Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei

Abstract

Ten putative Trichoderma reesei β-glucosidase (BGL) isozymes were heterologously expressed in Escherichia coli and Aspergillus oryzae and purified to homogeneity. Catalytic properties of nine enzymes which showed hydrolytic activity on cellobiose and p-nitrophenyl-β-D-glucopyranoside (pNPG) were investigated. Three BGLs, encoded by the genes cel3A, cel3B, and cel3E, contained a predicted signal peptide, showed higher hydrolytic activity on cello-oligosaccharides than on pNPG, and preferred longer oligosaccharides. Another three putative extracellular BGLs, Cel3B, Cel3F, and Cel3G, and two intracellular enzymes, Cel3C and Cel3D, exhibited preference for pNPG. Intracellular Cel1A showed the highest affinity for cellobiose as a typical cellobiase. Four BGLs, Cel3A, Cel3B, Cel3E, Cel1A, that showed high activity against cello-oligosaccharides were capable of catalyzing transglycosylation reactions from cellobiose, leading to formation of cellotriose and isomeric glucobioses. While Cel3A, Cel3B, and Cel3E synthesized mainly gentiobiose, glycosyl transfer reactions of Cel1A led mainly to sophorose and laminaribiose. Conversion of cellobiose to sophorose by Cel1A reached about 3.6 and 10 % at 1 and 10 % cellobiose concentration, respectively. The formation and persistence of individual cellobiose isomers in incubation mixtures of four BGLs (Cel3A, Cel3B, Cel3E, and Cel1A) with cellobiose correlated well with the k cat values for isomeric glucobioses. Cel1A also showed the lowest sensitivity to inhibition by glucose. Based on all studied catalytic properties, Cel1A appears to be unambiguously the best candidate for site-directed mutations or directed evolution toward improvement of activity, thermostability, and, eventually, efficiency of sophorose synthesis.

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production

“…cel3D, a putative intracellular β-glucosidase, was expressed at a lower level in a T. reesei mutant with the deletion of β-glucosidase regulator bglR than the parental strain PC-3-7, when cellobiose was used as the carbon source [20]. The purified cel3D that was heterologously expressed in E.coli displayed very low enzyme activity towards cellobiose and other oligosaccharides [15]. Clearly, very few studies have been performed on cel3D and the role of cel3D on the cellulase production remains unexplored.…”

“…Among them, cel3A (bgl1) is the major extracellular β-glucosidase in cellulase production [5], while cel1A (bgl2) [13] and cel1B [14] are intracellular. In addition, it is presumed that cel3B, cel3E, cel3F, cel3G and cel3H are extracellular, while cel3C, cel3D and cel3H are intracellular [15]. The involvement of β-glucosidases in cellulase production has been studied by characterization of mutants with knockout of different β-glucosidases.…”

The role of catalytic residue pKa on the hydrolysis/transglycosylation partition in family 3 β-glucosidases

“…18 For example, xylanases, for which this method has been successfully used, have a molecular weight of 20 kDa, 19,20 while the βgls secreted by Trichoderma reesei have estimated molecular weights ranging from 55 to 130 kDa. 21 Derivation of the apparent pK a values of the catalytic residues from the pHactivity profile 22 is also not trivial because interaction with other ionizable residues in the active site causes deviation from ideal titration behavior. 23 An alternative approach to these experimental methods is constant pH molecular dynamics (CpHMD), which captures fluctuations in the protonation state of ionizable residues caused by changes in solution pH and local conformation.…”

“…28,29 This study investigated the role of active site residue pK a s on the H/T partition of the Hypocrea jecorina (teleomorph of T. reesei) GH3 βgl, Cel3A (HjCel3A), using CpHMD/pH-REX. HjCel3A, along with HjCel3B, exhibits the highest activity against cellobiose among the H. jecorina GH3 βgls 21 and was found to improve the conversion of various cellulosic substrates by nearly 10%. 30 However, its hydrolytic activity decreases at high cellobiose or glucose concentration because of increased transglycosidic activity, 21 which appears to be prevalent among GH3 βgls.…”

Transglycosylation, a new role for multifunctional cellulase in overcoming product inhibition during the cellulose hydrolysis

“…5,6,[9][10]15 Several studies indicated that the downstream hydrolysis products (i.e., cellobiose, glucose and oligosaccharides) could be further consumed, and other disaccharides and/or oligosaccharides may synthesized by transglycosylation reaction of multifunctional cellulase. More specifically, during cellulose degradation process, the hydrolysis products of cellobiose and/or glucose are quickly consumed as substrates by transglycosylation to produce gentiobiose, spohorose, laminaribiose, and other disaccharides and oligosaccharides.…”