Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis.
Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis.
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提高假交替单胞菌蛋白酶的产量。
DOI：
10.1007/s10126-016-9721-9
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
He Hailun
中科院分区：
文献类型：
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作者：
Wu Cuiling;Liu Dan;Yang Xinghao;Wu Ribang;Zhang Jiang;Huang Jiafeng;He Hailun
假交替单胞菌属CSN 423是一株能分泌E423蛋白酶的海洋菌株。为了提高蛋白酶E423的产率，采用交替假单胞菌（Pseudoalteronassp.）使用UV照射对CSN 423进行诱变。筛选到一株蛋白酶产量提高5.1倍的突变菌株，命名为假交替单胞菌属（Pseudoalteronassp）。CSN 423-M。以酪蛋白为底物的酶谱分析结果表明，该菌株有3条蛋白酶谱带，质谱分析结果表明，其中2条低分子量蛋白谱带为同一蛋白酶，但成熟形式不同。对蛋白酶操纵子进行全序列测定，未发现突变，采用实时荧光定量聚合酶链反应（qRT-PCR）检测突变株蛋白酶合成和分泌相关基因表达水平的变化。突变株的fe 423表达量高于野生型。该结果与蛋白酶活性谱一致。此外，突变株的柠檬酸合成酶（cs）、α-酮戊二酸脱羧酶（kgd）、细胞色素c氧化酶亚基I（cox I）、tolC、hlyD（膜蛋白）、luxR 3、luxO和luxT（转录调节因子）的转录水平均有所提高。然而，在突变体中，己糖激酶（hk）、丙酮酸脱氢酶E1（pd-e1）、epsD（膜蛋白）和luxR 1保持不变，而luxR 2急剧下降。这些结果表明，氧化还原途径在突变株中得到促进，LuxR家族转录调节因子在假交替单胞菌中得到促进。可能在调节蛋白酶表达中起一定作用。同时，胞外蛋白酶的分泌与ABC转运系统密切相关。这些结果可能有助于阐明交替假单胞菌蛋白酶E423高产的分子机制。CSN 423-M。
Pseudoalteromonassp. 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,Pseudoalteromonassp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named asPseudoalteromonassp. 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 ofe423than 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, andluxT(transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1),epsD(membrane protein), andluxR1remained unchanged, andluxR2decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators inPseudoalteromonasspp. 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 fromPseudoalteromonassp. CSN423-M.