Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42
- 24 Downloads
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 (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.
KeywordsAnoxybacillus sp. WB42 Cysteine Disulfide bond Pullulanase Surficial residue Starch-debranching process
This work is financially supported by a Project Funded by the International S&T Innovation Cooperation Key Project (2017YFE0129600), the National Natural Science Foundation of China (21878125), the Natural Sciences Foundation of Jiangsu (BK20181206), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program, and First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-04).
Compliance with Ethical Standards
We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with submission to Applied Biochemistry and Biotechnology.
Conflict of Interest
The authors declare that they have no competing interests.
- 11.Liu, L., Deng, Z. M., Du, G. C., & Chen, J. (2014). In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica to improve thermostability. Applied and Environmental Microbiology, 80(3), 798–807.CrossRefGoogle Scholar
- 26.Rehman, H., Siddiqui, M., & Qayyum, A. (2018). Gene expression in Escherichia coli and purification of recombinant type II pullulanase from a hyperthermophilic archaeon, Pyrobaculum calidifontis. Pakistan Journal of Zoology, 50, 1381–1386.Google Scholar