High-level expression and characterization of a highly active hyaluronate lyase HylC with significant potential in hyaluronan oligosaccharide preparation.

Autor: Zhang YS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China., Jiang JY; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China., Gong JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China. Electronic address: jinsonggong.bio@hotmail.com., Su C; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China., Li H; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China., Kang CL; Shandong Engineering Laboratory of Sodium Hyaluronate and its Derivatives, Shandong Focusfreda Biotech Co., Ltd, Qufu 273165, PR China., Liu L; Shandong Engineering Laboratory of Sodium Hyaluronate and its Derivatives, Shandong Focusfreda Biotech Co., Ltd, Qufu 273165, PR China., Xu ZH; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi 214122, PR China., Shi JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China. Electronic address: shijs@163.com.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2023 Mar 20; Vol. 366, pp. 35-45. Date of Electronic Publication: 2023 Mar 15.
DOI: 10.1016/j.jbiotec.2023.03.003
Abstrakt: Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca 2+ , K + , Zn 2+ , Ni 2+ and Li + . Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 10 4 U/mL to 2.64 × 10 4 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×10 5 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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