Microbial Strain Development for Hyperproduction of β–glucosidase through γ–rays Mutagenesis and Expression Cloning

Abstract

The cellulose enzymatic hydrolysis is carried out by the synergistic action of cellulase enzyme complex, namely endo-glucanase, exo-glucanase and β-glucosidase (BGL). BGL is responsible for the regulation of the cellulolytic process as both endo- and exo-glucanase activities are often inhibited by cellobiose. In recent years, interest in BGL has gained momentum, owing to its potential use in various biotechnological processes including biomass degradation, fuel ethanol production, release of aromatic residues, extraction of fruit juices, production of fermented foods etc. A. niger is by far the most efficient BGL producer among the microorganisms investigated. To cope with the current industrial demand it is necessary to produce BGL with improved catalytic and kinetic properties. The current report deals with the development of A. niger strain through gamma rays treatment. Hyper producer mutant M-6 was selected on the basis of 2-deoxy-D-glucose resistance, which showed about two fold higher production of BGL as compared to control. Both the parent and mutant had shown best results with 3% wheat bran at 30 °C and pH 5. Random mutagenesis resulted in mutations within the BGL gene sequence, leading to overall conformational change in BGL secondary structure. The subunit and native molecular mass of the BGLs from parent and mutant were same i.e. 130 and 252 kDa, respectively. LC-MS/MS analysis also confirmed the identity of protein bands as BGL from both strains with a slight difference in molecular masses. The mutant BGL proved to be 1.6 fold efficient for cellobiose hydrolysis with lower activation energy (Ea) and thermodynamically more stable against denaturants than parent BGL. The mutant BGL gene was also expressed in Pichia pastoris and the recombinant BGL possessed similar characteristics as that of the mutant BGL. Effect of metals on parent and mutant BGLs showed that Ca2+ decreased the Ea, resulting in efficient formation of ES*-complex as compared to apo-BGL, while, Mn2+ decreased the Ea at lower metal concentrations only. The heat of ionization for the acidic and basic limbs of the apo- BGL reduced significantly in the presence of Ca2+ and Mn2+. The turnover (kcat) of apo-BGL showed an increase in the presence of low concentrations of Ca2+ and Mn2+. The BGL activation mechanism by Ca2+ was of mixed nature where Ca2+ was preferably binding with free enzyme, while Mn2+ was preferably binding with ES*-complex rather than free enzyme (partially un- competitive). Ca2+- and Mn2+- bound BGL also showed higher thermo-stability as compared to apo-BGL. Immobilization of BGL through entrapment on latex and silicone resulted in an increase in optimum temperature and thermal stability than the free enzyme. The immobilized BGL also increased the cellulose hydrolysis rate with more than 70% functional stability after multiple reuses. The current study has resulted in a highly stable and catalytically active BGL enzyme which may have substantial applications in biofuel and biorefinery industries.