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.

支链淀粉酶是一种常用的淀粉脱支酶，广泛应用于食品，化学和制药行业。由于淀粉脱支过程需要高温，因此需要热稳定的支链淀粉酶。在这项研究中，基于表面残留物置换和二硫键引入的策略，从无氧杆菌属的支链淀粉酶（Pul _A）衍生出突变体支链淀粉酶（Pul _AC）。分离出具有较高热稳定性和活性的WB42。来自野生型Pul _A的表面残基Lys419被精氨酸取代，并且在Thr245和Ala326以及Trp651和Val707之间引入了两个二硫键。比活度和k _cat / KPul _AC的m值分别达到98.20 U / mg和12.22 mL / mg / s，是野生型Pul _A的1.5倍。突变体Pul _AC的最佳温度为65℃。在65°C孵育30分钟后，Pul _AC保留了超过85％的活性，这比野生型Pul _A维持的活性高得多。由于其最高的最佳温度，热稳定性和比活性，本文报道的突变体Pul _AC可能在提高淀粉脱支过程的水解效率中起重要作用。