Cloning, Sequence Analyzing And Expression Of Gene Encoding A Thermostable α-amylase From Bacillus Licheniformis 0204

Thermostable a-amylse, catalyzing hydrolysis of starch at high temperatures, is one of the most important enzyme, being widely applied in sugar and fermentation industry. Bacillus licheniformis 0204 is one of the industrial strains, which secretes thermostable alpha-amylase at high level. However, little genetic information has been demonstrated. The aims of this research are focused on cloning, sequence analyzing and expression of the gene encoding a thermostable alpha-amylase from Bacillus licheniformis 0204.A pair of primers was developed based on the highly conserved region of known sequences of alpha-amylases by DNA alignment and applied for amplifying part of the structure gene encoding B. licheniformis 0204 a-amylase (BLA). A 1.0 kb amplified PCR product was obtained by PCR technique using the genomic DNA of B. licheniformis 0204 as template. The recombinant plasmid, pT-amyl', was constructed by directly inserting the amplified fragment into the cloning vector pGEM-T Easy and supplied to sequence. The nucleotide sequence of the PCR product released a fragment (amyl') of the gene shared a high identities with other gene sequences encoding alpha-amylase from Bacillus sp. The DNA sequences flanking amyl' were obtained by an inverse PCR techniques using the self-circulated chromosomal DNA fragments as template. The whole DNA sequence and its deduced amino acid sequence were strongly similar to those from B. licheniformis 584 and similar to those from bacillus species but B. circulans. These indicated that the DNA sequence (amyl) we obtained encoded an alpha-amylase of B. licheniformis 0204.To identify the function of amyl, the recombinant plasmid pET28a-amy/NEW was constructed by inserting the structure gene without signal sequence into pET28a in frame and expressed in Escherichia coli. The a-amylase activity was detected to 13.5 U/mg in E. coli with a 55 kD band displayed in SDS-PAGE gel and the recombinanta-amylase similar to the enzymatic properties of wild a-amylase of B. licheniformis 0204.In order to increase expression level of a-amylase in B. licheniformis 0204, the integrative fragment amyl-km was constructed simply by ligating amyl and kanamycin resistant cassette Km from pSKsymKm. The amyl-km was then introduced into to B. licheniformis 0204 by electro-transformation. Recombinants that integrated with amyl-km showed highest a-amylase activity 33 U/mL, which was 10% higher than the wild.Since the difficulty of transformation of B. licheniformis, the transformation system was modified and optimized. We studied the effect of hyperosmolarity on electrotransformation of B. licheniformis 0204. With the growth medium LB complemented 0.5 mol/L sorbitol, the recovery medium LB complemented 0.7 mol/L mannitol and the field strength was 20 kV/cm, the highest transformation efficiency of 9.8 X 102 CFU/ug was obtained.