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Enzyme and Microbial Technology

Ana C D Pfluck, Dragana P C de Barros, Luís P Fonseca, Eduardo P Melo
The exploitation of an efficient enzymatic system to perform biopolymers synthesis, namely polyesters from dicarboxylic acids and dialcohols, requires the evaluation of the enzyme operational stability. This becomes particularly relevant when non-conventional media, such as miniemulsions, are used due to the inhomogeneity of the reaction media and presence of surfactants and high concentrations of organic compounds which might be deleterious to the structure-function of the enzyme. The stability of three lipases, Candida sp...
July 2018: Enzyme and Microbial Technology
Seung Soo Lee, Hyojung Shin, Suah Jo, Sun-Mi Lee, Youngsoon Um, Han Min Woo
RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance production of value-added chemicals in Corynebacterium glutamicum as an industrial host. In this study, the RNA-guided CRISPR interference (CRISPRi) was applied to rapidly identify of unknown genes for native esterase activity in C. glutamicum. Combining with the carboxyl esterase (MekB) protein sequence alignment, two target genes (the cg0961 and cg0754) were selected for the CRISPRi application to investigate the possible native esterase in C...
July 2018: Enzyme and Microbial Technology
Cinzia Calvio, Fabio Romagnuolo, Francesca Vulcano, Giovanna Speranza, Carlo F Morelli
γ-Glutamyltransferase (GGT) catalyzes the transfer of the γ-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-γ-glutamic acid (γ-PGA) as a substrate...
July 2018: Enzyme and Microbial Technology
Folmer Fredslund, Martin S Borchert, Jens-Christian N Poulsen, Steen Bennike Mortensen, Mirjam Perner, Wolfgang R Streit, Leila Lo Leggio
Carbonic anhydrases (CAs) are extremely fast enzymes, which have attracted much interest in the past due to their medical relevance and their biotechnological potential. An α-type CA gene was isolated from DNA derived from an active hydrothermal vent chimney, in an effort to identify novel CAs with suitable properties for CO2 capture. The gene product was recombinantly produced and characterized, revealing remarkable thermostability, also in the presence of high ionic strength alkaline conditions, which are used in some CO2 capture applications...
July 2018: Enzyme and Microbial Technology
Zhiyan Wang, Chao Guo, Lin Liu, He Huang
Enterokinase is an ideal tool protease for cleaving fusion proteins in genetic engineering. The bovine enterokinase light chain (bEKL ) produced in Pichia pastoris was shown to be a glycoprotein. To study the effects of N-glycosylation on the biochemical properties of bEKL , the enzyme was deglycosylated via site-directed mutagenesis. The results showed that elimination of the N-glycosylation sites of bEKL (N64, N103 and N165) did not significantly affect the protein secretion level in P. pastoris, but it does greatly influence its enzymatic activity...
July 2018: Enzyme and Microbial Technology
Faseela Hamza, Ameeta Ravi Kumar, Smita Zinjarde
Coculturing microorganisms can lead to enhanced production of bioactive compounds as a result of cross-species or cross-genera interactions. In this study, we demonstrate improved production of the biosurfactant (BS-SLSZ2 with antibiofilm properties) by the marine epibiotic bacterium Staphylococcus lentus SZ2 after cross-genera interactions with an aquaculture pathogen Vibrio harveyi. In cocultures, growth of V. harveyi was completely inhibited and resultant biofilms were exclusively composed of S. lentus. The cell free supernatant (CFS) derived from cocultures displayed improved antibiofilm activity with enhanced contents of BS-SLSZ2 compared to monocultured S...
July 2018: Enzyme and Microbial Technology
Douglas B Jordan, J Rose Stoller, Rena E Kibblewhite, Victor J Chan, Charles C Lee, Kurt Wagschal
Two GH43 β-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His...
July 2018: Enzyme and Microbial Technology
Longbao Zhu, Fei Ge, Wanzhen Li, Ping Song, Hongjin Tang, Yugui Tao, Yi Liu, Guocheng Du
Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield β-phe. However, the final product mixture consists of both α- and β-phe owing to low regioselectivity, which is still a challenge to synthesize highly pure β-phe. Therefore, a modified TcPAM with high β-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn458 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroamination of t-CA and as promising candidates for mutagenesis to enhance β-selectivity and decrease α-selectivity...
July 2018: Enzyme and Microbial Technology
Yeo-Ul Park, Jong-Hyun Jung, Dong-Ho Seo, Dong-Hyun Jung, Jae-Han Kim, Ean-Jeong Seo, Nam-In Baek, Cheon-Seok Park
Desulfurococcus amylolyticus is an anaerobic and hyperthermophilic crenarchaeon that can use various carbohydrates as energy sources. We found a gene encoding a glycoside hydrolase family 57 amylolytic enzymes (DApu) in a putative carbohydrate utilization gene cluster in the genome of D. amylolyticus. This gene has an open reading frame of 1,878 bp and consists of 626 amino acids with a molecular mass of 71 kDa. Recombinant DApu (rDApu) completely hydrolyzed pullulan to maltotriose by attacking α-1,6-glycosidic linkages, and was able to produce glucose and maltose from soluble starch and amylopectin...
July 2018: Enzyme and Microbial Technology
Abdelaziz Bouchaib, Abdellatif Ben Abdellah, Mohamed Bakkali, Amine Laglaoui, Abdelhay Arakrak, Carlo Carboni, Abdelaziz Benjouad, Amal Maurady
The present work describes a method for detecting the ingress of gas phase oxygen into packed food. It uses the enzyme polyphenol oxidase (PPO)from Mushroom and Mediterranean dwarf palm. The PPO is incorporated into an indubiose film along with a non-toxic polyphenol such as gallic acid or chlorogenic acid. If exposed to oxygen, the test spot undergoes an irreversible and visible color change from pale to deep brown due to the PPO catalyzed oxidation of the respective polyphenol by oxygen. The color change can be detected visually or by spectrophotometry at 470 nm...
July 2018: Enzyme and Microbial Technology
Steven D Kane, Christopher E French
Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format...
June 2018: Enzyme and Microbial Technology
HongLing Liu, ShaoJie Yang, Qiang Liu, Ruiming Wang, Tengfei Wang
The process for production of trehalose using trehalose synthase (TreS) to convert maltose into trehalose in one step is highly desirable in the industry. Nonetheless, the studies on industrial-scale production of trehalose by recombinant TreS in Escherichia coli are still scarce. In this study, a TreS from Pseudomonas putida ATCC47054 was expressed in E. coli BL21(DE3) via plasmids pET15b and pET22b. pET15b-treS showed better plasmid stability and TreS expression, which revealed that the highest activity, 39866±1420U/(g dry cell weight) at the final lactose concentration of 4g/L for 7h at 27°C in a 5-L fermentor at pH 8...
June 2018: Enzyme and Microbial Technology
Cynthia Sanhueza, Gonzalo Carvajal, Javiera Soto-Aguilar, Maria Elena Lienqueo, Oriana Salazar
Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan...
June 2018: Enzyme and Microbial Technology
A K M Kafi, M Naqshabandi, Mashitah M Yusoff, Maxwell J Crossley
A new 3-dimensional (3D) network of crosslinked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface has been described in order to build up the effective electrical wiring of the enzyme units with the electrode. The synthesized 3D HRP/CNT network has been characterized with cyclic voltammetry and amperometry which results the establishment of direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the high biological activity and stability is exhibited by the immobilized HRP and a quasi-reversible redox peak of the redox centre of HRP was observed at about -0...
June 2018: Enzyme and Microbial Technology
Haikun Zhang, Xiaoke Hu
Recovery of noble metals using marine bacteria is becoming an attractive research area because the marine microbes can better adapt to unfavorable environment than terrestrial microorganisms. In this study, we first reported that a marine Bacillus sp. GP was capable of producing Pd and Au NPs in the presence of sodium lactate. Ultraviolet visible spectrometer (UV-vis), transmission electron microscopy (TEM), X-ray diffraction patterns (XRD), X-ray photoelectron spectroscopy (XPS) and fourier transform infrared spectroscopy (FTIR) analyses were employed to explain the process and mechanism of Pd(II)/Au(III) reduction through GP...
June 2018: Enzyme and Microbial Technology
Zhe Xu, Ting Cai, Neng Xiong, Shu-Ping Zou, Ya-Ping Xue, Yu-Guo Zheng
Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability...
June 2018: Enzyme and Microbial Technology
Vinay Narwal, Parveen Kumar, Pooja Joon, C S Pundir
An amperometric sarcosine biosensor was fabricated based on covalent immobilization of sarcosine oxidase (SarOx) onto the nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT)/chitosan (CHIT) and copper nanoparticles (CuNPs), electrodeposited on gold (Au) electrode. The SarOx/CHIT/CuNPs/c-MWCNT/Au electrode was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The enzyme electrode exhibited optimum current within 2 s at a potential of 0...
June 2018: Enzyme and Microbial Technology
Xuemei Zhang, Jie Ren, Peiyuan Yao, Rui Gong, Min Wang, Qiaqing Wu, Dunming Zhu
Reversible benzoic acid decarboxylases are versatile biocatalysts by taking advantage of both decarboxylation and carboxylation reactions, especially for the biocatalytic Kolbe-Schmitt reaction. In the course of developing a benzoic acid decarboxylase tool-box, a putative benzoic acid decarboxylase gene from Fusarium oxysporum was heterologously over-expressed in Escherichia coli, the recombinant protein was purified and characterized. The purified enzyme exhibited relatively high catalytic efficiencies for the decarboxylation of 2, 3-dihydroxybenzoic acid and carboxylation of catechol (kcat /Km  = 2...
June 2018: Enzyme and Microbial Technology
Sumangala Darsandhari, Dipesh Dhakal, Biplav Shrestha, Prakash Parajuli, Joo-Hyun Seo, Tae-Su Kim, Jae Kyung Sohng
A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp...
June 2018: Enzyme and Microbial Technology
Gonzalo de Gonzalo, Antonio Franconetti
The recently discovered cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) was employed in the preparation of different optically active sulfoxides. This novel Baeyer-Villiger monooxygenase (BVMO) shows a similar substrate specificity and selectivity when compared with the prototype CHMO from Acinetobacter calcoaceticus. Interestingly, TmCHMO is able to catalyse the asymmetric sulfoxidations at relatively high temperatures, substrate concentrations and in the presence of organic solvents while maintaining or even increasing its selectivity...
June 2018: Enzyme and Microbial Technology
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