title: Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis.
abstract: Pseudoalteromonas sp. CSN423, a marine strain, can express a major protease designated as E423 and it was secreted into the supernatant. To improve the protease E423 yield, Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named as Pseudoalteromonas sp. CSN423-M. Three protease bands were detected by zymography with casein as substrate, and results of mass spectrometry (MS) showed that two lower molecular weight protein bands were the same protease but with different mature forms. The entire protease operon was sequenced and no mutation was found. Mutant strain-associated changes of expression levels of protease synthesis and secretion-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Mutant strain had higher expression of e423 than wild-type strain. Such result was consistent with protease activity profiles. Moreover, the mutant strain had higher transcriptional levels of citrate synthase (cs), α-ketoglutarate decarboxylase (kgd), cytochrome c oxidase subunit I (coxI), tolC, hlyD (membrane protein), luxR3, luxO, and luxT (transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1), epsD (membrane protein), and luxR1 remained unchanged, and luxR2 decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators in Pseudoalteromonas spp. may play some role in regulating protease expression. Meanwhile, the secretion of extracellular protease was closely related to ABC transport system. These results may shed some light on the molecular mechanism underlying higher yield of protease E423 from Pseudoalteromonas sp. CSN423-M.
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.|Department of Biochemistry, Changzhi Medical College, Changzhi, 046000, China.
authors.forename: Cuiling
authors.initials: C
authors.lastname: Wu
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.
authors.forename: Xinghao
authors.initials: X
authors.lastname: Yang
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.
authors.forename: Ribang
authors.initials: R
authors.lastname: Wu
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China.
authors.forename: Jiafeng
authors.initials: J
authors.lastname: Huang
authors.affiliation: School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, 410013, China. helenhe@csu.edu.cn.
authors.forename: Hailun
authors.initials: H
authors.lastname: He
mesh_terms: D059001:Aquatic Organisms; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D053858:Metabolic Networks and Pathways / Q000235:genetics / Q000528:radiation effects*; D008970:Molecular Weight; D016296:Mutagenesis / Q000528:radiation effects*; D009876:Operon; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D031161:Pseudoalteromonas / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D010447:Peptide Hydrolases
keywords: Extracellular protease; Marine bacteria; Mutagenesis; Pseudoalteromonas sp.