Enhanced pullulanase production through expression system optimization and biofilm-immobilized fermentation strategies

Peifang Ren; Qiwei Dong; Chaowei Zhou; Tianpeng Chen; Wenjun Sun; Yong Chen; Hanjie Ying

doi:10.1016/j.ijbiomac.2025.139933

Enhanced pullulanase production through expression system optimization and biofilm-immobilized fermentation strategies

Int J Biol Macromol. 2025 Jan 15:139933. doi: 10.1016/j.ijbiomac.2025.139933. Online ahead of print.

Authors

Peifang Ren¹, Qiwei Dong¹, Chaowei Zhou¹, Tianpeng Chen¹, Wenjun Sun², Yong Chen³, Hanjie Ying⁴

Affiliations

¹ National Engineering Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China; State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China.
² National Engineering Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China; State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China. Electronic address: [email protected].
³ National Engineering Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China; State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China. Electronic address: [email protected].
⁴ National Engineering Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China; State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, PR China; Soochow University, Suzhou, Jiangsu 215123, PR China.

PMID: 39824400
DOI: 10.1016/j.ijbiomac.2025.139933

Abstract

Pullulanase (PUL) plays a crucial role in breaking down α-1,6-glycosidic bonds in starch, a key process in starch processing and conversion. Based on PulB with high enzymatic activity, the expression of PUL in Bacillus subtilis was enhanced by plasmid screening, double promoter optimization, and signal peptide engineering. Furthermore, we innovatively employed a mussel foot protein to enhance the cell adhesion to carriers and utilized biofilm-based cell immobilization technology to optimize the fermentation process and stimulate biofilm formation. This approach led to a notably elevated enzyme activity, reaching 2233.56 U mL^-1. The PUL crude enzyme solution, capable of generating high glucose syrup and resistant starch, paves the way for new avenues of exploration and advancement in research and industrial biotechnology.

Keywords: Biofilm; Immobilized fermentation; Pullulanase.