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Cloning, Sequence Analysis, and Expression in Escherichia coli of a Gene Coding for an Enzyme from Bacillus circulans K-1 that Degrades Guar Gum
- Source :
- Bioscience, Biotechnology, and Biochemistry. 62:514-520
- Publication Year :
- 1998
- Publisher :
- Informa UK Limited, 1998.
-
Abstract
- 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 Mr 55,242. 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 SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, 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.
- Subjects :
- Sequence analysis
Molecular Sequence Data
Gene Expression
lac operon
Bacillus
Molecular cloning
Biology
medicine.disease_cause
Galactans
Polymerase Chain Reaction
Applied Microbiology and Biotechnology
Biochemistry
Analytical Chemistry
law.invention
Mannans
Bacterial Proteins
law
Mannosidases
Plant Gums
Escherichia coli
medicine
Amino Acid Sequence
Cloning, Molecular
Molecular Biology
Polyacrylamide gel electrophoresis
Sequence Homology, Amino Acid
Organic Chemistry
beta-Mannosidase
Nucleic acid sequence
General Medicine
Molecular biology
Recombinant Proteins
Genes, Bacterial
Bacillus circulans
Recombinant DNA
Biotechnology
Subjects
Details
- ISSN :
- 13476947 and 09168451
- Volume :
- 62
- Database :
- OpenAIRE
- Journal :
- Bioscience, Biotechnology, and Biochemistry
- Accession number :
- edsair.doi.dedup.....35b52bad4a07a4b1cfffadcb1a08552d