Screening And Identification Of Protease-producing Bacteria From Litopenaeus Vannamei Intestinal And Study On Enzymatic Properties

Protease is mainly comes from microbes, plants and animals. Degradation of small molecular peptides or amino acids and other substances by catalytic the peptide bond hydrolysis of proteins and peptides. Controlling hydrolysis of protein by protease to produce functional peptides, functional proteins and flavor seasoning has become an important way to achieve high-value utilization of protein resources. With the mature of enzyme hydrolysis technology and the rapid development of controllable protein enzymatic hydrolysis industry, exploring proteases with novel characteristics is becoming more and more urgent. Protease has been widely applied in aquatic protein by enzymatic hydrolysis, but not all of protease is suitable for aquatic protein. The hydrolysis ability of different protease on the same substrate showed significant difference. Therefore,choose the suitable protease used in hydrolyze aquatic protein is crucial. At present, the commonly used goods protease is mainly with papain, pepsin, trypsin, terrestrial microbial protease. But the research of marine source protease in the application of aquatic protein processing is lack. Microbial source protease is mostly exogenous enzymes and not restricted by resources, space and environment, has greater advantages than animal and plant sources of protease. The marine source microbial protease used in aquatic protein processing may be possesses advantages of homology, have better hydrolysis effect of aquatic protein, and the hydrolysis products with different tastes and functional performance contrast with terrestrial microbial protease.In order to develop marine microbial protease, Bacterial strains were isolated and purified from the intestine of Litopenaeus vannamei by 2216 E culture medium. 26protease-producing bacterial strains were isolated with hydrolysis circle of protease, and preliminary estimate the enzyme production ability by determinate the D/d of difference strains.Then A high yield protease strain was isolated by detect the protease activity of fermentation broth and identified as LVi-6. The strain LVi-6 was identified as Vibrio anguillarum by morphology, physiological and biochemical identification, 16 S r DNA gene sequence analysis and phylogenetic tree analysis.The crude protease was then purified by ammonium sulfate precipitation,Q-Sepharose FF anion-exchange chromatography and Superdex S-100 gel filtration chromatography. The protease was finally purified to 2.71-fold with a specificity activityfrom 4035.11 U/mg to 10925.27 U/mg protein and a total yield of 47.03%. The purified protease was medium temperature neutral protease, exhibited optimum activity at 50℃ and p H 7.5. It was stable below 40℃and p H 9~12, heat-sensitive above 60℃.The protease could be activated in the presence of Zn2+and Co2+, especially Co2+、Na+、K+、Ca2+、Mg2+、Ba2+ and Fe2+ reflected no obvious effect on enzyme activity. The protease was slightly inhibited in the presence of Cu2+, however, Hg+ reflected strong inhibitory effect on enzyme activity. It was inhibited by EDTA, indicating that it is a metalloproteinases. The enzyme activity was maximum When its concentration to 28 g/L with casein as substrate,while the Km and Vmax values were 8.99 g/L and 5.90 μg/(m L?min), respectively. The chemical modification of protease with DTNB、NBS、Br Ac、acetylacetone、PMSF and formaldehyde showed that amino and tryptophan residues involved in the composition of the enzyme active center could be considered as the essential functional groups of protease.However, the sulfydryl, histidine residues, arginine residues and serine residues were not directly related to the activity of protease, suggesting that they were not essential for the enzyme active center. The characteristic absorption peak of the 278 nm had no change,which showed the tertiary structure was not destroyed in spite the protease was modified with NBS.The protease was identified through combined utilize MALDI-TOF/TOF-MS,LC/MS/MS mass spectrometry and N-terminal amino acid sequence determination of Edman degradation. Through the bioinformatics analysis learn that the PI and molecular weight is 5.70 and 66725.61 Da, respectively, and it is composed of 611 amino acids, 196 ones of which are hydrophobic and 146 ones are polar exogenous amino acids. The homology of the protease and Vibrio anguillarum metalloproteinases reach to 99% by homology analysis, which shows that the protease have Vibrio anguillarum protease sequence structure characteristics and similar functions. The Vibrio anguillarum metalloproteinases crystal structure was used to homology modeling and structure prediction, which showed that there is a conservative area in the central concave position,and the conservative area is its active site which is combined with. Preliminary enzyme properties study was speculated that Zn2+ binding sites should be existed in the active center and the result is both match with each other, therefore, the homology modeling and structural prediction has high theoretical value and practical significance.In order to assess the protease hydrolysis characteristics, the difference of DH, free amino acid contents and molecular weight distribution was evaluated through comparing with commercially available neutral protease, trypsin and carboxypeptidase with casein as substrate, which showed that the DH and free amino acid contents in the enzymatichydrolysate of Vibrio anguillarum protease were both higher, and its molecular weight mainly concentrated in the range of 1000~5000 Da, and less than 1000 Da. The enzymatic hydrolysis ability of Vibrio anguillarum protease was evaluated through comparing with neutral protease with Shrimp protein as substrate. The results showed that the DH、free amino acid contents and DPPH radical scavenging in the enzymatic hydrolysate of Vibrio anguillarum protease were higher than neutral proteases.