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Enzyme and Microbial Technology

Cun-Duo Tang, Hong-Ling Shi, Qing-Hai Tang, Jun-Shi Zhou, Lun-Guang Yao, Zhu-Jin Jiao, Yun-Chao Kan
Two novel glycosyl hydrolase family 5 (GH5) β-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9K(M). The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3...
November 2016: Enzyme and Microbial Technology
Khadijeh Alishah, Sedigheh Asad, Khosro Khajeh, Neda Akbari
Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention...
November 2016: Enzyme and Microbial Technology
Fiyinfoluwa A Adesioye, Thulani P Makhalanyane, Peter Biely, Don A Cowan
Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect...
November 2016: Enzyme and Microbial Technology
Xunyan Dong, Yue Zhao, Jinyu Hu, Ye Li, Xiaoyuan Wang
The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C...
November 2016: Enzyme and Microbial Technology
Chao Wang, Xianhong Ouyang, Sisi Su, Xiao Liang, Chao Zhang, Wenya Wang, Qipeng Yuan, Qiang Li
NAD(+)-dependent Cα-dehydrogenase LigD and glutathione-dependent β-etherase LigF which selectively cleave the β-O-4 aryl ether linkage present in lignin, are key-enzymes for the biocatalytic depolymerization of lignin. However, the catalytic efficiency of the two enzymes is low when they are used to break down the β-aryl ether linkage in natural lignin. When sulfonated lignin was added to LigF hydrolysis reactions, the conversion rate of MPHPV decreased significantly from 99.5% to 32.6%. On the contrary, sulfonated lignin has little affection on LigD, which the conversion rate of GGE only decreased from 41...
November 2016: Enzyme and Microbial Technology
Nannan Zhang, Jinbao Wang, Yang Zhang, Haijun Gao
1,2,4-Butanetriol (BT) and related derivatives have been widely used in many fields, especially in the military and in medicine. In this paper, we systematically optimized the BT biosynthetic pathway. We first investigated the activities of various NADH dependent aldehyde reductases (ALRs), which catalyze the fourth reaction in the four-step pathway for BT production from xylose in E. coli, and found that a combination of multiple endogenous enzymes catalyzed aldehyde reduction in the BT production bioprocess and that YqhD in E...
November 2016: Enzyme and Microbial Technology
Le Minh Tran, Seung Hyuck Bang, Jihee Yoon, Yang-Hoon Kim, Jiho Min
In this study, this protein was overexpressed in yeast cells grown on trehalose-containing medium to assess its impact on yeast vacuolar activity. ATH was confirmed to be located in both cell surface and vacuoles and the overexpression of ATH was observed to decrease vacuolar activity. Therefore, an assumption was suggested to explain this phenomenon as follows: when grown on containing trehalose medium, the ATH localization at cellular periplasm, but not the vacuole, is prioritized to utilize the extracellular trehalose for cell growth...
November 2016: Enzyme and Microbial Technology
Anna M Knapinska, Sabrina Amar, Zhong He, Sandro Matosevic, Claudia Zylberberg, Gregg B Fields
Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs)...
November 2016: Enzyme and Microbial Technology
S Akram Shirdel, Khosrow Khalifeh, Bijan Ranjbar, Abolfazl Golestani, Khosro Khajeh
We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp(689) was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25°C for 3 weeks indicates that all mutants remain considerably active at 4°C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2min keeping at 40°C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate...
November 2016: Enzyme and Microbial Technology
Zhuotao Tan, Hongmin Ma, Qing Li, Lingling Pu, Yang Cao, Xudong Qu, Chenjie Zhu, Hanjie Ying
The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of enzymes in reaction, economic, ecological issues. Many carbonyl reductases for producing chiral alcohols have been reported but there is still a lack of good catalytic efficacies. Herein, five carbonyl reductases from different Streptomyces were discovered by the strategy of genome mining. These reductases were overexpressed, and we chose SgCR for further study as it owned better enzyme activity...
November 2016: Enzyme and Microbial Technology
Yu-Rong Cheng, Zhi-Jie Sun, Gu-Zhen Cui, Xiaojin Song, Qiu Cui
Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp...
November 2016: Enzyme and Microbial Technology
Mohsen Mohammadi, Zargham Sepehrizadeh, Azadeh Ebrahim-Habibi, Ahmad Reza Shahverdi, Mohammad Ali Faramarzi, Neda Setayesh
Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis...
November 2016: Enzyme and Microbial Technology
Roswanira Abdul Wahab, Mahiran Basri, Raja Noor Zaliha Raja Abdul Rahman, Abu Bakar Salleh, Mohd Basyaruddin Abdul Rahman, Thean Chor Leow
Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity...
November 2016: Enzyme and Microbial Technology
Dal Rye Kim, Hee Kyung Lim, Kee In Lee, In Taek Hwang
The model 3-D structure of xylanase KRICT PX3 (JF320814) identified by DNA sequence analysis revealed a catalytic domain and CBM4-9 which functions as a xylan binding domain (XBD). To identify its role in xylan hydrolysis, six expression plasmids were constructed encoding the N-terminal CBM plus the catalytic domain or different glycosyl hydrolases, and the biochemical properties of the recombinant enzymes were compared to the original structure of PX3 xylanase. All six of the recombinant xylanases with the addition of CBM in the pIVEX-GST expression vector showed no improved PX3 hydrolytic activity...
November 2016: Enzyme and Microbial Technology
Thi Thanh Hanh Nguyen, Seong-Bo Kim, Nahyun M Kim, Choongil Kang, Byoungsang Chung, Jun-Seong Park, Doman Kim
Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer...
November 2016: Enzyme and Microbial Technology
Mi-Ji Yu, Sun-Hee Yoon, Young-Wan Kim
Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs...
November 2016: Enzyme and Microbial Technology
Bassam Alkotaini, Nam Soo Han, Beom Soo Kim
Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar...
November 2016: Enzyme and Microbial Technology
Scott M Plummer, Mark A Plummer, Patricia A Merkel, Moira Hagen, Jennifer F Biddle, Lisa A Waidner
Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production...
November 2016: Enzyme and Microbial Technology
Eric Lorenz, Maximilian Schmacht, Martin Senz
Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S...
November 2016: Enzyme and Microbial Technology
Kwabena O Duedu, Christopher E French
Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii...
November 2016: Enzyme and Microbial Technology
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