Molecular Modification And Efficient Expression Of B. Stearothermophilus Maltogenic Amylase

High maltose syrups, which are characterized by their low sweetness, low viscosity in solution, and good heat stability, are widely used in the fileds of food, makeups, and pharmaceuticals. In recent years, enzymatic preparation of maltose syrups has drawn rising interest for its mild reaction conditions, high specificity, and high catalytic efficiencies, and has developed to be the main method in the production of high maltose syrups.Maltogenic amylases(EC 3.2.1.133) are capable of catalyzing the hydrolysis of α-1,4-glycosidic bond in maltotriose, starch and related dextrin, which enables them to participate in the two-step saccharification process to improve the content of maltose. In this study, the maltogenic amylase from Bacillus stearothermophilus was expressed in Escherichia coli, and its enzymatic properties were studied. Site-directed mutagenesis was performed and the mutants were applied in the preparation of maltose syrups. One of the mutants with the best performance was further expressed in Brevibacillus choshinensis. The media and the fermentation conditions of the recombinant mutant were optimized in the flasks and in a 3-L fermentor, which improves the expression level of the mutant.The main results are demonstrated as follows:(1) A recombinant strain of E. coli BL21(DE3)/opt-amyM/pET-24a(+) was constructed and cultured for 48 h. The enzyme activities of the media reached 4379 U·mL-1. The recombinant enzyme was purified through heat treatment, ammonium sulfate precipitation and DEAE ion-exchange. The enzyme properties were studied, exhibiting a p H optimum of 5.5, a temperature optimum of 60 °C, and a half-life of 325 h for thermostability determination. Incubation of the enzyme with different metal ions showed that the enzyme was metal ion-independent, and its hydrolysis activity was suppressed in varied level under the effects of some metal ions.(2) On the foundation of sequence alignment and protein structure analysis, the Trp 177 on the subsite +2 of B. stearothermophilus maltogenic amylase was substituted to Phe, Tyr, Leu, Asn, and Ser, respectively, to generate the mutants of W177 F, W177 Y, W177 L, W177 N, and W177 S. The enzyme profiles of the mutants were studied. The mutants were subsequently used for two-step saccharification. The constitution and the content of the product syrups were analyzed. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%(W177S), compared with 3.2% for the wild-type maltogenic amylase. The transglycosylation activities of the mutant enzymes were measured. It turned out that the transglycosylation activities of the mutant enzymes decreased to 82.3%-47.6% of that of the wild-type enzyme. It was found that the lower the transglycosylation activity of the enzyme was, the lower the content of the maltotriose content in the maltose syrups. The effect of the hydrolysis of the subsite +2 to transglycosylation and to the preparation of maltose syrups was analyzed.(3) The effects of the DE value of the liquefied starch, the temperature, the start pH, and the enzyme dosage were explored. The optimal condition was determined as follows: DE value of the liquefied starch was 8.63; the temperature of the two-step saccharification was controlled at 60 °C; the start pH was 5.5; and the enzyme dosage was 6.67 μg·g-1 dry starch.(4) The gene of W177 S was expressed in B. choshinensis, and the expression level was optimized through fermentation optimization. The recombinant strain of B. choshinensis/w177s/pNCMO2 was constructed. When the strain was cultured in the TM medium for 48 h, the enzyme activity of W177 S reached 80 U·m L-1. In the flasks, the medium composition and the fermentation conditions were optimized. The optimized medium contains 5 g·L-1 yeast extract, 15 g·L-1 soy peptone, 40 mM sodium phosphate buffer, and 1 mM Al3+. The enzyme activity reached 999 U·m L-1 when the fermentation was conducted in the optimized medium, under the optimal conditions of 5% inoculation volume, start pH 6.5, 30 °C, and 40 mL volume. The pH optimum and the dissolved oxygen optimum were subsequently studied in a 3-L fermentor, which were determined to be pH 6.5 and 30%, respectively. The enzyme activity reached 1429 U·mL-1 finally, which was 18-fold than that of the unoptimized conditions.