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Subcloning and Nucleotide Sequencing of the pul Gene of Thermus sp. AMD‐33
Author(s) -
Sashihara Nobuhiro,
Nakamura Nobuyuki,
Horikoshi Koki
Publication year - 1993
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/star.19930450407
Subject(s) - subcloning , thermus , genetics , thermus thermophilus , biology , gene , nucleotide , microbiology and biotechnology , computational biology , plasmid , escherichia coli , bacteria , thermophile
The entire sequence of a thermostable pullulanase gene pul of Thermus sp. AMD‐33 was determined. A single open reading frame of 2.154 bp was found which encodes a protein with a molecular mass of 80.327. Pullulanse purified from an E. coli transformant harbouring the plasmid encoded pul gene had a molecular mass of 81.000 as determined by SDS‐PAGE. The plasmid‐encoded enzyme was inhibited by pCMB, however, the activity was recovered after treatment with 2‐mercaptoethanol. The N‐terminal region was almost identical to that deduced from the DNA sequence. No signal peptide‐like sequence could be detected. Amino acid sequence analysis revealed that 4 regions out of the 6 regions which are characteristically found in amylases were conserved in the plasmid‐borne pullulanase. but no amylase activity could be detected.