Purification And Gene Expression Of A Novel Intermediately-thermostable α-amylase From Bacillus Sp. YX-1

α-Amylases (EC3.2.1.1, 1,4-α-D-glucan-glucanohydrolase) are starch-degrading enzymes that catalyze the hydrolysis of internalα-1,4-O-glycosidic bonds in polysaccharides with the retention ofα-anomeric configuration in the products. It is one of the most important industrial enzymes, which can be used in wide number of industrial processes including biofuel, traditional brewing, food and textile.Now most of the commercial bacterialα-amylases used in the liquefaction step have pH optima at around 6.5 and unstable at low pH, however, natural pH of starch slurry is usually around 4.5, therefore, it is necessary to improve the activity and stability of the enzymes at low pH values for omitting the pH adjustment step. Furthermore, it could supply the theoretical basis for elucidating the acid-stability mechanism of the microorganisms and its metabolites, developing and utilizing the new products by protein engineering. This study reports the purification, characterization and gene expression of a novelα-amylase from a newly isolated Bacillus sp. YX-1.On the basis of the hydrolysis ability in the starch agar plate (pH 4.5), an isolate YX-1 was selected. The newly isolated strain was identified as a Bacillus species according to the characteristic of this strain and the 16S rDNA sequence. Moreover, a comparison of 16S rDNA sequence of this strain with other related Bacillus sp. shows that the 16S rDNA sequence of this strain has no high similarity with B. subtilis, B. licheniformis, B. stearothermophilus and B. amyloliquefaciens, which are the usualα-amylase-producing strains for industrial applications. The 16S rDNA sequence of the novel isolatedα-amylase-producing strain YX-1 has been deposited into the GenBank database under accession number DQ883446.YX-1 was used as the parent strain to increase its enzyme production capacity by the method of UV mutation. After several screen procedures, one mutant strain with higher enzyme-producing ability was selected. The activity was about 200% higher than that of the parent strain. The crude enzyme of the mutant was similar with that of the parent strain which could hydrolyze starch substrate in acidic condition.The amylase from Bacillus sp. YX-1 was purified reaching 34 folds of electrophoretic homogeneity by sequential ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. The enzyme had a molecular weight of 58kDa estimated by SDS-PAGE. The action mode of the enzyme was evaluated by studying the hydrolysis performance of soluble starch at pH 4.5 and 60℃. The purified amylase could convert starch substrate to low molecular oligosaccharides with rapid blue color reduction in iodine-starch reaction and appearance of reducing sugar, indicating an endo-type activity of this amylase.The purifiedα-amylase exhibited the maximum activity at pH 5.0 and 40-50℃. Furthermore, the enzyme performed stable over the pH range of 4.5-11.0 and at 60℃. The enzyme preparation could hydrolyze various raw starches very well at pH 5.0, which was around the saccharification pH of industrial application. It is worth noting that the enzyme exhibits a high digestibility towards raw corn starch granule of concentrations ranging from 15 to 20%, omitting an energy intensive gelatinization step. These features are very important for industrial starch liquefaction. From the viewpoints of application, the optimum activity of the enzyme at low pH values and its suitable thermostability made it favorable in industrial operations for traditional fermentation and food processing. The amino acid sequences of two peptides from the purified enzyme were analyzed by LC-MASS-MASS, and the enzyme did not show strict identity to theα-amylases reported for some differences in amino acid residues.To further investigate this enzyme, theα-amylase from Bacillus sp. YX-1 was subsequently cloned, sequenced and expressed in E. coli BL21. Theα-amylase gene (amy) was amplified using genomic DNA from Bacillus sp. YX-1 as a template and the specific primers based on theα-amylase genes of Bacillus species from the GenBank database. After cloning the gene encoding the enzyme, sequencing analysis revealed that the sequence contains one complete open reading frame with a length of 1545 base pairs and encodes a protein of 514 amino acid residues with a calculated molecular weight of 58 kDa plus a signal peptide of 31 amino acids. The nucleotide sequence of the Bacillus sp. YX-1α-amylase has been deposited in the GenBank database under accession number EU159580. The amino acid composition of the sequence and its relevant non-covalent action, the secondary structure analysis, the three dimensional structure prediction were utilized respectively to elucidate the enzymatic properties of this enzyme. The principle on the development of enzyme activity with UV mutation was also analyzed.An expression vector, pET-28a-amy, containing theα-amylase gene (amy) inserted into pET-28a was constructed, and E. coli BL21 (DE3) was transformed with the ligation mixture to yield the transformant E. coli BL21 (DE3) (pET-28a-amy). SDS-PAGE analysis of expressed recombinant protein revealed that most of recombinant protein existed as inclusion body when expressed at 37℃with 0.5 mmol/L IPTG induction, while at 30℃, the amount of soluble protein increased, and thus the expression conditions were determined as cultivation temperature 30℃and IPTG 0.5 mmol/L. Under those conditions, the cell-free extract from recombinant E. coli showed a specificα-amylase activity of 1.52 U/mg.The recombinant enzyme of N-terminal His6-tag protein was purified from the cell-free extract of E. coli transformant using a HisTrap HP affinity column to an apparent homogeneity on SDS-PAGE. The recombinant enzyme displayed physical-chemical characteristics similar to those observed for the native enzyme from Bacillus sp. YX-1.