title: Pilot-Scale Production and Thermostability Improvement of the M23 Protease Pseudoalterin from the Deep Sea Bacterium Pseudoalteromonas sp. CF6-2.
abstract: Pseudoalterin is the most abundant protease secreted by the marine sedimental bacteriumPseudoalteromonassp. CF6-2 and is a novel cold-adapted metalloprotease of the M23 family. Proteases of the M23 family have high activity towards peptidoglycan and elastin, suggesting their promising biomedical and biotechnological potentials. To lower the fermentive cost and improve the pseudoalterin production of CF6-2, we optimized the fermentation medium by using single factor experiments, added 0.5% sucrose as a carbon source, and lowered the usage of artery powder from 1.2% to 0.6%. In the optimized medium, pseudoalterin production reached 161.15 ± 3.08 U/mL, 61% greater than that before optimization. We further conducted a small-scale fermentation experiment in a 5-L fermenter and a pilot-scale fermentation experiment in a 50-L fermenter. Pseudoalterin production during pilot-scale fermentation reached 103.48 ± 8.64 U/mL, 77% greater than that before the medium was optimized. In addition, through single factor experiments and orthogonal tests, we developed a compound stabilizer for pseudoalterin, using medically safe sugars and polyols. This stabilizer showed a significant protective effect for pseudoalterin against enzymatic thermal denaturation. These results lay a solid foundation for the industrial production of pseudoalterin and the development of its biomedical and biotechnological potentials.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. yangjie199102@163.com.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. yangjie199102@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. yuyang15643177899@163.com.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. yuyang15643177899@163.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. tangbailu@yeah.net.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. tangbailu@yeah.net.
authors.forename: Bai-Lu
authors.initials: BL
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. servetonentsh@hotmail.com.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. servetonentsh@hotmail.com.
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhong
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. clubstone@sdu.edu.cn.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. clubstone@sdu.edu.cn.
authors.forename: Mei
authors.initials: M
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. xbb@sdu.edu.cn.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. xbb@sdu.edu.cn.
authors.forename: Bin-Bin
authors.initials: BB
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. zhangxiying@sdu.edu.cn.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. zhangxiying@sdu.edu.cn.
authors.forename: Xi-Ying
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. bczhou@qdio.ac.cn.
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. zhangyz@sdu.edu.cn.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. zhangyz@sdu.edu.cn.|Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China. zhangyz@sdu.edu.cn.
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. cxl0423@sdu.edu.cn.|Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. cxl0423@sdu.edu.cn.
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D004795:Enzyme Stability; D005285:Fermentation; D045726:Metalloproteases / Q000096:biosynthesis / Q000737:chemistry*; D010865:Pilot Projects; D031161:Pseudoalteromonas / Q000201:enzymology*; D012623:Seawater / Q000382:microbiology; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D012996:Solutions; D045726:Metalloproteases
keywords: M23 proteases; cold-adapted enzymes; fermentation; marine bacteria; optimization; stability