Thermostable Alpha-amylase Gene Cloning And Expression In Pichia Pastoris

Thermostable alpha-amylase is currently one of the most important industrial enzymes, which has been widely used in food, fermentation, pharmaceutical, textile, oil exploration and other industries,and which accounts for more than 25% of the market yield of the enzyme preparation. The wild strain of anzyme which is isolated directedly from nature has a low productin capacity and can not meet the needs of industry. Therefore, how to get a thermostable alpha-amylase strain has been the key point to solve the amylase preparation industry. Although the way of mutation breeding can improve the ability of enzyme production strains, to obtain the desired mutant strains, it must be repeated mutagenesis and screening, which has a big workload, a low mutagenic rate, a large blindness, a limited improvement of enzyme production capacity.With the development of genetic engineering techniques, it has become possible by means of cloning thermostable alpha-amylase gene and constructing genetical engineering bacteria to significantly increase the enzyme production capacity. This can not only overcome many traditional breeding problems existed, but also can effectively change some features of the thermostable alpha-amylase. So it has become a new hotsport and has important theoretical pratical value by means of genetic engineering methods to improve alpha-amylase production and the optimization of enzymatic properties.Thermostable alpha-amylase which’ derives from Bacillus licheniformis has excellent features such as high temperature, high activity, good stability and so on, therefore those strains becomes the first choice for the production of the thermostable alpha-amylase. In this study, Bacillus licheniformis L7 was the experimental material, which was saved and screened by our laboratory, thermostable alpha-amylase gene was cloned, expressed, researched about enzymatic properties with the way of genetic engineering techniques, the main research results were as follows:According to the sequence of thermostable alpha-amylase gene (GenBank Accession No.:X03236.1) derived from Bacillus licheniformis, a pair of primers were designed and synthesized. Sequence analysis revealed that the gene had no introns, and a high-fidelity DNA polymerase was used for amplifying thermostable alpha-amylase amy gene by using Bacillus licheniformis genomic DNA as a template, PCR amplified a specific band about 1500bp, then this band was recycled, puried, connected to pMD19-T-simple-vector, and transformed into E.coli DH5a, screened positive colonies, sequenced Sequencing showed that the length of amy gene was 1536bp, including the initial condon, terminal condon, and a complete ORF, the ORF was 1536bp, encoding 512 amino acids residues of the polypeptide chains, and its 5’end had a signal peptide sequence encoding 29 amino acids residues. Subsequently the enzyme protein sequences of encoding 483 amino acids residues, predicted molecular weight of the enzyme protein 55.2KDa. Sequence alignment of thermostable alpha-amylase amy gene with GenBank database showed it shared a 99% sequence similarity with the amy gene from Bacillus licheniformis reported (GenBank, Accession No:X03236.1), named the gene as thermostable alpha-amylase amy L7.The thermostable alpha-enzyme gene amy L7 was cloned, connected with the expression vector pPICZaA, built the recombinant expression vector, then the pPICZaA-amy plasmid was linearized and electroporated into yeast competent cells of Pichia pastoris X33 host strains, and about 40 positive recombinant yeasts was screened,then induced by methanol, and the enzymatic activity of the expressed protein was tested in vitro. SDS-PAGE analysis showed that a molecular weight of the expressed protein was about 58KDa. In the condition of shake flask culture, fermentation conditions of yeast expression engineering strain were optimized, the results showed that the optimal concentration of methanol induction was 1%, the culture temperature was 28℃, and a rotation speed was 220 rpm,three-day induction culture with 250mL flask reached the highest activity 330 U/mL, (the original enzyme activity was 60U/mL)which was nearly 5.5 times as big as the original one.The enzymatic properties of the recombinant enzyme showed that:the optimum pH and optimum temperature of the recombinant enzyme were 7.0 and 70℃, and it could be maintain above 80% of the highest enzyme activity between the range from 5.5 to 8.0 and the range from 55℃ to 70℃; when 4mmol/L of the metal ion was added to the reaction system, it showed that Mg2+, Li+, Ca2+, Mn2+, Cu2+,Co2+ and Ba2+ had activating effect in the activity of the recombinant enzyme, and Ca2+ played the strongest role of activation, Ag+, Zn2+, Hg2+, Fe3+, Al3+ had inhibition effect in activity of the recombinant enzyme.