Expression, biochemical and structural characterization of high-specific-activity ß-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis.

BACKGROUND: ß-amylase (EC 3.2.1.2) is an exo-enzyme that shows high specificity for cleaving the α-1,4-glucosidic linkage of starch from the non-reducing end, thereby liberating maltose. In this study, we heterologously expressed and characterized a novel ß-amylase from Bacillus aryabhattai. RESULTS: The amino acid-sequence alignment showed that the enzyme shared the highest sequence identity with ß-amylase from Bacillus flexus (80.73%) followed by Bacillus cereus (71.38%). Structural comparison revealed the existence of an additional starch-binding domain (SBD) at the C-terminus of B. aryabhattai ß-amylase, which is notably different from plant ß-amylases. The recombinant enzyme purified 4.7-fold to homogeneity, with a molecular weight of ~ 57.6 kDa and maximal activity at pH 6.5 and 50 °C. Notably, the enzyme exhibited the highest specific activity (3798.9 U/mg) among reported mesothermal microbial ß-amylases and the highest specificity for soluble starch, followed by corn starch. Kinetic analysis showed that the Km and kcat values were 9.9 mg/mL and 116961.1 s- 1, respectively. The optimal reaction conditions to produce maltose from starch resulted in a maximal yield of 87.0%. Moreover, molecular docking suggested that B. aryabhattai ß-amylase could efficiently recognize and hydrolyze maltotetraose substrate. CONCLUSIONS: These results suggested that B. aryabhattai ß-amylase could be a potential candidate for use in the industrial production of maltose from starch.

Production of recombinant beta-amylase of Bacillus aryabhattai.

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai ß-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for ß-amylase production, containing glucose (7.5 g·L-1), pig bone peptone (40.0 g·L-1), Mg2+ (0.05 mol·L-1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the ß-amylase yield reached 925.4 U mL-1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4 g L-1. Through this cultivation approached 30 °C with 0 g·L-1 initial glucose concentration, the maximum ß-amylase activity reached 5371.8 U mL-1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.

Efficient production of aggregation prone 4-α-glucanotransferase by combined use of molecular chaperones and chemical chaperones in Escherichia coli.

In this study, a combined optimization strategy, based on co-expression of molecular chaperones and supplementation of osmolytes, was used to reduce the formation of inclusion bodies and enhance the expression of the soluble form of 4-α-glucanotransferase. The 4-α-glucanotransferase yield was enhanced by co-expression with pGro7 and supplementation of trimetlylamine oxide. Subsequently, the effects of process conditions (temperature, inducer concentration, and arabinose concentration) on cell growth and 4-α-glucanotransferase production were also investigated in shake flasks. In addition, a modified high-cell-density fermentation approach was proposed and applied in 3-L fermentor supplied with l-arabinose and trimetlylamine oxide, which achieved a dry cell weight of 65.92 g·L-1. Through this cultivation approach at 28 °C, the activity of 4-α-glucanotransferase reached 332.5 U·g-1 dry cell weight, which was 24.6-fold greater than the initial activity in shake flask cultivation. This combined strategy is expected to provide an efficient and economical approach to overproduction of aggregation prone proteins on a large scale.