| Pectinase is a class of enzymes which can degrade pectin, it belong to the third generation of feed enzyme which is develop rapidly. By decomposing pectin, pectinase can break down the cell wall. After the cell wall is broken, the nutrients such as protein and starches is released from the cell, this increase the reaction time of digestive juices and nutrients which can improve the utilization rate of feed. Due to the feed processing require to high temperature stage, thermal stability is an important criterion for the quality of feed enzyme. At present, thermostable enzyme mainly comes from hermophilic which is living in high temperature circumstance. In this paper, we have cloned the pectinase gene from Thermostable Bacillus coagulas, then constructde the expressing recombinant cells and realized the prokaryotic expression. At last, we studied the enzymatic properties of expressed recombinant proteins, this lay theoretical basis for the application of pectinase. The main research contents are as follows:1. According to the NCBI databases of Bacillus Bacillus 2-6 gene sequence, a pair of specific primers for cloning pec gene was designed. Using the pair of primers and the thermostable Bacillus Bacillus SR06 which are keeping by our lab as template, the pectinase gene was cloned from the thermostable Bacillus coagulans SR06, and then the gene was clone in pMD18-T vector to recombine a pMD-pec. Successfully,the pectinase gene was cloned into the exprsssion vector pGEX-4T-3, and the the recombinat plamid pGEX-4T-3-pec was transformed into E.coli JM109. We named the recombinat expression fugus as JM109-pGEX-4T-3-pec.2. It show that the pectinase gene was 1404 bp in size. The result of NCBI Blast show the pectinase is similar to Bacillus coagulans(2-6)pectin lyase like gene, and the identify of the pectinase and pectin lyase like gene is 98.7%, and the identify of the gene encoding amino acid sequence of them reach to 99%. The predict results of hypothesis protein of the pectinase gene was as follows: the formula of the hypothesis protein was C2298H3565N675O660S11, the Molecular was 51.56 kDa, its Theoretical pI was 9.37, the hypothesis protein was Hydrophilic protein, its had no transmembrane regions, but has4 chain Helix, and 38 chain sheet.3. The recombinant fugus JM109-pGEX-4T-3-pec was cultured in LB medium, and was induced with 0.07 mM IPTG for 4 hours at a 30?. In this condition, the protein was soluble. The SDS-PAGE electrophoresis result shows the expression protein was about51 kDa,which is with the consistent of predict. According to chromatographic purification,we can purified the the protein.4. In order to know the character of the pectinase, we had taken the enzymology properties research. The enzyme showed optimal activity at pH 8.0 and temperature of55?. The crude pectinase was loss less 10% activity after 120 min incubation at 40-60?.It retained more than 70% activity after 120 min incubation at 70?. It also retained more than 45% activity after incubation 120 min at 80-90?. Amazing, it also retained more than 65% activity after incubation 20 min at 100?. In the meanwhile, the crude pectinase also show very stable at different pH. the enzyme activity also retained more than 75%.Besides, the enzyme also show a good tolerance of heavy metal ion, and it could be activate by some ion such as Li+,Ca2+and Ca2+. Moreover, it also show a tolerance of the stomach and intestine environment. The result show the crude pectinase which was incubated at stomach and intestine environment about 120 min, the activity of pectinase also retain 78.71% and 98.45%. At last, we taken a experiment about different substrate,the result shows that the optimal substrate was 50% DE pectin, the next was polygalacturonic acid sodium salt, and the last was 70% DE pectin. Above all, the the characterization of the crude enzyme result shows that the enzyme was thermal stability and acid and alkali stability, it also shows a tolerance twards the stomach and the intestine conditions in vitro. |
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