Cloning, Sequence Analysis, and Expression in Escherichia coli of a Gene Coding for an Enzyme from Bacillus circulans K-1 that Degrades Guar Gum
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概要
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A 2, 048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking.This G-gene consisted of 1, 551 nucleotides coding for a protein with Mr55, 242.The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19.The molecular mass of recombinant G-enzyme estimated by SDSPAGE was 62 KDa, close to that from strain K-1.Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Gle, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides.Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band.The results suggested that the sugars accounted for the differences in the molecular masses.The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr.Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme.Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E.coli after translation.The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.
- 社団法人日本農芸化学会の論文
- 1998-03-23
著者
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Yoshida Seiji
Research Institute Of Technology Konoike Construction Co. Ltd.
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Yoshida S
Laboratory Of Cancer Cell Biology Research Institute For Disease Mechanism And Control Nagoya Univer
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SAKO Yoshihiko
Laboratory of Marine Microbiology, Department of Applied Bioscience, Graduate School of Agriculture,
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UCHIDA Aritsune
Laboratory of Marine Microbiology, Department of Applied Bioscience, Graduate School of Agriculture,
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Sako Y
Graduate School Of Agriculture Kyoto University
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Sako Yoshihiko
Laboratory Of Microbiology Department Of Fisheries Faculty Of Agriculture Kyoto University
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Sako Yoshihiko
Laboratory Of Marine Microbiology Division Of Applied Biosciences Graduate School Of Agriculture Kyo
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Uchida A
Graduate School Of Agriculture Kyoto University
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Uchida Aritsune
Laboratory Of Marine Microbiology Department Of Applied Bioscience Graduate School Of Agriculture Ky
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Sako Yoshihiko
Laboratory Of Marine Microbiology Department Of Applied Bioscience Graduate School Of Agriculture Ky
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