Screening Of High Yield Of α-amylase Bacillus Licheniformis And Construction Of A Recombinant Plasmid

In this study,16thermophilic Bacillus strains were screened from Baiyunbianhigh-temperature Daqu as starting strains, from which we screened Bacillus subtiliswith high yield of α-amylase. And its enzyme-production properties was studied. Toresearch the aroma-production or enzyme-production mechanism of Bacillus subtilis atmolecular level, this paper construct a plasmid which containing the ampicillinresistance gene transposon.The main results were as follows:(1) Through a preliminary inspection method of starch-iodine plate to examine theα-amylase activity of16thermophilic Bacillus strains, screening the highest yield of α-amylase activity strain, then the α-amylase activity of this strain was further accuratelydetermined through improved Yoo method. The optimal temperature of the enzymeactivity was70℃, which showed that the enzyme was a high temperature α-amylase.The colony morphology of the Bacillus producing α-amylase was observed, combinedwith its16S rDNA sequence comparison result the strain was identified as Bacilluslicheniformis, which was named BG14.(2) On the characteristics of α-amylase activity of BG14were studied. Its growthcurve and enzyme-production curve showed that:0h~2h was the slow growth phase ofthe BG14,2h~18h was the logarithmic phase, and its high temperature α-amylase wasmaximum at the time of its growth to42h. The optimum conditions of producing theenzyme by Bacillus licheniformis BG14were as follows: pH7.5, temperature40℃,liquid volume20%, speed180rpm. Optimal medium of Bacillus licheniformis BG14producing the enzyme was determined by orthogonal experiments: soybean meal0.3%,wheat bran0.4%, soluble starch0.7%, peptone0.8%, K2HPO40.5%, pH7.0~7.5, inwhich the enzyme activity could reach334.32U/mL,α-amylase activity was increasedby43.09%than the initial fermentation medium.(3) Solid state fermentation process was simulated by enhanced functional bran ofBG14. The physical and chemical parameters results showed that total acid and totalester content of the wine producing with BG14strengthen bran were higher thanwithout it, liquor ratio was relatively higher. And its flavor components such as furfural,isovaleraldehyde and β-benzyl alcohol were also more abundant than that without BG14strengthen bran showed by gas chromatographic analysis.(4) The results of experiment showed that BG14was not sensitive for spectinomycin,but it was very sensitive for ampicillin. This study constructed the recombinant plasmidpMarA-4T-2which with ampicillin and erythromycin double tolerance gene,and BG14could not grow in concentration of50μg/mL ampicillin plates or25μg/mL erythromycinplates. Recombinant plasmid pMarA-4T-2has Genetic Stability in E.coli/DH5α,pMarA-4T-2could use to research the enzyme-production mechanism and construct themutant of BG14.