Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42

Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (Pul_AC) derived from the pullulanase (Pul_A) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type Pul_A was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and k_cat/K_m value of the Pul_AC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type Pul_A. The optimum temperature of the mutant Pul_AC was 65 °C. The Pul_AC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type Pul_A. Due to its high optimum temperature, thermostability, and specific activity, the mutant Pul_AC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.