD production up to 86.56 ± 3.88 mg L-1 at 120 h incubation was obtained compared to wild type. While there was a marginal increase in riboflavin synthesis by the clone, FMN accumulation was not detected and could be attributed to other metabolic fluxes channeling FMN. This is the first report on development of FAD overproducing strain in A.gossypii.
       
  • Cascade synthesis of rare ketoses by whole cells based on
           L-rhamnulose-1-phosphate aldolase
    • Abstract: Publication date: Available online 24 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Zhou Chen, Zijie Li, Fen Li, Mayan Wang, Ning Wang, Xiao-Dong GaoAbstractDihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.
       
  • The boosting effect of recombinant hemicellulases on the enzymatic
           hydrolysis of steam-treated sugarcane bagasse
    • Abstract: Publication date: Available online 19 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Lorena Cardoso Cintra, Isabelle Cristine da Costa, Izadora Cristina Moreira de Oliveira, Amanda Gregorim Fernantes, Syd Pereira Faria, Rosália Santos Amorin Jesuíno, Maria Cristina Ravanal, Jaime Eyzaguirre, Luiz Pereira Ramos, Fabrícia Paula de Faria, Cirano José UlhoaAbstractTo increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), β-xylosidases (XYL) and α-L-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant β-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-L-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.
       
  • Lipase-mediated synthesis of ricinoleic acid vanillyl ester and evaluation
           of antioxidant and antibacterial activity
    • Abstract: Publication date: Available online 16 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Chae Gyeong Park, Jin Ju Kim, Hyung Kwoun KimCastor oil extracted from castor bean has antibacterial property, and has been used in various folk remedies. The major structural component of castor oil, ricinoleic acid, has actual antibacterial activity. Some phenolic compounds derived from plants have antioxidant property. Among them, vanillyl alcohol from vanilla bean has strong antioxidant activity. As vanillyl alcohol has low solubility in hydrophobic solvents and castor oil has low solubility in hydrophilic solvents, there is practical difficulty in using them. We performed lipase-mediated transesterification using vanillyl alcohol and castor oil, and synthesized ricinoleic acid vanillyl ester (RAVE). 2,2-Diphenyl-1-picrylhydrazyl assay and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay revealed that RAVE had a strong antioxidant activity in various organic solvents. RAVE also had antibacterial activity against some food spoilage bacteria. It showed more powerful antibacterial activity for gram positive bacteria than for gram negative bacteria. The critical micelle concentration of RAVE was measured at 7.36 μM and it partitioned exclusively into emulsion phase in water-emulsion system. Zeta potential measurement, membrane release test, and fluorescent microscopy showed that RAVE inserted itself into the bacterial cell membrane, destroyed membrane permeability, and induced cell death. As such, RAVE is a novel multi-functional compound with antioxidant and antibacterial activity, so it can be used as a functional material in the food and cosmetic industries.Graphic abstractGraphical abstract for this article
       
  • Modulatory Effect of low-shear modeled microgravity on stress resistance,
           membrane lipid composition, virulence, and relevant gene expression in the
           food-borne pathogen Listeria monocytogenes
    • Abstract: Publication date: Available online 15 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Sunirmal Sheet, Yesupatham Sathishkumar, Kuntal Ghosh, Mi-Sook Choi, Kwan Seob Shim, Yang Soo LeeAbstractThe present study investigated the influence of low-shear modeled microgravity (LSMMG) conditions on Listeria monocytogenes stress response (heat, cold, and acid), membrane fatty acid composition, and virulence potential as well as stress-/virulence-associated gene expression. The results showed that LSMMG-cultivated cells had lower survival rate and lower D-values under heat and acid stress conditions compared to cells grown under normal gravity (NG). Interestingly, the cold resistance was elevated in cells cultivated under LSMMG conditions when compared to NG conditions. A higher amount of anteiso-branched chain fatty acids and lower ratio of iso/anteiso were observed in LSMMG cultured cells, which would contribute to increased membrane fluidity. Under LSMMG conditions, upregulated expression of cold stress-related genes (cspA, cspB, and cspD) was noticed but no change in expression was observed for heat (dnaK, groES, clpC, clpP, and clpE) and acid stress-related genes (sigB). The LSMMG-grown cells showed inferior virulence capacity in terms of infection, cell cycle arrest, and apoptosis induction in Caco-2 cells compared to those grown under NG conditions. Approximately 3.65, 2.13, 4.02, and 2.65-fold downregulation of prfA, hly, inlA, and bsh genes, respectively, in LSMMG-cultured cells might be the reason for reduced virulence. In conclusion, these findings suggest that growth under LSMMG conditions stimulates alterations in L. monocytogenes stress/virulence response, perhaps due to changes in lipid composition and related genes expression.
       
  • Isolation and subunit compositions of the xylanosome complexes produced by
           Cellulosimicrobium species
    • Abstract: Publication date: Available online 15 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Tong-Yi Dou, Jing Chen, Chenglin LiuAbstractCellulosimicrobium cellulans, which is type species of the genus Cellulosimicrobium, produces xylanase predominant nanoscale multienzyme complexes, i.e., xylanosomes, when grown on water-insoluble polysaccharides. Here, we report on the isolation of similar multienzyme complexes (MECs) produced by two other species in genus Cellulosimicrobium (Cellulosimicrobium funkei and Cellulosimicrobium terreum). Functional studies and subunit structure identifications using genomic sequencing and proteomic techniques were also performed. When compared with the xylanosomes produced by C. cellulans F16, the isolated MECs showed a larger particle size and shared at least three conserved multidomain proteins. In addition, they also exhibited different enzymatic activities and subunit compositions, which indicates diverse capability and strategies in degrading hemicelluloses.
       
  • Development of glutaric acid production consortium system with
           α-ketoglutaric acid regeneration by glutamate oxidase in Escherichia coli
           
    • Abstract: Publication date: Available online 12 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Soo-Yeon Yang, Tae-Rim Choi, Hye-Rim Jung, Ye-Lim Park, Yeong-Hoon Han, Hun-Suk Song, Ranjit Gurav, Shashi Kant Bhatia, Kyungmoon Park, Jung-Oh Ahn, Yung-Hun YangAbstractGlutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced L-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5⿿mM glutaric acid was produced from 800⿿mM 5-aminovaleric acid using only 120⿿mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.
       
  • Designing a cellulolytic enzyme cocktail for the efficient and economical
           conversion of lignocellulosic biomass to biofuels
    • Abstract: Publication date: Available online 10 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Mukund Adsul, Simranjeet Kaur Sandhu, Reeta Rani Singhania, Ravi Gupta, Suresh K. Puri, Anshu MathurConcerns about dwindling fossil fuels and their unfavorable environmental impacts shifted the global focus towards the development of biofuels from lignocellulosic feedstocks. The structure of this biomass is very complex due to which variety of enzymes (cellulolytic, hemicellulolytic, auxiliary/AA9) and proteins (e.g. swollenin) required for efficient deconstruction. Major impediments in large-scale commercial production of cellulosic ethanol are the cost of cellulases and inability of any single microorganism to produce all cellulolytic components in sufficient titers. In the recent past, various methods for reducing the enzyme cost during cellulosic ethanol production have been attempted. These include designing optimal synergistic enzyme blends/cocktail, having certain ratios of enzymes from different microbial sources, for efficient hydrolysis of pretreated biomass. However, the mechanisms underlying the development, strategies for production and evaluation of optimal cellulolytic cocktails still remain unclear. This article aims to explore the technical and economic benefits of using cellulolytic enzyme cocktail, basic enzymatic and non-enzymatic components required for its development and various strategies employed for efficient cellulolytic cocktail preparation. Consideration was also given to the ways of evaluation of commercially available and in-house developed cocktails. Discussion about commercially available cellulolytic cocktails, current challenges and possible avenues in the development of cellulolytic cocktails included.Graphical abstractGraphical abstract for this article
       
  • Development of sequential and simultaneous bacterial cultures to hydrolyse
           and detoxify wood pre-hydrolysate for enhanced acetone-butanol-ethanol
           (ABE) production
    • Abstract: Publication date: Available online 4 October 2019Source: Enzyme and Microbial TechnologyAuthor(s): Mariem Theiri, Hassan Chadjaa, Mariya Marinova, Mario JolicoeurAbstractThe use of microorganisms is a promising option for an eco-efficient and successful conversion of hardwood hemicelluloses to biofuels. The focus of this work is the treatment of hemicellulosic pre-hydrolysate by flocculation, followed by simultaneous or separate detoxification with Ureibacillus thermosphaericus and Cupriavidus taiwanensis co-culture, and hydrolysis with Paenibacillus campinasensis. A reduction of phenolic compounds up to 56% was achieved mainly after flocculation, applied as a first detoxification step, but no increase in sugars concentration was observed. The ABE fermentation of the hydrolysate obtained from the simultaneous hydrolysis and detoxification produced 6.8 g L-1 of butanol after 116 h, which was higher than that generated with xylose synthetic medium. The higher biofuel concentration in the hydrolysate is attributed to the existence of carbon sources, other than xylose.
       
 
 
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