Study On Zymologic Characteristics, Genetic Cloning And Expression Of Alkaline Protease In Bacillus Pumilus

Along with the sustainable development of animal husbandry, protein feeds is continuing shortage in our country, so exploitation of the new type protein feed resources become more and more urgent. The technology of the new type protein feed exploitation is to utilize modern biotechnoliques to take some low available feeds or uncommon feeds into the new type protein feeds, in order to enhance their availability. The application of the modern biotechniques is to produce fermentated feeds with fermentation engineering and enzymolysis feeds with enzyme engineering. No matter fermentation engineering or enzyme engineering is involved in the development and utilization of the microbial resources or microbial enzyme resources. A number of microorganisms such as bacteria, fungi, actinobacteria.have been isolated from exploitation of the new type feather protein feeds, Most of them belong to Bacillus which produces the alkaline protease. A number of alkaline proteases have been commercially exploited to assist protein degradation in various industrial processes. Many researchs are still only in the separation and purification of protease in aspect of degradation of feather keratin. How to improve the production of protease by technique for gene engineering is hot issue in present research. the comparative study of protease production characteristics between hemoglobin-degrading bacteria NJM4 and keratin-degrading bacteria WHK4 based on the previous research in our laboratory. WHK4 was selected for separating the protease and investigating the characteristics of the protease because of the high protease production. In order to improve the protease production of NJM4, the expression of alkaline protease in Escherichia. coli and Bacillus pumilus was studied by genetic recombination. The details are divided into six parts as follows:ExperimentⅠThe comparison of biological characteristics between Bacillus pumilus NJM4 and Bacillus pumilus WHK4 Comparison of biological characteristics between Bacillus pumilus NJM4 and Bacillus pumilus WHK4 were studied systematically, and the differences of protease production betweent them were analyzed. Firstly, the cultural, physiological and biochemical characteristics were compared. Then, the difference of protease production in the same medium and protease activity using the same protein as substrate were compared. The sequences of the alkaline protease gene and the promoter gene of alkaline protease were aligned in gene level. The results showed that there were no differences of physiological and biochemical characteristics between them. However, the growth velocity of WHK4 was faster than the NJM4. The spore of WHK4 was formted in 24 h in the same medium with NJM4,but the NJM4 did not. In aspect of protease production, the protease production of WHK4 was higher than NJM4 in hemoglobin and feather meal fermentation medium and lower than NJM4 in LB medium. The protease activity of WHK4 was always higher than NJM4 using different protein as substrate. There was only one base difference in gene level. It was suggest that WHK4 is a Superior strain for protease production.ExperimentⅡOptimization of protease production by Bacillus pumilus WHK4 using the feather meal as subtrateTo obtain the maximum protease production the optimal conditions were studied. The effects of feather meal concentration, initial pH, inoculum size, the additional carbon source and nitrogen sources on protease activity were studied by single-factor test. Then the feather meal concentration, temperature, initial pH, inoculum size, the additional Ammonium Sulfate and maltose were optimized by orthogonal test based on the single-factor test. The optimal conditions were achieved at initial pH 7.38, inoculum size 5% with 16 h old inoculum, incubation temperature 37℃, and the medium composed of 40 g feather meal,10.0 g (NH4)2SO4,10.0 g maltos in 1000mL based fermentation medium. Maximum protease production attained at the optimal conditions was (90U·mL-1) after 24 h cultivation. Those conditions have established the base for further seperation and purification of protease of WHK4ExperimentⅢStudy on seperation, purification and enzymological characteristics of protease in Bacillus pumilus WHK4The aim of the experiment was to isolate and investigate the protease of Bacillus pumilus. The crude enzyme was achieved in optimal medium at optimal conditions. The protease was purified from crude enzyme by 50% Ammonium Sulfate, Sephdex G-100 chromatography and analyzed by SDS-PAGE and zymography. The effect of protease activity on temperature and pH was detected and the characteristics of protease were investigated. It showed that there are two proteases in crude enzyme. One protease with molecular weights of 50 KD showed an optimal activity at 60℃and pH8.5. The protease activity was inhibited in presence of Fe3+, Cu2+, SDS and EDTA identifying it as a metalloprotease. There was no effect on protease activity in presence of Ba2+, Ca2+, Mg2+, Fe2+, DMSO, isopropanol, glycerine and Triton X-100. It was suggest that the protease obtained will be potentially applied in organic solvent tolerant biocatalysis and detergent industry.Experiment IV Genetic expression and bioactivity of recombinant protein of alkaline protease in Bacillus pumilus NJM4The aim of this experiment is to express the alkaline protease in E. coli. Primers were designed by homology-based cloning to clone the alkaline protease gene use pfu DNA Polymerase. The DNA fragment was cloned into pUC57 clone vector by double digest with XbaI and BamHI, respectively, ligated together by T4 DNA ligase, Recombinant plasmid was named pUC57-AP and transformed into E.coli TOP 10. The positive clones with white color were selected from LB/Amp/X-gal/IPTG plate and identification by PCR. Sequencing of pUC57-AP was conducted using both the forward and reverse primers. The DNA fragment was cloned into pET-28b expression vector by double digest with Ncol and BamHI, respectively, ligated together by T4 DNA ligase, Recombinant plasmid was named pET-28b and transformed into E.coli BL21 (DE3). The positive clones with hydrolytic zone were selected from LB/Kan/casein plate and identification by PCR. The protease activity of the positive clone in medium was detected after inducing with IPTG. An 1152 bp length alkaline protease gene which encoded 383 amino acids was successfully cloned. A strain expressing the protease activity was named with BL21 (DE3)-28b-AP. The protease activity by BL21 (DE3)-28b-AP in medium was 37.3 times as large as the initial strain (BL21 (DE3)). The highly expression genetic engineering microorganism laid the groundwork for production of alkaline protease.Experiment V Study on the protoplast transformation of Bacillus pumilus NJM4 The conditions of protoplast formation and feasibility of protoplast transformation by plamid in Bacillus pumilus NJM4 was studied. The protoplast was prepared using lysozyme solution to remove the cell wall. The effects of lysozyme concentration, enzymolysis time and enzymolysis temperature on the formation ratio of protoplast were studied according to microscopy. The pUC57-AP was transformed into NJM4 by the induction by PEG 6000 with the presence of Ca2+ in reaction. DM3 medium (containing ampicillin 5μg·mL-1) was used for selected transformants. Amicillin resistance strains without protease activity were selected from DM3 medium. It was suggested that Bacillus pumilus NJM4 is a kind of potential genetic engineering recipient.Experiment VI Preliminary Study on Process of Protoplast Formation Monitored by SpectrophotometerTo explore correlation between optical density and protoplast formation rates in process of bacillus protoplast formation. OD600nm were evaluated by spectrophotometer in process of protoplast formation, meanwhile protoplast formation rates were determined under microscope. Optical density curves and protoplast formation curve were drawn, respectively and analyzed correlation of them by statistical software(SPSS). It was shown that there were highly significant correlations between optical density and protoplast formation rates. It was suggested that quantitative analysis of protoplast formation rates could carried out by monitored the optical density of protoplast solution.