Improvement Of Thermostable α-amylase Production By Genetic Integrated Amplification

In order to improve production of thermophilic alpha-amylase (BLA) by industrial Bacillus licheniformis strains, in this studies, a integrated expression plasmid, pBli16s-amyL- Ery^R, was developed by combination of the genetic elements, amyL encoding a thermophilicα-amylase, 16S rDNA recovered from Bacillus licheniformis genome by polymerase chain reaction and an erythromycin resistance cassette (Ery^R) into the vector pLakr. Plasmid pBli16s-amyL-Ery^R was further genetically transformed into the host B. licheniformis CBBD302 by using protoplast transformation and selected out by regeneration medium containing erythromycin. Twenty positive transformants were randomly picked and subsequently inducted in LB medium with 1, 2, 4, 8, 12, 16, 20, 24μg/mL erythromycin respectively to increase the exchange of the purpose gene in new offspring homologous chromosomes, thereby to increase the gene number of the purpose gene. With the increasing concentration of erythromycin, enzyme activity improved obviously, beyond a certain range, enzyme activity declined. Resistance sensitivity of different transformations were different, including, the enzyme activity of transformation NO.16 was highest when the final concentration of erythromycin was 16μg/mL, increasing about 80.3%.The optimum conditions for the enzyme producted by transformant No. 16 were further investigated in the shake flask. The optimal medium composed of 2.5% cottonssed meal, 2.5% ammonium sulfate, 4% lactose and 40 mmol/L phosphates, pH6.0. The optimum fermentation conditions were: 50 mL of working volumein a 250 mL of flask, 2% inoculum capacity with 16 h-aged inoculum and grew at 42℃and 220 r/min. The enzyme level produced by the transformant No.16 in 120 h reached to 1582.1 U/mL, which is about 84.6% more comparison to that of the parent strain (857.7 U/mL). The enzymatic properties of BLA produced by the transformant No.16 were investigated, the maximum activity was obtained under the temperature of 90℃and pH of 6.0. No significant inactivation reaction was obtained under 75⁸0℃and pH5.0⁶.0. Ca²⁺, Mg²⁺, Li⁺, K⁺ and Na⁺ ions promoted the activity while Al³⁺, Zn²⁺, Fe²⁺, Cu²⁺ and Mn²⁺ions as well as chemicals Tris and EDTA inhibited it.It showed that transformation NO.16 obtained from random integration had further research potential through fermentation optimization and enzymatic properties, meanwhile, it indicated that random genetic integration using 16S rDNA was feasible.