Characterization Directed Evolution And Medium Optimization Of A New Pullulanase

Pullulanases belong to a family of 13 glycosyl hydrolase (so called a-amylase family). These enzymes are widely distributed in animals, plants, fungi, yeasts and G(+) or G(-) bacteria. According to substrate specificity and the different types of product, pullulanase are divided into three types:type Ⅰ pullulanase, type Ⅱ pullulanase and neopullulanase.In this work, a new pullulanase PulA (F) from Anoxybacillus sp. LM18-11 was used as the object, the length of the pullulanase gene was 2121 bp, encoding 707 amino acids and the molecular weight was 82.737 kDa, and about 82.94% homology with Anoxybacillus flavithermus WKl by amino acid sequence analysis. By exogenous express and studied its enzymatic properties, It showed maximum activity at 60℃ and pH 6.0. PulA(F) has a half-life of 70,48,1.5 hours at 55℃,60℃,65℃, respectively. Kinetic study showed that the specific activity and Km for pullulan of PulA(F) are 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far, so we regarded it as a new pullulanase. From the crystal structures of PulA, the results showed PulA(F) structure comprises four domains (N1, N2, A, and C) and A is the catalytic domain. Without domain N1 only, PulA (-N1) can form the correct folding soluble protein, and the results of enzymatic properties shoued maximum activity at 55℃ and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA(F) are 324 U/mg and 1.95 mg/mL, respectively. PulA(-N1) has a half-life of 10,2,0.5 hours at 55℃,60℃,65℃, respectively.Although the PulA had higher thermal stability for the optimal pH 6.0, there also existed a certain gap during the process compared with pH 4.5 in industrial production. Therefore, in order to reduce its optimum reaction pH, error-prone PCR method is used to realize the PulA directed evolution. In the end, Mg2+3 mmol/L、Mn2+ 0.05 mmol/L was selected as the optimal conditions, and the mutation rate is average of 0.24% at this conditions. And set up high throughput screening method of PulA mutant strains.The experiment successfully constructed expression plasmid (pBE980-pulA) for exogenous expression of PulA, and successfully conversion Bacillus subtilis WB600. Through optimizations of the fermentation culture medium of PulA,in the fermentation condition of PulA in Bacillus subtilis WB600, PulA yield was 333.198 U/mL in 100 mL flask.