Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for an enzyme from Bacillus circulans K-1 that degrades guar gum.
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.
Yoshida
S
S
Research Institute of Technology, Konoike Construction Co., Ltd., Ibaraki, Japan.
Sako
Y
Y
Uchida
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005685:Galactans / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D008351:Mannans / Q000378:metabolism*; D008361:Mannosidases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D053149:Plant Gums; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044902:beta-Mannosidase
D001426:Bacterial Proteins; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D011994:Recombinant Proteins; C007894:guar gum; D008361:Mannosidases; D044902:beta-Mannosidase; C112065:mannan endo-1,4-beta-mannosidase, Bacillus circulans