文献类型：期刊文献

英文题名：A Type I Pullulanase from Geobacillus subterraneus: Functional Expression in Escherichia coli, Enzyme Characterization, Truncation, and Application

作者：Chen, Mianhui[1];Zhang, Jin[1];Wang, Jingjing[1];Lin, Lin[2,3];Wei, Wei[1];Shen, Yaling[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Newworld Inst Biotechnol, Shanghai 200237, Peoples R China;[2]Shanghai Inst Technol, Sch Chem & Environm Engn, Shanghai 201418, Peoples R China;[3]Natl Engn Res Ctr Nanotechnol, Res Lab Funct Nanomat, Shanghai 200241, Peoples R China

年份：2022

卷号：74

期号：11-12

外文期刊名：STARCH-STARKE

收录：;EI(收录号：20223712718844);WOS:【SCI-EXPANDED(收录号:WOS:000851985400001)】；

基金：M.C. and W.W. received funding from the Shanghai Natural Science Foundation (No. 20ZR1415400), the Shanghai outstanding technical leaders plan 19XD1431900, 19XD1431800, the National Natural Science Foundation of China (Grant No. 81830052, 81530053), and Shanghai Key Laboratory of Molecular Imaging (18DZ2260400).

语种：英文

外文关键词：Geobacillus subterraneus; pullulanase; structure truncation

摘要：This study clones a pullulanase gene (pul13552), encoding a thermostable type I pullulanase, from a Geobacillus subterraneus strain KCTC 3922 and the characteristics are probed. The gene encodes a 722 amino acid pullulanase with an open reading frame of 2169 bp. T The purified recombinant Pul13552 hydrolyzes the alpha-1,6 linkages specifically in pullulan to release maltotriose as the major product. Specific activity is detected to be 64.75 U mg(-1) and the K-m and V-max values of purified Pul13552 are 4.37 mg mL(-1) and 0.096 g (L min)(-1), respectively. The optimal activity of purified Pul13552 is exhibited at pH 6.0 and 60 degrees C, and the immobilized Pul13552 shows several different characteristics from the mobilized one. Substrates screening shows that Pul13552 hydrolyzes pullulan, amylopectin, and soluble starch, but not amylose. Substrate preference and product analysis indicate that the pullulanase from G. subterraneus strain KCTC 3922 is the type I pullulanase. Subsequently, the N-terminal of the Pul13552 is truncated and the Pul13552T shows different substrates specificity and stability. Furthermore, the saccharification maize starch is employed as the substrate to produce maltotriose by the Pul13552 and the yield reaches 8.5 g L-1. Generally, the study identifies an unreported pullulanase with appropriate thermostability and optimum pH, and the truncated Pul13552, Pul13552T, demonstrates the N-terminal of the Pul13552 is important for substrates recognition.