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)) ofAnoxybacillussp. 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 andk(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 degrees C. The Pul(AC)retained more than 85% activity after incubation at 65 degrees 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.