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Enzyme and Microbial Technology

Theerasak Rojanarata, Wanida Laiwattanapaisal, Tanasait Ngawhirunpat, Praneet Opanasopit, Kalvalin Worrawethanakul, Takron Chantadee, Thitirat Fuangwattana
A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d-4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l-glutamic acid using d-phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration...
September 2018: Enzyme and Microbial Technology
Maria G Savvidou, Alexandra Katsabea, Pavlos Kotidis, Diomi Mamma, Theopisti V Lymperopoulou, Dimitris Kekos, Fragiskos N Kolisis
The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol-1 . The enzyme's enthalpy (ΔΗ*), entropy (ΔS*) and Gibb's free energy (ΔG*) were in the range of 65...
September 2018: Enzyme and Microbial Technology
Malwina Ewa Sosnowska, Urszula Jankiewicz, Marta Kutwin, André Chwalibog, Agnieszka Gałązka
Cells of Escherichia coli Rosetta, containing plasmid pET-28a with sequences of DNA of chitinase from Stenotrophomonas maltophilia N4, were used for the efficient synthesis of recombinant chitinolytic enzyme. The objective of this study was to improve thermal stability of the recombinant chitinase by salts and metal nanoparticles (NP). The studied chitinase was thermolabile and largely lost its activity in the first minutes of storage at 50 and 60 °C. The optimum temperature for colloidal chitin hydrolysis by the enzyme was 50 °C...
September 2018: Enzyme and Microbial Technology
Fidal V T, Chandra T S
A new approach to deposition of electroactive ZnO thin films have been carried out, by one-pot chemical bath deposition with Al dopant and incorporation of neutral red as organic mediator. The morphological, structural and functional characterization of the neutral red incorporated, Al-doped ZnO (NR-AZO) film was carried out using electron microscopy, FTIR, XRD and EIS respectively. The incorporated neutral red was found to induce strain in the crystal of AZO proportional to the concentration used in depositing solution which further affected the charge transfer resistance of the films in solution...
September 2018: Enzyme and Microbial Technology
Line Munk, Mogens Larsen Andersen, Anne S Meyer
Laccases (EC catalyze oxidation of phenolic groups in lignin to phenoxyl radicals during reduction of O2 to H2 O. Here, we examine the influence on this radical formation of mediators which are presumed to act by shuttling electrons between the laccase and the subunits in lignin that the enzyme cannot approach directly. Treatments of three different lignins with laccase-mediator-systems (LMS) including laccases derived from Trametes versicolor and Myceliophthora thermophila, respectively, and four individual mediators, 1-hydroxybenzotriazole (HBT), N-hydroxyphthalimide (HPI), 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were assessed by real time electron paramagnetic resonance measurements...
September 2018: Enzyme and Microbial Technology
Gisela Ibáñez-Redín, Tiago Almeida Silva, Fernando Campanhã Vicentini, Orlando Fatibello-Filho
Carbon Black (CB) has acquired a prominent position as a carbon nanomaterial for the development of electrochemical sensors and biosensors due to its low price and extraordinary electrochemical and physical properties. These properties are highly dependent on the surface chemistry and thus, the effect of functionalization has been widely studied for different applications. Meanwhile, the influence of CB functionalization over its properties for electroanalytical applications is still being poorly explored. In this study, we describe the use of chemically functionalized CB Vulcan XC 72R for the development of sensitive electrochemical biosensors...
September 2018: Enzyme and Microbial Technology
An Su, Abhijit Shirke, Joshua Baik, Yi Zou, Richard Gross
Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields >98% were achieved for all three cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 °C for 24 h...
September 2018: Enzyme and Microbial Technology
Shruti Bindal, Vikas Kumar Dagar, Meenu Saini, Yogender Pal Khasa, Rani Gupta
Increasing demand of microbial γ-glutamyl transpeptidase (GGT) in food and pharmaceutical sectors raised the need for process development for high level production of the enzyme. In this respect, GGT from Bacillus licheniformis ER15 (SBLGGT) was cloned along with its native secretion signal and expressed in E. coli using different expression vectors. Native signal of the enzyme assistedits extracellular translocationin E. coli.Maximum enzyme expression was shown by construct pET51b-sblggt,in comparison to other clones, in E...
September 2018: Enzyme and Microbial Technology
Maria Bawn, Fabiana Subrizi, Gary J Lye, Tom D Sheppard, Helen C Hailes, John M Ward
The use of biocatalysis for the synthesis of high value added chemical building blocks derived from biomass is becoming an increasingly important application for future sustainable technologies. The synthesis of a higher value chemical from l-arabinose, the predominant monosaccharide obtained from sugar beet pulp, is demonstrated here via a transketolase and transaminase coupled reaction. Thermostable transketolases derived from Deinococcus geothermalis and Deinococcus radiodurans catalysed the synthesis of l-gluco-heptulose from l-arabinose and β-hydroxypyruvate at elevated temperatures with high conversions...
September 2018: Enzyme and Microbial Technology
Quang Anh Tuan Le, Hee Gon Kim, Yong Hwan Kim
The electro-biocatalytic conversion of CO2 into formic acid using whole-cell and isolated biocatalysts is useful as an alternative route for CO2 sequestration. In this study, Shewanella oneidensis MR-1 (S. oneidensis MR-1), a facultative aerobic bacterium that has been extensively studied for its utility as biofuel cells as well as for the detoxification of heavy metal oxides (i.e., MnO2 , uranium), has been applied for the first time as a whole-cell biocatalyst for formic acid synthesis from gaseous CO2 and electrons supplied from an electrode...
September 2018: Enzyme and Microbial Technology
Se Jun Kim, Gyong Sik Ha, Gyunghwa Lee, Sung In Lim, Chung Min Lee, Yoo Hee Yang, JaeMin Lee, Ju Eun Kim, Jae Hee Lee, YongUk Shin, Chan-Wha Kim, Dong Eok Lee
Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity...
August 2018: Enzyme and Microbial Technology
Hiroyuki Okano, Misato Baba, Ryota Hidese, Kei Iida, Tongyang Li, Kenji Kojima, Teisuke Takita, Itaru Yanagihara, Shinsuke Fujiwara, Kiyoshi Yasukawa
We evaluated fidelity of various reverse transcriptases (RTs) by a novel method with modified next-generation sequencing (NGS). In the optimized condition, one NGS run could handle cDNA products from multiple cDNA synthesis reactions performed at different conditions. This was achieved using a primer containing not only the tag of 14 randomized bases to label each cDNA molecule but also a tag of five bases to label each reaction condition. With this method, we quantitated the error rates of 44 cDNA synthesis reactions by retroviral RTs or genetically engineered DNA polymerases with RT activity under different conditions...
August 2018: Enzyme and Microbial Technology
Paz García-García, Javier Rocha-Martin, Gloria Fernandez-Lorente, Jose M Guisan
Diamine oxidase (DAO) from Pisum sativum is an enzyme that catalyzes the degradation of biogenic amines (BA) present in wine, producing harmless aldehydes and hydrogen peroxide (H2 O2 ). H2 O2 promotes a rapid inactivation of the immobilized enzyme. At first glance, co-immobilization of DAO and catalase (CAT) could improve the elimination of the released hydrogen peroxide. Two different co-immobilized derivatives were prepared: (a) both enzymes co-localized and homogeneously distributed across the whole structure of a porous support, and (b) both enzymes we de-localized inside the porous support: DAO immobilized on the outer part of the porous support and catalase immobilized in the inner part...
August 2018: Enzyme and Microbial Technology
Xue Cai, Ines Seitl, Wanmeng Mu, Tao Zhang, Timo Stressler, Lutz Fischer, Bo Jiang
A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41-67% identity to each other). The encoded open reading frames were used as templates for further in silico screening...
August 2018: Enzyme and Microbial Technology
Xiaoyi Li, Yan Xiao, Yingang Feng, Bin Li, Wenli Li, Qiu Cui
Low-cost saccharification is one of the key bottlenecks hampering the further application of lignocellulosic biomass. Clostridium thermocellum is a naturally ideal cellulose degrading bacterium armed with cellulosomes, which are multienzyme complexes that are capable of efficiently degrading cellulose. However, under controlled condition, the inhibition effect of hydrolysate cellobiose severely restricts the hydrolytic ability of cellulosomes. Although the addition of beta-glucosidase (Bgl) could effectively relieve this inhibition, the spatial proximity effect of Bgl and cellulosomes on cellulose degradation is still unclear...
August 2018: Enzyme and Microbial Technology
Ling Lin, Ying Wang, Mianbin Wu, Li Zhu, Lirong Yang, Jianping Lin
Fumarases have been successfully applied in industry for the production of l-malate. However, the industrialization of fumarases is limited by their low thermostability. In this study, the thermostability of fumarase C from Corynebacterium glutamicum was enhanced through directed evolution, simulated mutagenesis, site-directed mutagenesis and saturated mutagenesis. Mutant 2G (A411V) was initially constructed through directed evolution. Its half-life at 50 °C (t1/2 , 50°C ) increased from 1 min to 2.2 min, and the T50 15 (temperature at which the activity of enzyme decreased by 50% in 15 min) increased from 44...
August 2018: Enzyme and Microbial Technology
Birgitte Zeuner, Marlene Vuillemin, Jesper Holck, Jan Muschiol, Anne S Meyer
The α-1,3/4-l-fucosidases (EC; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs...
August 2018: Enzyme and Microbial Technology
Yuanqing Lin, Wenhui Jin, Jindan Wang, Zhengwen Cai, Shuyu Wu, Guangya Zhang
We generated a bifunctional enzyme chimera containing the xylanase and lichenase coupled with SpyTag between them. Meanwhile, we generated another chimera containing SpyCatcher and elastin-like polypeptides (ELPs). As ELPs could bond to the xylanase-lichenase chimera through SpyTag/SpyCatcher spontaneous reaction in mild condition, which would lead to the formation of a 3-arm star multifunctional chimera. We purified the xylanase-lichenase by the non-chromatographic purification tag of ELPs. Interestingly, 57...
August 2018: Enzyme and Microbial Technology
Xian Wu, Long Zou, Yunhong Huang, Yan Qiao, Zhong-Er Long, Haoping Liu, Chang Ming Li
The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c-type cytochromes (OM c-Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c-Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact...
August 2018: Enzyme and Microbial Technology
Dhivya Ramakrishnan, Manish K Tiwari, Gomathi Manoharan, Thiagarajan Sairam, Rajesh Thangamani, Jung-Kul Lee, Jeya Marimuthu
Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions...
August 2018: Enzyme and Microbial Technology
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