Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae

Arch Microbiol. 2014 Jan;196(1):25-33. doi: 10.1007/s00203-013-0935-1. Epub 2013 Nov 12.

Abstract

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-D-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2-5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-D-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cellulomonas / enzymology*
  • Cellulomonas / genetics*
  • Diploidy
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Glucose / metabolism
  • Glucose / pharmacology
  • Hydrolysis
  • Oligosaccharides / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics*
  • beta-Glucosidase / genetics*
  • beta-Glucosidase / metabolism*

Substances

  • Enzyme Inhibitors
  • Oligosaccharides
  • Recombinant Proteins
  • beta-Glucosidase
  • Glucose