Isolation, Fermentation And Gene Clone Of A Strain Producing Acid Stable Alpha-amylase

In low pH condition, acid alpha-amylase cuts off the starch molecule randomly within the alpha-1,4-glycosidic bond under acidic conditions to meet some of the deep processing of raw starch process requirements. In addition, acid alpha-amylase is widely applied in numerous areas, such as alcoholic fermentation, silage, medicine and other fields, and has great application potential and development prospect. At present, the research of acid alpha-amylase is mostly at the laboratory stage, the enzyme activity of fermentation production is relatively low, the production cost is relatively high, which restrict the acid alpha-amylase widely used in food and fermentation industries.A high-yield acid stable alpha-amylase strain was screened from soil samples from starch and sugar refinery through trypan-blue media. The strain, named xm-1, individual strain elongated rod-shaped, bacillus in the middle, Gram-positive, cell size is 0.5～1.1×2.2～3.2μm, catalase positive, VP-positive, nitrate reduction positive, don't produce indole, urease negative, use glucose and sucrose to produce acid but no gas, no acid production using lactose gas. Submitted the bacterium 16S rDNA gene sequence to Genbank, the gene accession number is FJ600486. The strain was identified as Bacillus subtilis by means of morphology and physiological-chemical characterization as well as 16S rDNA sequencing.The optimum growth temperature of the strain is 37℃, the initial pH of the medium is 4.5, and the highest activity under optimal condition is 242U/mL at the 13th hour. The media volume has less effect on amount of amylase production. Use strain xm-1 as the starting strain, by single factor experiments and response surface analysis, it's revealed that the optimal fermenta- tion medium consists of soluble starch, 8.1g/L, peanut cake 34.3 g/L, NH₄NO₃ 6g/L, KH₂PO₄ 3g/L, CaCl₂·2H₂O 2.94g/L. The highest enzyme activity is 537.1U/mL, about 2.2 times of the former.The acid alpha-amylase from Bacillus subtilis xm-1 was purified and characterized. The crude amylase was purified 32.5 times and 10.0% recovery of enzyme activity through 85% saturation ammonium sulfate precipitation and Sephadex G-75 gel chromatography. The study of the enzyme properties showed that the molecular weight of the purified alpha-amylase is approximate 60KDa, and the optimum pH and temperature was 5.0 and 45℃respectively. The enzyme was stable at pH3.4⁶.0, poor tolerance to high temperature. Metal ion of Cu²⁺, Zn²⁺ and EDTA inhibited the amylase activity at various extent, while Ca²⁺ and Mn²⁺ can distinctly enhanced the activity of about one time, K+ and Na+on the activity of some role in promoting.Cloned the strain xm-1 alpha-amylase gene by PCR, determined its sequence, and submitted to Genbank, the gene accession number is GQ153530. By gene sequence alignment with the alpha-enzyme sequence has been reported in NCBI , the similarity of gene sequence as high as 98%. While in the amino acid level, there are sevel differences, these differences may have the majority influence on changing the nature of the enzyme characteres.