Chuan Zhang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, ChinaYue Zhang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, ChinaXiaohu Ding
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, ChinaZhongyuan Li
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, ChinaYajian Song
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, ChinaXuegang Luo
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology, Tianjin 300457, China[Objective] To clone and recombinant express the gene Heparinase I from Bacteroides thetaiotaomicron, and then characterize the recombinant SUMO-Bt-HepI and Bt-HepI. [Methods] Codon optimization was done on the gene sequence of B. thetaiotaomicron heparinase I. The target gene was obtained by PCR amplification, inserted into the expression vectors pET-28a and pE-SUMO, and then transformed into E. coli Rosetta (DE3) to obtain recombinant products Bt-HepI and SUMO-Bt-HepI. Heparin sodium was used as substrate to study the enzymatic properties of the recombinant proteins. [Results] SDS-PAGE analysis showed that the molecular weights of Bt-HepI and SUMO-Bt-HepI were about 42.5 kDa and 55 kDa, respectively. Compared with Bt-HepI, the specific enzyme activity of Heparinase I increased by 48.9% after fusion SUMO-Tag. The enzymological properties showed that the optimum pH and temperature of Bt-HepI and SUMO-Bt-HepI were pH 9 and 45℃, and both recombinant enzymes were stable at pH 5-9, while the acid resistance of SUMO-Bt-HepI was obviously higher than Bt-HepI when the pH value was lower than 5. Besides, SUMO-Bt-HepI also showed higher activities than Bt-HepI under 50℃. In addition, Ca2+ and Mg2+ have obvious promoting effect on the recombinant heparinase I, while Cu2+, Mn2+ and Zn2+ show certain inhibiting effect, suggesting that in addition to the well-known Ca2+ binding site, Mg2+ binding sites may aslo exist in the structure of B. thetaiotaomicron Heparinase I. [Conclusion] Recombinant Heparinase I in B. thetaiotaomicron using SUMO fusion system significantly improved its specific enzyme activity for potential production and application.
Chuan Zhang, Yue Zhang, Xiaohu Ding, Zhongyuan Li, Yajian Song, Xuegang Luo. SUMO-Fused expression and characterization of Heparinase I from Bacteroides thetaiotaomicron. [J]. Acta Microbiologica Sinica, 2019, 59(7): 1318-1330
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