Characterization of a Multimodular Endo-β-1,4-Glucanase (Cel9K) From Paenibacillus Sp. X4 With a Potential Additive for Saccharification

J Microbiol Biotechnol. 2018 Apr 28;28(4):588-596. doi: 10.4014/jmb.1712.12047.

Abstract

An endo-β-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9). The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison to the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and 50°C and showed a half-life of 59.2 min at 50°C. The CMCase activity was increased to more than 150% in the presence of 2 mM Na⁺, K⁺, and Ba²⁺, but decreased significantly to less than 50% by Mn²⁺ and Co²⁺. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4 and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

Keywords: GH9; Multimodular endo-β-1,4-glucanase; Paenibacillus sp. X4; saccharification.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Bacillus / enzymology
  • Bacillus / genetics
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Cellulase / chemistry*
  • Cellulase / genetics*
  • Cellulase / isolation & purification
  • Cellulase / metabolism*
  • Cloning, Molecular
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lignin / metabolism
  • Metals / metabolism
  • Molecular Weight
  • Oryza
  • Paenibacillus / enzymology*
  • Paenibacillus / genetics*
  • Protein Sorting Signals
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Substrate Specificity
  • Sugars / metabolism
  • Temperature
  • Time Factors

Substances

  • Bacterial Proteins
  • Metals
  • Protein Sorting Signals
  • Sugars
  • lignocellulose
  • Lignin
  • Cellulase