Heterologous Expression Of α-amylase In Bacillus Licheniformis With Genetic Engineering Modification

-amylase from Bacillus amyloliquefaciens is an important liquefying enzyme whoseoptimum temperature lies between60-70℃. The enzyme could be quickly inactivated at90-95℃and is therefore widely used in small starch processing enterprises that cannot affordpressure equipment. The enzyme is also essential in special processes in which theliquefaction degree must be strictly controlled.-amylase in China is produced by B.amyloliquefaciens BF7658as well as mutant or recombinant bacteria derived from the strain.These strains show excellent productive performance but present the disadvantage of lowfermentation temperatures. The growth and fermentation temperatures of B. licheniformis,who is the ideal host to realize high temperature fermentation, are higher than those of B.amyloliquefaciens. In the present work, heterologous expression of BaA from B.amyloliquefaciens M23was studied using B. licheniformis CBBD302as host cells. Importantfindings in this research are detailed below:1. Heterologous-amylase expression was observed in B. licheniformis. Two primerswere designed according to the nucleotide sequence from B. amyloliquefaciens and the BaAgene which encodes the mature-amylase peptide was amplified from the B.amyloliquefaciens M23genome. The PCR product was ligated with pHY-WZX to constructthe pHY-WZX-BaA (abbreviated as pBaA) recombinant plasmid. The recombinantmedium-temperature amylase was successfully expressed in B. licheniformisCBBD302/pBaA, who produced294U·mL-1amylase activities in flasks.2. The protoplast transformation method was optimized by addition of DNase inhibitor.The transformation efficiency was highest at10μg DNA concentration and0.8mmol·L-1PLPdosage in450μL protoplast. The amy and apr genes in B licheniformis, which encodeamylase and alkaline proteases, respectively, were deleted through homologous recombination,and two gene defective mutants, B. licheniformis D402(△amy) and B. licheniformis D502(△amy::△apr), were obtained.3. Two recombinant strains, D402/pBaA and D502/pBaA, were obtained by transformingthe two defective gene mutants with pBaA. The optimum fermentation temperature of therecombinant B. licheniformis was42℃, which is8℃higher than the optimum B.amyloliquefaciens M23fermentation temperature. The recombinant B. licheniformisD402/pBaA and D502/pBaA strains respectively produced301U·mL-1and364U·mL-1amylase activities in flasks and527U·mL-1and619U·mL-1amylase activities in a30Lfermentor. Amylase activity of D502/pBaA could reach746.69U·mL-1under optimizedconditions in a30L fermentor.4. We tested the application performance of crude BAA from different bacteria. Thecrude recombinant BAA from D402/pBaA and D502/pBaA held the perfect property of easyinactived in95℃for20min, while the crude recombinant BAA from D302/pBaA was notinactived in the same condition as it had some thermophilic-amylase. The enzymologyproperties of pure recombinant BAA is similar with reported BAA from B. amyloliquefaciens.