Screening and Identification of Elastase-High-Producing Strain and Preliminary Study on Its Fermentation Conditions

Abstract: Elastase is one of the extend-spectrum endopeptidases with the characteristic of hydrolyzing insoluble elastin. In medicine, food, agriculture, chemical industry and other areas, it has been widely used, and has great potential development and commercial value, Elastase can degrade insoluble protein of the insect body surface or virus coat protein. According to the characteristic, we can develop a new environmentally friendly high-performance enzyme, antiviral agents and pesticide,especially producing elastase by microbial fermentation has many advantages, such as low cost, high yield, simple extracting technology and so on. Therefore obtaining the high-yielding strains and improving the fermentation process have great significance to promoting the development of enzyme preparation industry. The study was proceed from breeding high-yield strains, and the use of effective primary screening and secondary screening system, finally an elastase-high-yield strain LSF-97 was isolated from the straw, and was identified by composite methods. And then, the fermentation conditions were further optimized , moreover the enzymatic properties were studied preliminarily, the result of the study were as follows:(1) 1308 strains were isolated from the straw samples which were collected from the farm near the Qinling Taibai Mountain Zone, first the casein (skim milk) plate selecting was used to screening ,furthermore the strains whose HC (the ratio of diameter of elastin hydrolysis circle and colony) were greater would be screened by elastin (beef tendon ), and then the effect on the decomposition activity of the best strains woud be re-tested. Finally, the strain LSF-97 which had a excellent decomposing ability to beef tendon was obtained.(2) The morphological, physiological and biochemical characteristics as well as 16S rDNA sequence homology of the selected strain were studied as a comprehensive identification of the strain LSF-97. The results showed that: the strain LSF-97 is relative to the Bacillus pumilus with more than 99 % similar in 16S rDNA sequence homology, and the morphological and physiological and biochemical characteristics are also consistent with pattern of bacteria . So it was identified as Bacillus pumilus var. qinlingensis.(3) The strain LSF-97 was studied with orthogonal test, as the research object, to determine the optimum fermentation medium as follows: glucose 3%, casein 2%, K2HPO4 0.1%, MgSO4 0.01%. Further study shows that the optimum fermentation conditions were as follows: fermentation time 32h, the initial pH 8.0, temperature 38℃and 10% medium cubage, which were beneficial to its enzyme production and stability, the enzyme activity was as high as to 113U/mL on the conditions.(4) The elastase of LSF-97 strain had a broad action spectrum. It could not only degrade casein, but also hydrolyze soluble silk fibroin, keratin, bovine serum albumin and gelatin.It could be used to applied to exploit new biological and chemical pesticides or antiviral agents…
Key words: elastase; Bacillus; screening; identification; fermentation

This entry was posted in Master Thesis. Bookmark the permalink.