Construction Of Genetic Engineering Bacteria Of Thermostable α-amylase From Bacillus Licheniformis And Study On Its Fermentation Condition

Alpha-Amylase (1,4-α-d-glucan glucanohydrolase;EC3.2.1.1) hydrolyzes1,4-α-d-glucosidic linkages in starch and related substrates, and it is one of the mostimportant industrial enzymes in the world. Bacillus licheniformis α-amylase (BLA) is athermoenzyme with unique characteristics, including chemical, temperature, and pHstability, and is thus often used in high-temperature industrial enzymatic processes. Thisenzyme has extensive industrial applications, such as in the starch processing, brewing,baking, textile, and paper sectors. Moreover, it is an essential component for biofuelproduction. With the development of molecular biology, random mutagenesis, site-directedmutagenesis, and DNA shuffling methods have been widely applied to change andimprove the properties of enzymes to meet industrial requirements. In this study, we focuson the previously constructed prokaryotic expression of genetically engineered bacteria E.coli BL21(DE3)-pET22b-amy and eukaryotic expression genetically engineered bacteria P.pastoris GS115-pPIC9-amy. In addition to, comparing the enzymatic properties of twogenetically engineered bacterias, and then filtering out the best industrial productionstrains.For the prokaryotic expression of genetically engineered bacteria E.coli BL21(DE3)-pET22b-amy use biotechnology method to make thermostable α-amylases gene α-AMYsite-directed mutagenesis, so that to obtain the mutant enzymes which enzymatic propertiesmaybe improved. Firstly, the thermostable α-amylases gene α-AMY from B. licheniformiswas detected by nucleotide sequence analysis and aligned with the published amylase gene(GenBank Accession No. A23402), which revealed99.9%sequence identity. α-AMY wasconnected to pMD-18T vector, three amino acids (Asp194, His235, Tyr302) which locatearound the active center and participate in the enzyme-catalyzed reaction are mutated toAla194, Asp235, Asn302. The mutant α-AMY genes are respectively connected to thepET-22b vector, constructed in three different recombinant plasmid pET-22b-α-AMY. Therecombinant plasmid pET-22b-α-AMY is used to transform E. coli BL21(DE3), the E. coli transformants carrying the recombinant plasmid are highly expressed α-amylase.Secondly, the enzymatic properties of the mutant α-amylases are measured, all mutantα-amylases isoelectric point are5.5. The activity of the Asp194Ala mutant amylase is236.3U/mg, and His235Asp mutation is754.3U/mg, the Tyr302Asn mutation is563U/mg,respectively. Compared with other mutant amylases His235Asp mutation is more suitable forthe industrial production.For eukaryotic expression genetically engineered bacteria P. pastorisGS115-pPIC9--amy we focus on its fermentation conditions. Constructed of regression model ofrecombinant Pichia pastoris production thermostable α-amylase by used four-linearregression with orthogonal design methods. The Optimization of fermentation conditionwas that inducing96h, adding0.5%methanol, inoculation150mL, and shaking speed200r/min. In these optimum conditions, the thermostable α-amylase activity was217.2U/mL.Finally, the enzymatic property of thermostable α-amylase is also measured. Thisα-amylase protein isoelectric point is5.8, tolerance pH range is4.0-10.0, the optimum pHis4.5-6.0, the optimum temperature is80℃-90℃, further more the α-amylase at90℃,after20min completely loss its activity.Comparing several characteristics of the thermostable α-amylase that produce bymutation prokaryotic expression of genetically engineered bacterias and eukaryoticexpression of genetically engineered bacteria.Other features of those strains of geneticallyengineered bacterias are similar, however comparied with other strains the mutantHis235Asp has highest activity of α-amylase. So that, His235Asp mutant geneticallyengineered bacteria is the most suitable for the industrial production.