The Preliminary Study For Cloning, Expression Of AmyF Gene From A Bacillus Sp. WS06 And Biological Activity Of The Enzyme

A Bacillus sp. WS06, which produces an extracellular α-amylase, was isolated from the cecum in a piglet. An amyF gene from this Bacillus strain was cloned and its nucleotide sequence was determined. An open reading frame composed of 1581 bases, which encodes 526 amino acid residues was found. The amyF gene show high sequence homologies with other microbial amylase genes, such as Bacillus megaterium and Bacillus polymyxa (93% and 53% identity). The deduced amino acid sequence revealed that four highly conserved regions of the a-amylase family. The amyF gene was overepressed using the pET21a vector and Escherichia coli BL21DE3. The recombinant enzyme (AmyF) was purified 22.2 fold to electrophoretic homogeneity and had a molecular mass of 57 kDa (by SDS-PAGE). The enzyme was optimally active at pH 7.0 and 55-6C℃ and showed stability at pH range of 2.2 to 12.8 at the temperature below 55℃. The enzyme activity was inhibited by Cu²⁺, Fe³⁺, Fe²⁺, Zn²⁺, Citrate and EDTA, etc. It was stable by K⁺, Ca²⁺ and SDS. This enzyme efficiently hydrolyzed various types of starch to yield a series of malto-oligosaccharides by endo-cleavage mode. AmyF may has an important influence on utilization of resistant starch in piglet intestine.Another study was designed to investigate the effects of the amylase and RS hydrolysis in vitro. The fresh contents were collected from the rat cecum and incubated into some special culture. It was found that the RS was digested, the amylases were produced, reducing sugars were increased and the bacteria that can utilize starch were increased. The concentration of ammonia and pH were determined and the structure of duodenum, cecum and colon were observed. It was investigated that the effect of RS on intestinal health from the intestinal structure and micro-environmental change.