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Enzyme and Microbial Technology

Guangming Tian, Qin Wang, Xuetuan Wei, Xin Ma, Shouwen Chen
Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway...
April 2017: Enzyme and Microbial Technology
Meenu Saini, Shruti Bindal, Rani Gupta
Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5...
April 2017: Enzyme and Microbial Technology
Christoph Gonaus, Daniel Maresch, Katharina Schropp, Peter Ó Conghaile, Dónal Leech, Lo Gorton, Clemens K Peterbauer
Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N(75) and N(175), near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold...
April 2017: Enzyme and Microbial Technology
Fouzia Tanvir, Atif Yaqub, Shazia Tanvir, William A Anderson
In this study we report a method for the rapid and sensitive estimation of bacterial cell concentration in solution based on a colorimetric enzyme/gold nanoshells conjugate system. The CTAB capped gold nanoshells are electrostatically attracted by both the bacterial surface and the enzyme β-galactosidase. The preferential binding of cationic (CTAB)-functionalized gold nanoshells to the more negative bacterial surfaces leaves active β-galactosidase in solution, providing an enzyme-amplified colorimetric response of the binding event...
April 2017: Enzyme and Microbial Technology
Christina Andreeßen, Vanessa Gerlt, Alexander Steinbüchel
3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy...
April 2017: Enzyme and Microbial Technology
Jian Gao, Jian-Wen Huang, Qian Li, Weidong Liu, Tzu-Ping Ko, Yingying Zheng, Xiansha Xiao, Chih-Jung Kuo, Chun-Chi Chen, Rey-Ting Guo
1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1...
April 2017: Enzyme and Microbial Technology
Hui Lin, Amanda Hildebrand, Takao Kasuga, Zhiliang Fan
In a cellulosic biorefinery, the cellulase enzymes needed for hydrolysis are one of the major contributors to high processing costs, while the hydrolysis product, cellobiose, has strong inhibition to the cellulases. In this study, we report engineering recombinant Neurospora crassa strains which are able to produce cellobionate, an organic acid, from cellulose without any enzyme addition. Recombinant strains were constructed by heterologously expressing laccase genes from different sources under different promoters in N...
April 2017: Enzyme and Microbial Technology
Daniel Bakonyi, Werner Hummel
A gene encoding a novel 7α-specific NADP(+)-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition...
April 2017: Enzyme and Microbial Technology
Dong-Xu Jia, Lin Zhou, Yu-Guo Zheng
Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs...
April 2017: Enzyme and Microbial Technology
Archana Vimal, Awanish Kumar
l-asparaginase is an enzyme of medical prominence and reputable as a chemotherapeutic agent. It also has immense potential to cure autoimmune and infectious diseases. The vast application of this enzyme in healthcare sector increases its market demand. However, presently the huge market demand is not achieved completely. This serves the basis to explore better producer microbial strains to bridge the gap between huge demand and supply of this therapeutic enzyme. The present study deals with the successful screening of potent microorganisms producing l-asparaginase...
March 2017: Enzyme and Microbial Technology
Agustín Castilla, Paola Panizza, Diego Rodríguez, Luis Bonino, Pilar Díaz, Gabriela Irazoqui, Sonia Rodríguez Giordano
Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain...
March 2017: Enzyme and Microbial Technology
Shotaro Matsumoto, Hiroyuki Yamada, Yuika Kunishige, Shigeo Takenaka, Masami Nakazawa, Mitsuhiro Ueda, Tatsuji Sakamoto
Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78...
March 2017: Enzyme and Microbial Technology
Alireza Madjid Ansari, Keivan Majidzadeh-A, Behrad Darvishi, Hassan Sanati, Leila Farahmand, Dariush Norouzian
In this study attempts were made to investigate the effect of extremely low frequency magnetic field (ELF-MF) on expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris GS115 at optimal environmental conditions. GOX encoding gene from A. niger was expressed in P. pastoris GS115 employing methanol inducible AOX1 promoter. From resistant transformants to high zeocin concentrations, maximum GOX expressing transformant was isolated and used throughout the study. Optimal environmental conditions for achieving maximum glucose oxidase (GOX) production was determined by varying temperature (20-30°C) and final methanol concentration from 0...
March 2017: Enzyme and Microbial Technology
Brian C Pierce, Jane Wittrup Agger, Jesper Wichmann, Anne S Meyer
The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography...
March 2017: Enzyme and Microbial Technology
Lei Zhao, Li He, Shujuan Chen, Likou Zou, Kang Zhou, Xiaolin Ao, Shuliang Liu, Xinjie Hu, Guoquan Han
Biochemical oxygen demand (BOD) sensors based on Zr (IV)-loaded collagen fiber (ZrCF), a novel material with great porous structure, were developed. This novel material shows adsorbability by microorganisms. Saccharomyces cerevisiae and Escherichia coli were used for the construction of BOD sensors. Factors affecting BOD sensor performance were examined. The ZrCF-based BOD sensor showed different sensitivities and linear response ranges with different biofilm densities. The amount of microorganisms strongly affected the performance of the BOD sensor...
March 2017: Enzyme and Microbial Technology
A S Zaitseva, V A Arlyapov, N Yu Yudina, S V Alferov, A N Reshetilov
We investigated the use of one- and two-mediator systems in amperometric BOD biosensors (BOD, biochemical oxygen demand) based on the yeast Debaryomyces hansenii. Screening of nine mediators potentially capable of electron transfer - ferrocene, 1,1'-dimethylferrocene, ferrocenecarboxaldehyde, ferroceneacetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methylene blue and potassium ferricyanide - showed only ferrocene and neutral red to be efficient electron carriers for the eukaryotes studied...
March 2017: Enzyme and Microbial Technology
Peng-Cheng Chen, Pu Zheng, Xiang-Yu Ye, Fan Ji
A new applicability of cell-immobilized system for biological production of target chemical was reported in this work. A. succinogenes CCTCC M2012036 was immobilized on positively charged polypropylene microfiber membrane by physical interaction and were used for converting glucose into succinic acid. Glucose consumption and succinic acid production kinetics were investigated for optimizing the operational parameters. The cell-immobilized membrane presented good reuse stability, and six cycles of fermentation without activity loss were realized with an average succinic acid yield of 0...
March 2017: Enzyme and Microbial Technology
David Gervais, Justin Hayzen, Charlotte Orphanou, Alexandra McEntee, Christine Hallam, Rossalyn Brehm
During Erwinia chrysanthemil-asparaginase (ErA) manufacture, minor conformational variants are formed that elute in the acidic region of the analytical ion-exchange HPLC chromatogram. These variants retain enzymatic activity and form part of the biopharmaceutical product, but must be kept within acceptable limits through controlled operation of the manufacturing process. The high isoelectric point of the ErA native tetramer (8.6) leads to certain process steps being operated in the alkaline pH region. Previously, the formation of these species during processing was not fully understood...
March 2017: Enzyme and Microbial Technology
Laura Fernandez-Lopez, Sara G Pedrero, Nerea Lopez-Carrobles, Beatriz C Gorines, Jose J Virgen-Ortíz, Roberto Fernandez-Lafuente
Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one...
March 2017: Enzyme and Microbial Technology
Ishak Muhammad Syafiq, Kai-Hee Huong, K Shantini, S Vigneswari, Nursolehah Abd Aziz, Al-Ashraf Abdullah Amirul, Kesaven Bhubalan
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction...
March 2017: Enzyme and Microbial Technology
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