Screening Of An Alkaline Protease Producing Bacterium And Expression Of Apr Gene And Study On Enzymatic Properties

In the study, one bacterium producing alkaline protease was isolated from soil and its taxonomic state was conformed by morphological observation, physiological and biochemical identifications and16S rRNA gene analysis, and orthogonal experimental design was used to explore the effect of fermentation improving feed quality of blood meal and soybean meal. Furthermore, expression plasmid of alkaline protease gene was constructed and its expression conditions and characteristics were studied, and enzymatic properties of this recombinant enzyme were determined.1. Isolation and identification of a bacterium producing alkaline protease. To screen a bacterium producing alkaline protease and study its fermentation effect on protein feeds, a new strain was isolated from topsoil surface of meat power plant and purified using skim milk media. Its taxonomy was established according to its morphological features, physiological and biochemical characteristics as well as phylogenetic analysis of16S rRNA gene sequences. This strain was most closely related to Bacillus subtilis, with their homology of99.5%, and named Bacillus subtilis strain D-2.2. Effect Bacillus subtilis strain D-2of on fermenting protein feeds. Effects of inoculation (3%,6%and12%), culture time (12h,24h and48h) of seed liquid and solid-state fermentation time (12h,24h and48h) on fermented performance of soybean meal and blood powder were investigated by orthogonal experiments. The results showed that the optional fermentation conditions as follows:the inculum of6%, seed liquid culturing time of24h, and solid-state fermentation time of48h. Under optional fermentation conditions, the contents of small peptides were significantly increased to17.06%and12.73%in fermented soybean meal and blood powder, respectively. These results indicated that alkaline protease produced by Bacillus subtilis strain D-2effectively decomposed protein in both soybean meal and blood powder, which improved dietary performance of protein feeds.3. Clone and expression of alkaline protease gene from Bacillus subtilis strain D-2. To establish the expression system of high yield of Bacillus subtilis alkaline protease gene, alkaline protease gene was amplified by PCR and ligated to the prokaryotic expression vector pET-32a. After identification by sequence, the recombinant protein was expressed in E. coli BL21. The homology of this gene and code protein was compared and inducing conditions were analyzed. The results showed that the size of amplified gene was1149bp, with382amino acids coded. The coded protein molecular weight was60.4kDa by SDS-PAGE. Alkaline protease was expressed as soluble protein at25℃and the part of the protein existed as inclusion body at37℃when expressed for4hours in the concentration of IPTG0.2mmol/L. The alkaline protease activity was determined in this soluble protein.4. Purification and enzymatic characteristics of the recombinant bacteria. To study precipitation properties and enzymatic characteristics of the recombinant enzyme, the crude enzyme, purified by ammonium sulfate precipitation and dialysis from fermentation liquid were determined under different temperature, pH value, metal ions and chemical reagents. The results indicated that the whole recombinant protease was almost precipitated through60%ammonium sulfate fractionation, The optimum reaction temperature was50℃.The optimum reaction pH was10.5and the optimum reaction pH range of protease was in the range of8.0-12.0. Ca2+and Mg2+showed activation effect on the activity of the protease to a certain extent, Hg+and Ag+showed obvious inhibition effect, PMSF and DFP showed extensive inhibition effect.5. Analysis of nucleic acid and protein structures of alkaline protease from Bacillus subtilis strain D-2. To investigate coded gene and bioinformatics of alkaline protease from Bacillus subtilis strain D-2, this sutdy used different programs of NCBI database, BioEdit software, DNAstar software, ProtScale program, NetPhos2.0Server program, SCRATCH Protein Predictor program, TMHMM prediction, Hopfield neural network prediction, NetTurnP1.0program, CBS Prediction Servers program, Pfam prediction, SMART prediction, PSORTb program to analyze nucleotide sequence, base homology and open reading frame of the gene. After translated into amino acid sequence, the primary, secondary and tertiary structures of this protein were predicted and analyzed, which included amino acid sequence analysis, signal peptide prediction, hydrophobic characteristics analysis, transmembrane region analysis, subcellular localization, domain and spatial structure prediction. The results showed that this alkaline protease have signal peptides of1-30amino acids with high hydrophobicity and transmembrane region, extracellular region of30～382amino acids. The spatial structure of this alkaline protease, a secreted protease, had many domains like a-helix, P-sheet and (3-turn and was predicted.