Catalytic properties of the cloned amylase from Bacillus licheniformis.
A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.
Kim
I C
IC
Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon, Korea.
Cha
J H
JH
Kim
J R
JR
Jang
S Y
SY
Seo
B C
BC
Cheong
T K
TK
Lee
D S
DS
Choi
Y D
YD
Park
K H
KH
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
D005936:Glucans; C009109:pullulan; D000681:Amylases