High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

Abstract

In this study, a novel beta-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-beta-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular beta-1,3-1,4-glucanase. The recombinant beta-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l(-1) with an activity of 55,300 U ml(-1) in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS-PAGE. It was optimally active at pH 7.0 and a temperature of 70 degrees C. Furthermore, the enzyme exhibited strict specificity for beta-1,3-1,4-D: -glucans. This is the first report on the cloning and expression of a beta-1,3-1,4-glucanase gene from Paecilomyces sp.

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

“…J18 and Paecilomyces sp. FLH30, respectively [15,16], it exhibited obviously distinct biochemical properties. PsBgl16A and PtLic16A exhibited their highest activity at neutral pH (7.0) and 70°C, whereas Bgl16C1 was optimally active at acidic pH 5.0 and 55°C and lost all activity under alkaline conditions.…”

“…FLH30 (ADZ46179) [16] and 58% identity with b-1,3-1,4-glucanase from Paecilomyces sp. J18 (ADK55597) [15]. The deduced Bgl16C1 contained a catalytic domain of glycosyl hydrolase family 16 and no carbohydrate binding domain.…”

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

“…he mixed-linked ␤-1,3-1,4-glucan (␤-glucan) is the predominant cell wall polysaccharide in the endosperm of cereals, such as oat, wheat, and barley, and accounts for up to 5.5% of the dry weight of grains (18). ␤-1,3-1,4-Glucanase (EC 3.2.1.73; ␤-glucanase, lichenase) is able to specifically cleave ␤-1,4-glycosidic linkages adjacent to a 3-O-substituted glucose residue in mixed-linked ␤-glucans (29).…”

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

“…Using thermostable cellulases can increase the production efficiency in industries that prefer high-temperature reaction conditions, such as brewing and bioethanol production (Blumer-Schuette et al 2008;Szijártó et al 2011;Vieille and Zeikus 2001;Viikari et al 2007). To exploit the high catalytic efficiency at elevated temperatures, scientists in both academic and industrial organizations have searched for suitable enzymes by screening the unknown in nature or modifying the known in the laboratory (Dumon et al 2008;Dutta et al 2008;Hua et al 2010;Kapoor et al 2008;Sandgren et al 2003b;Yang et al 2010). In general, there are two strategies of enzyme modification: (1) directed evolution by randomly mutating the enzyme-encoding gene and subsequent selection for desirable properties and (2) rational engineering by specifically mutating the gene, based on the structural information of the enzyme (Bornscheuer and Pohl 2001;Böttcher and Bornscheuer 2010;Percival-Zhang et al 2006;Schülein 2000).…”