Expression and characterization of a novel mesophilic protease from metagenomic library derived from Antarctic coastal sediment |
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Authors: | Yingjing Zhang Jing Zhao Runying Zeng |
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Institution: | (1) Key Lab of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, Daxue Road 178#, Xiamen, 361005, China;(2) College of Oceanography and Environmental Science, Xiamen University, Xiamen, 361005, China; |
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Abstract: | A metagenomic cosmid library was constructed, in which the insert DNA was derived from the coastal sediment near Antarctic
China Zhongshan Station. One clone (ACPRO001) expressing protease activity was isolated from the library using milk agar plates.
Sequencing of the clone revealed a novel protease gene. The amino acid sequence comparison and phylogenetic analysis indicated
that it could be classified as a subtilisin-like serine protease, though the highly conserved residue Asp was replaced by
Ala. The ACPRO001 protease gene was expressed in pET-His and purified for characterization. The optimal temperature and pH
for the activity of the ACPRO001 protease were 60°C and pH 9.0, respectively. The enzyme retained about 73% of residual activity
after 2 h incubation at 50°C in the presence of Ca2+. The presence of Ca2+ increased the thermostability of ACPRO001 protease obviously. The enzymatic activity was inhibited by 1 mM phenylmethyl sulfonylfluoride
(PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. |
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Keywords: | |
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