Cloning, characterization and heterologous expression of the firstPenicillium echinulatumcellulase gene

Abstract

Aims: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe‐egl1) encoding a putative endoglucanase. Methods and Results: Pe‐egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5·0–9·0), and an optimal temperature of 60°C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre‐incubation at 70°C). Calcium exerted a strong stimulatory effect over EGL1 activity. Conclusions: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. Significance and Impact of the Study: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro‐organism.

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

“…However, the activity of Spr Cel8A increased in the presence of Co 2+ , Mn 2+ , Li 1+ and Na 1+ (1 and 5 mM), and other metallic ions such as Cu 2+ , Ni 2+ and Fe 2+ at 1 mM, and Ca 2+ 5 mM. This suggests that this endoglucanase manifests a conformational stability, because the enzyme retained its activity even in the absence of metallic ions (Welfle et al 1995; Rubini et al 2010). An increase of 139.5 % in the activity of an endo-1,4-β-glucanase from B. subtilis was observed in the presence of Co 2+ (Au and Chan 1987); whereas Spr CelA from S. proteamaculans increased by 108 %.…”

“…4b). Enzyme inhibition by metallic cations usually suggests the presence of at least one sulfhydryl group in the active site, usually a cysteine amino acid (Rubini et al 2010). This group oxidation by the cations destabilizes the conformational folding of the enzyme (Rouvinen et al 1990) or leads to the formation of disulfide bonds at an irregular position of the protein (Ohmiya et al 1995).…”

High-level expression of a novel Penicillium endo-1,3(4)-β-d-glucanase with high specific activity in Pichia pastoris

“…Several endo-b-1,4-glucanases from Penicillium pinophilum KMJ601 [19], P. purpurogenum [22], and P. echinulatum [32] have been reported, but all of them only have the ability to hydrolyze the b-(1 ? 4) glycosidic bonds of glucan.…”

Cloning, expression and characterization of a novel GH5 exo/endoglucanase of Thermobifida halotolerans YIM 90462T by genome mining

“…Compared to other groups of extremophilic microorganisms, such as thermophiles and alkaliphiles, halophiles have not often been utilized in biotechnological processes, even though researchers recently predicted that halophiles may be used to break down biomass material and form biofuel products (25). The ThCel5A we cloned from T. halotolerans YIM 90462 T was tolerant to most ions, especially to Pb 2þ and Ag þ , which are well-known to be toxic to enzymes by oxidizing their sulfhydryl groups (26). In contrast, Ca 2þ and Mn 2þ stimulated the activity of ThCel5A, which was most likely due to the ability of Ca 2þ to stabilize an enzyme conformation with a higher affinity for the substrate, while Mn 2þ hyper-stabilized the catalytic core (26,27).…”

“…The ThCel5A we cloned from T. halotolerans YIM 90462 T was tolerant to most ions, especially to Pb 2þ and Ag þ , which are well-known to be toxic to enzymes by oxidizing their sulfhydryl groups (26). In contrast, Ca 2þ and Mn 2þ stimulated the activity of ThCel5A, which was most likely due to the ability of Ca 2þ to stabilize an enzyme conformation with a higher affinity for the substrate, while Mn 2þ hyper-stabilized the catalytic core (26,27). However, Endo et al found that the activity of Egl-252, an alkaline GH5 endoglucanase from an alkaliphilic Bacillus isolate, was more stable in the absence of CaCl 2 (28).…”