Cloning, sequencing, and expression of Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase in Escherichia coli.
The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with FO1F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-beta-N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.
Takegawa
K
K
Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Japan. takegawa@ag.kagawa-u.ac.jp
Yamabe
K
K
Fujita
K
K
Tabuchi
M
M
Mita
M
M
Izu
H
H
Watanabe
A
A
Asada
Y
Y
Sano
M
M
Kondo
A
A
Kato
I
I
Iwahara
S
S
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase