Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca

Eur J Biochem. 2000 Aug;267(16):4988-97. doi: 10.1046/j.1432-1327.2000.01546.x.

Abstract

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinomycetales / enzymology*
  • Actinomycetales / genetics
  • Amino Acid Sequence
  • Base Sequence
  • Catalytic Domain
  • Cellulase / chemistry
  • Cellulase / genetics*
  • Cellulase / metabolism*
  • Cellulose 1,4-beta-Cellobiosidase
  • Cloning, Molecular
  • Clostridium / enzymology
  • Escherichia coli
  • Isoenzymes / chemistry
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Streptomyces
  • Substrate Specificity
  • Thermodynamics

Substances

  • Isoenzymes
  • Recombinant Proteins
  • Cellulase
  • Cellulose 1,4-beta-Cellobiosidase