Nucleotide sequencing, purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

J Dairy Sci. 2003 Aug;86(8):2547-57. doi: 10.3168/jds.S0022-0302(03)73849-1.


An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carboxylic Ester Hydrolases / chemistry*
  • Carboxylic Ester Hydrolases / genetics*
  • Carboxylic Ester Hydrolases / isolation & purification
  • Cheese / microbiology
  • Chromatography, Affinity
  • Gene Library
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lacticaseibacillus casei / enzymology*
  • Lacticaseibacillus casei / genetics
  • Molecular Sequence Data
  • Molecular Weight
  • Open Reading Frames
  • Sequence Analysis
  • Sodium Chloride
  • Substrate Specificity
  • Temperature


  • Sodium Chloride
  • Carboxylic Ester Hydrolases
  • arylesterase