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Enzyme and Microbial Technology

Amir Sk Hossain, Renata Teparić, Vladimir Mrša
In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to β-1,3-glucan...
April 2019: Enzyme and Microbial Technology
Mansi Malik, Reeti Chaudhary, Chandra Shekhar Pundir
The nanoparticles of commercially available pyruvate oxidase (POx) from Aerococcus species were prepared by desolvation method, which were then characterized and covalently immobilized onto gold electrode (AuE) to construct an improved model of amperometric pyruvate biosensor. The POxNPs/Au electrode was analyzed morphologically by scanning electron microscopy (SEM). On the other hand, cyclic voltammetry studies (CV) and electrochemical impedance spectroscopy (EIS) helped in deciphering the electrochemical properties of the electrode at different stages of construction...
April 2019: Enzyme and Microbial Technology
Rahman Rahman Pour, Austine Ehibhatiomhan, Yuling Huang, Ben Ashley, Goran M Rashid, Sharon Mendel-Williams, Timothy D H Bugg
Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7-8 fold enhanced kcat /KM towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin...
April 2019: Enzyme and Microbial Technology
Hu Xu, Qian Wang, Zhiwei Zhang, Li Yi, Lixin Ma, Chao Zhai
Human rhinovirus 3C protease (HRV 3C protease) is commonly used as a tool to remove fusion tags from recombinant proteins in gene engineering due to its distinguished specificity and high activity at low temperature. This paper is aimed to simplify the strategy of removing epitope tags from target proteins with HRV 3C protease. Fusion proteins composed of a xylanase from Bacillus sp. HBP8 (xylHB) and double tags (MBP/Nus and 6×His, with an HRV 3C protease recognition site between them) were applied as substrates...
April 2019: Enzyme and Microbial Technology
Justyna Bzura, Robert Koncki
Two fully mechanized flow analysis systems for urease activity assays have been developed, characterized and compared. Both of them are based on almost the same compact system of solenoid micropumps and microvalves controlled and actuated by highly effective, low-power and economic Arduino microcontroller. For photometric detection of ammonia formed in the course of enzymatic hydrolysis of urea, the Berthelot method and the Nessler reaction have been examined. For both these detection schemes very simple dedicated optoelectronic flow-through detectors made of paired light emitting diodes have been developed...
April 2019: Enzyme and Microbial Technology
Divya Sharma, Geeta Sharma, Ritu Mahajan
The aim of this study is to enhance the production of industrially valuable xylanase and pectinase enzymes in short duration, using agrowaste extracted substrates. Conventional cum statistical multifactor analysis approaches were used in order to evaluate the effect of crude extracted substrates, supplemented for the production of xylanase-pectinase enzymes. Incorporation of crude extracted xylan (1.2 mg/ml of inoculum) and pectin (4.8 mg/ml of inoculum) substrates in inoculum resulted in maximal xylanase (320 ± 15) and pectinase titre (90 ± 8) after 48 h, using 2% wheat bran and 2% citrus peel in production medium with 48 h of fermentation time, with one variable factor at a time approach...
March 2019: Enzyme and Microbial Technology
Yefeng Yao, Qingbao Ding, Ling Ou
(Deoxy)guanosine-5'-triphosphate (5'-(d)GTP), the precursor for synthesizing DNA or RNA in vivo, is an important raw material for various modern biotechnologies based on PCR. In this study, we investigated the application of whole-cell catalysts constructed by bacterial cell surface display in biosynthetic reactions of 5'-(d)GTP from (deoxy)guanosine-5'-monophosphate (5'-(d)GMP). By N-terminal or N- and C-terminal fusion of the ice nucleation protein, we successfully displayed the GMP kinase of Lactobacillus bulgaricus and the acetate kinase of E...
March 2019: Enzyme and Microbial Technology
Samkelo Malgas, Brett I Pletschke
Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation...
March 2019: Enzyme and Microbial Technology
Harshini Pakalapati, Mohammad Asad Tariq, Senthil Kumar Arumugasamy
Recently enzymatic catalysts have replaced organic and organometallic catalysts in the synthesis of bio-resorbable polymers. Enzymatic polymerization is considered as an alternative to conventional polymerization as they are less toxic, environmental friendly and can operate under mild conditions. In this research, the enzymatic ring-opening polymerization (e-ROP) of e-caprolactone (e-CL) using Candida Antartica Lipase B (CALB) as catalyst to produce the Polycaprolactone. Two modelling techniques namely response surface methodology (RSM) and artificial neural network (ANN) have been used in this work...
March 2019: Enzyme and Microbial Technology
Valentina Perna, Andreas Baum, Heidi A Ernst, Jane W Agger, Anne S Meyer
Laccases (EC are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis...
March 2019: Enzyme and Microbial Technology
Huahua He, Chao Zhai, Meng Mei, Yi Rao, Yao Liu, Fei Wang, Lixin Ma, Zhengbing Jiang, Guimin Zhang, Li Yi
Porcine interferon-α (pIFN-α) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-α in Pichia pastoris GS115. The codon optimized pIFN-α shared 76% sequence identity with the original pIFN-α, which was inserted into the P. pastoris genome under AOX1d1-2x201 promoter and MF4I secretion sequence...
March 2019: Enzyme and Microbial Technology
Seyyed Soheil Rahmatabadi, Issa Sadeghian, Younes Ghasemi, Amirhossein Sakhteman, Shiva Hemmati
The enzyme phenylalanine ammonia lyase (PAL) is of special importance for the treatment of phenylketonuria patients. The aim of this study was to find a stable recombinant PAL with suitable kinetic properties among all natural PAL producing species using in silico and experimental approaches. To find such a stable PAL among 481 natural isoforms, 48,000 of 3-D models were predicted using the Modeller 9.10 program and evaluated by Ramachandran plot. Correlation analysis between Ramachandran plot and the energy of different thermodynamic components indicated that this plot could be an appropriate tool to predict protein stability...
March 2019: Enzyme and Microbial Technology
Xueqiao Xie, Peng Luo, Juan Han, Tong Chen, Yun Wang, Yunfeng Cai, Qian Liu
In the present study, a novel and efficient immobilization for horseradish peroxidase (HRP) had been developed by using 6-arm magnetic composite microsphere (Fe3 O4 @PAA-6-arm-PEG-NH2 ) containing 6-arm polyethylene glycol (6-arm-PEG-NH2 ) and Fe3 O4 . The morphology and chemical properties of Fe3 O4 @PAA-6-arm-PEG-NH2 were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FTIR), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA)...
March 2019: Enzyme and Microbial Technology
Gema Núñez-López, Azucena Herrera-González, Lázaro Hernández, Lorena Amaya-Delgado, Georgina Sandoval, Anne Gschaedler, Javier Arrizon, Magali Remaud-Simeon, Sandrine Morel
Fructosylation can significantly improve the solubility, stability and bioactivity of phenolic compounds, increasing their health benefits. Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC was found to transfer the fructosyl unit of sucrose to different classes of phenolic compounds. Among the various acceptors tested, the isoflavone puerarin and the phenol coniferyl alcohol were the most efficiently fructosylated compounds, with conversion rates of 93% and 25.1%, respectively. In both cases, mono-, di-, and trifructosides were synthesized at a ratio of 37:14:1 and 32:8:1, respectively...
March 2019: Enzyme and Microbial Technology
Shujin Pu, Xuan Zhang, Chengli Yang, Sidra Naseer, Xutong Zhang, Jie Ouyang, Dali Li, Junfang Yang
Metal-organic framework (MOF) has generated a lot of research interests for it can be employed as immobilization material for enzyme. There are many small molecules in enzyme solution during the extraction process, such as NaCl. It is important to study the effects of the small molecules on MOFs. Here we report a facile and efficient method to encapsulate (R)-1-phenylethanol dehydrogenase ((R)-PEDH) into zeolitic imidazolate framework-8 (ZIF-8). In this work, the effects of NaCl on shape of ZIF-8 and enzyme encapsulation have been investigated...
March 2019: Enzyme and Microbial Technology
Yao Chen, Hao Chu, Wei Liu, Wei Feng
3,4 Dihydroxy phenyl l-alanine (L-DOPA) is the gold standard Parkinson's disease therapy. A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA using H2 O2 as the co-substrate. d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids (e.g. d-alanine), and H2 O2 is evolved. However, both the enzymes DAAO and HDP can be inactivated by H2 O2 during the catalysis. In situ generation and utilization of H2 O2 can siginificatly reduce the inactivation by H2 O2 ...
February 2019: Enzyme and Microbial Technology
Weixin Zhao, Guocheng Du, Song Liu
Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK)2 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions...
February 2019: Enzyme and Microbial Technology
Zohreh Nowroozi-Nejad, Bahram Bahramian, Saman Hosseinkhani
We investigated the mechanism of luciferase immobilization on a solid surface through therphtalaldehyde in order to understand the role of this linker on stability and activity of luciferase. Metal organic frameworks (MOFs) are capable supports for the stabilization of some proteins and biomolecules, therefore, in this context for the first time, we report a light emmiting enzyme immobillization on one of these supports and then outline chemical developments in this process. To this end, Fe-MIL-88(NH2) was used to immobilize native luciferase and therphtalaldehyde linker was attached to the framework as an activated group...
February 2019: Enzyme and Microbial Technology
Yuping Shen, Man Wang, Yufei Chen, Lili Xu, Yi Lu, Yiying Zhou, James P Tam, Feng Han, Huan Yang, Xiaobin Jia
Sagittatoside B, a rare secondary flavonol glycoside in Epimedii Folium, has much better in vivo bioactivities than its original glycoside epimedin B. Its preparation methods, such as acidic hydrolysis, are of low efficiency, and byproducts are generated. The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on recyclable and integrated biphase enzymatic hydrolysis. β-glucanase was selected from five commercial enzymes based on the best catalysis performance...
February 2019: Enzyme and Microbial Technology
Christin Fischer, Thomas Kleinschmidt
In this study the effect of glucose depletion using glucose oxidase and catalase, simultaneously to the synthesis of prebiotic galactooligosaccharides (GOS) by β-galactosidase was studied. Considering total GOS yield, a strong dependency on the source of β-galactosidase was found. Using an Aspergillus oryzae lactase, a small increase in GOS yield (from 50.0 ± 1.3 g/L to 54.1 ± 1.9 g/L) was noted. Due to the decreased rate of GOS disaccharide formation by the Kluyveromyces lactis enzyme, the total GOS yield was diminished (from 47...
February 2019: Enzyme and Microbial Technology
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