JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
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2016
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IF 2.189
Min Li,Zheng Zhou,Zhi-Jun Zhang,Hui-Lei Yu,Jian-He Xu
Nitrile reductase is a newly discovered enzyme class that can catalyze the reduction of nitriles directly to amines, a very important reaction in synthetic organic chemistry. However, little progress has been made towards the application of this biocatalysis reaction for organic synthesis since the initial discovery of nitrile reductase for over ten years. One of the potential reasons may be due to the lack of structural information and biochemical properties, in addition to its inherent narrow substrate scope. Meanwhile, the highly conserved active site residues make it a great challenge to widen the substrate spectrum of nitrile reductase by means of protein engineering approaches. To explore more nitrile reductases with different characteristics ( e.g . specific activity, thermostability, etc ) for potential synthetic applications, a new nitrile reductase, designated as Pc NRed, was discovered from the genome of Pectobacterium carotovorum , with a moderate specific activity of 366 U/g protein towards its natural substrate, preQ 0 . The Pc NRed was subsequently overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and then its biochemical properties were examined. The optimal reaction temperature and pH were determined to be 40 °C and 7.4, respectively. The thermal deactivation process of Pc NRed obeys first-order kinetics, with half-lives of 47.2, 13.6 and 3.9 h measured at 30, 40, and 50 °C, respectively. The kinetic constants were also determined with respect to both of the substrates NADPH and preQ 0 , giving k cat of 0.025 s −1 and 0.024 s −1 , and K M of 0.229 mM and <0.25 μM. To shed some light on the structural information of Pc NRed, a homology model was built and compared with its counterparts, which is helpful for further engineering of this novel but less-elucidated enzymes.
研究领域:
生物化学
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酶工程
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合成生物学
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产品关联
DOI
高影响因子
CHEMBIOCHEM
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2017
·Vol. 19
·期 5
·521-526
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IF 2.847
Zheng Zhou,Min Li,Jian-He Xu,Zhi-Jun Zhang
Graphical Reduced to clear : The nitrile reductase from Pectobacterium carotovorum is able to catalyze the reduction of nitriles to primary amines with the consumption of two molecules of NADPH. A single mutation at the entrance of the substrate channel increases both the activity and stability of the nitrile reductase. Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot −1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3- d ]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3- d ]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.
研究领域:
生物化学
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合成生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY
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2021
·Vol. 56
·期 7
·3495-3506
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IF 3.713
Hongbin Pu,Yiwen Xu,Lian Lin,Da-Wen Sun
Summary During vacuum cooling, polypropylene turnover boxes are repeatedly used for containing vegetables for cooling, while Pectobacterium carotovorum subsp. carotovora ( Pcc ) can form a biofilm on polypropylene adhesion surface. This study evaluated the effects of multiple cycles of vacuum cooling on the morphology and composition of Pcc biofilm formed on polypropylene turnover boxes. Results showed that Pcc biofilm in different growth stages including adhesion, development and mature phases displayed different responses to vacuum cooling. Pcc biofilm in adhesion stage decreased from 10 6 to 10 5 CFU cm −2 , indicating the positive interference of vacuum cooling on biofilm formation in the initial developmental stage. However, with the increasing of the number of vacuum cooling cycles from 0 to 3, the biomass of Pcc biofilm in the development stage increased from 10 7 to 10 8 CFU cm −2 and the polysaccharide secretion increased from 7.96 to 16.7 mg cm −2 , showing the promotion of biofilm formation and the secretion of extracellular polymeric substances. Therefore, regular cleansing and sterilisation of turnover boxes are necessary to prevent cross-contamination during vacuum cooling.
研究领域:
微生物学
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食品科学
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生物技术
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DOI
高影响因子
TEXTILE RESEARCH JOURNAL
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2020
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IF 1.926
Yawei Wang,Yun Bai,Tong Shu,Pei Fan,Huashan Zhang,Ossi Turunen,Hairong Xiong,Longjiang Yu
A novel versatile glycoside hydrolase Cel5M was identified from Pectobacterium carotovorum and used in ramie degumming. The full-length Cel5M (GenBank: MH544226) containing the catalytic domain Cel5MD and its carbohydrate-binding module (CBM3) were expressed in E. coli. Both Cel5M and Cel5MD showed a broad substrate spectrum cleaving konjac glucomannan (KGM), carboxymethylcellulose (CMC) and oat spelt xylan. The optimal temperature of Cel5MD was 55oC with CMC and KGM, whereas the optimum temperature of Cel5M was 65oC with KGM and 60oC with CMC. The improved thermal activity with KGM was probably reflected also in the much lower Km with KGM than with CMC, indicating stronger binding of the full-length enzyme with KGM. Both enzymes were highly stable over a broad pH range from pH 5.0 to 9.0 with KGM. The use of the Cel5M enzyme together with the wild-type P. carotovorum HG-49 strain showed significant improvement when compared to using the mere strain HG-49 for application in ramie degumming. Moreover, the whole P. carotovorum Cel5M gene was over-expressed in P. carotovorum HG-49 itself to improve the ramie-degumming capacity. Altogether, this study indicates that in situ production of processing enzymes could be utilized in the textile industry.
研究领域:
生物技术
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酶学
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纺织科学
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产品关联
DOI
高影响因子
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