The 45th Academic Congress of Taiwan Association for Food Science and Technology

Cloning and expression of a raw starch binding α-amylase gene from Clostridium butyricum in Escherichia coli

JF Lai, WZ Huang, HZ Zeng, HJ Lin, MC Tsai, ZW Chen, JH Lin, JP Wang

Amylases are among the most important of enzymes that are capable of digesting the glycosidic linkages found in starches. Though amylases originate from different sources which include bacteria, fungi, animals and high starch containing plants, the microbial amylases are the most produced and used in a variety of industries such as food, textile, paper, detergent and alcohol industries. Previous studies had showed that Clostridium butyricum, a butyric acid producing Gram-positive anaerobe, has the ability to produce raw starch-binding α-amylase. However, the gene encoding the raw starch-binding α-amylase has not yet been cloned. The objective of this study was to clone and express the gene encoding raw starch-binding α-amylase from Clostridium butyricum in Escherichia coli, and to purify the recombinant enzyme for enzyme activity assay. In this study, the α-amylase gene was amplified by polymerase chain reaction (PCR) from Clostridium butyricum genomic DNA. Then, the PCR product was cloned into the expression vector pET29a and transformed into E. coli BL21(DE3), E. coli C41, E. coli star, respectively. The recombinant α-amylase was expressed after induction with IPTG and could be purified by immobilized metal-ion affinity chromatography. The specific activity of the purified recombinant α-amylase towards soluble starch was 0.11 U/mg. In the near future, the characterization and potential application of this recombinant enzyme will be further investigated.