Construction Of Engineering Bacteria And Yeast Which Product Thermostable α-Amylase And Study On Enzymatic Properties Of Their Products

The thermostable alpha-amylase can decompose the starch (natural substrate usually has alpha -1,4 sugar glucoside key), products are alsace gum, oligose and glycose. In general, the wild strains which exist in the natural world have some problems ,for example,they have very low enzyme output and it is hard to control the working contition. It is enzyme preparation profession universal of concern how to solve these problems. The research of this enzyme is quite active in recent years, because it has the following characteristic: (1)at the high temperature it has optimize enzyme reaction condition, it saves or does not use the cooling water; (2)it can cut down the consistency of wine-viscosity and reduce dynamia analosis when transport; (3) the mixed fungus'pollution opportunities are fewer; (4) the thermostability is good,and need fewer calciumion. Thus in many realms of production, especially producing amylaceum ,glucose molasses with enzyme, the thermostable alpha-amylase has taken the place of common temperature alpha-amylase. The thermostable alpha- amylase is not only used in producing amylaceum ,glucose molasses,all kinds of slarch and Alsace gum but also used in the colliquation of alcohol ,beer and proferment in brewage industry, desize of textile products, manufacture of digestant.At the same time ,it is used in fruice production and vegetable elaboration and so on.At present the wild strains which are mutated repeatedly are mostly used in industrialization production ,but it had discovered platform effect in mutation breeding. They have been restricted that further enhancing the enzyme activity and improving working condition, Bacillus Licheniformis and Bacillus subtilis are used in the production, the latter was restricted in the practical application because of the low tolerated temperature. The development of molecular biology opened a new way for solving the above problems. It is the aim of this experiment to construct engineering bacteria and yeast which product thermostable alpha-amylase by genetic engineering and explore a suitable path to industrialized production.In this experiment we taked Bacillus Licheniformis mAn-612 genome as a template, designed amplification primers according to the gene scequence which had been published, the thermostable alpha-AMY gene of Bacillus Licheniformis was cloned by PCR .The ligation mixture was trasformed into the competent cell E.coliDH5α(the cloning host)after it was ligated with pMD-18T vectors. The homologies are 99.9%and 99.8% compared with the published squence.We cleaved pMD-18T-alpha-AMY and pET-22b with BamHⅠand HindⅢ,and then ligated the positive cloning and pET-22b cleaved with BamHⅠand HindⅢ,the ligated DNA was first transformed into cloning host E.coli DH5α,and then DE3(The expression host). The homology reached 99.9%,and the reading frame was correct.At last,we got the recombinant plasmid for expressing.Massively genetic engineering bacteria pET-22b-alpha-AMY had been induced and fermned with IPTG, and the expression condition had been optimized. The SDS-PAGE finally demonstrated, it was expression of IPTG induced at 30℃8h later that DE3 had the highest quantity of expressive.The weight of the protein was similar to the predicted's (53ku),and 1mmol/L IPTG led to the highest expressive quantity.Otherwise, most of protein excisted in the form of soluble protein, 20.7% of tropina was foreign protein.We cleaved pMD-18T - alpha -AMYand pPIC9 with EcoR I and Not I,and then ligated the positive cloning and pPIC9 cleaved with EcoR I and Not I.The ligated DNA was first transformed into cloning host E.coli DH5α.The positive recombinant plasmid which had been linearized with SacI was concentrated, and then was trasformed into yeast( 1 500V electric shock ).The target gene was integrated with GS115 genome in the way of insertion .By nutrition phenotype screening,PCR identification with consensus primer and specific primer ,and enzmatic activity screening,we determine the recombinant engineering yeast constructed successfully.The massively genetic engineering yeast pPIC9-alpha-AMY had been induced and fermned with the methyl alcohol, and the expression condition had been optimized. The SDS-PAGE finally demonstrated, it was the highest expression quantity in 30℃, methyl alcohol induced 96h later that yeast had.The weight of the protein was bigger than the predicted's (53ku)by13ku,and 1 % methyl alcohol led to the highest expressive quantity.Otherwise most of protein were excreted into the supernatant.We suspensed engineering bacteria with TE which had been freeze thawed, took it carry on supersonic quassation after being added lysozyme and then loaded supernatant into the dialysis belt whose outer layer was PEG.Taked the concentrated solution to carry on the non-denatured vertical board PAGE, and dyed by the starch -idodine activeness, cuted the gel to purify anticipated thermostable alpha- amylase.Recombination engineering yeast were induced with methanol.We precipitated the supernatant of culture medium with 70% saturated ammonium sulfate, and suspensed the precipitation with TE, and then load it into the dialysis belt whose outside was PEG. Taked the concentrated solution to carry on the DEAE-cellulose chromatographic analysis to obtain purified thethermostable alpha– amylase.Determined the relative molecular weight of the both proteins,respectively with SDS-PAGE and coomassie brilliant blue staining method.As a result ,we found the molecular weight of the eukaryotic expression apozymase was bigger than the anticipated's. Their PI were tested with isoelectric focusing electrophoresis PAGE, and their PI were5.5 and 5.8; the enzyme activity was separately determined in 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, 110℃0.05mol/L pH7 KH2PO4-NaOH buffer, then we found both of the apoenzymes had the highest enzyme activity in 90℃;we tested the enzyme activity in the range of pH3-12, the result indicated that the optimum pH was 6.0-6.5; the heat stability of eukaryotic expression apozymase was higher than another by determination of heat stability.The engineering bacteria and yeast which had been constructed have the better industrial production value. The enzyme produced has the good zymology characters, and it is suitable for industry.