Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli / 生物工程学报

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.

Application of bioinformatics in researches of industrial biocatalysis / 生物工程学报

Industrial biocatalysis is currently attracting much attention to rebuild or substitute traditional producing process of chemicals and drugs. One of key focuses in industrial biocatalysis is biocatalyst, which is usually one kind of microbial enzyme. In the recent, new technologies of bioinformatics have played and will continue to play more and more significant roles in researches of industrial biocatalysis in response to the waves of genomic revolution. One of the key applications of bioinformatics in biocatalysis is the discovery and identification of the new biocatalyst through advanced DNA and protein sequence search, comparison and analyses in Internet database using different algorithm and software. The unknown genes of microbial enzymes can also be simply harvested by primer design on the basis of bioinformatics analyses. The other key applications of bioinformatics in biocatalysis are the modification and improvement of existing industrial biocatalyst. In this aspect, bioinformatics is of great importance in both rational design and directed evolution of microbial enzymes. Based on the successful prediction of tertiary structures of enzymes using the tool of bioinformatics, the undermentioned experiments, i.e. site-directed mutagenesis, fusion protein construction, DNA family shuffling and saturation mutagenesis, etc, are usually of very high efficiency. On all accounts, bioinformatics will be an essential tool for either biologist or biological engineer in the future researches of industrial biocatalysis, due to its significant function in guiding and quickening the step of discovery and/or improvement of novel biocatalysts.

Study on production of acrylamide by microbial method (I)--Culture of bacterium cells and expression of high activity of nitrile hydratase / 生物工程学报

The cultural conditions for the growth of Norcardia cell were studied in this paper. Controlling pH value, adding nutrient and optimizing the quantity of inducer during cultivation, the activity of nitrile hydratase reached 6567 u/mL (culture medium), which was the highest value appeared in native journals. In the farther hydratase experiments, no by-product, crylic acid, was detected. It showed that the activity of amidase was not promoted obviously while the activity of nitrile hydratase was increased greatly. The results set a strong foundation for the industrial application and the research on new technology.

Study on production of acrylamide by microbial method (II)--enzyme catalytic kinetics and de-active dynamics of nitrile hydratase / 生物工程学报

The hydration reaction by microbial method is the crisis of the procedure of acrylamide production from acrylonitrile. This research studied the enzyme catalytic kinetics and de-active kinetics of nitrile hydratase in the type of free cell. Firstly, the effects of the concentration of cells, the temperature, pH value, the concentration of acrylonitrile and the concentration of acrylamide on the activity of nitrile hydratase was investigated. The result is that the temperature and the concentration of acrylamide are the most important among these factors. The activity of the nitrile hydratase was 5659 u/mL (broth) at 28 degrees C; the counterpart was only 663 u/mL (broth) at 5 degrees C. And the activity of NHase in solution of 45% acrylamide was just about half of that in solution of 5% acrylamide. After study on the relation of temperature and the reaction speed, It was found that the activation energy of the hydration of NHase was 65.57 kJ.mol-1. This paper studied the effects of concentration of cells, temperature, pH value, concentrations of acrylonitrile and acrylamide on the deactivation of Nhase, as well as the related enzyme de-active kinetics. The result also showed that the temperature and the concentration of acrylamide are the most important among these factors. In solution of 35% acrylamide, the residual activity was about 0% of the original value after 55 h; but in solution of 10% acrylamide, after the same period of time, the residual activity was 50% of the original one. It was also found that the concentration of acrylonitrile had little effect on the stability of NHase. The coefficient of deactivation at 28 degrees C was 21.77 times of the one at 5 degrees C. Correlating the temperature and the coefficient of deactivation, the activation energy of the de-active reaction was found to be 92.28 kJ.mol-1.