Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillussp. S09

Abstract

Backgroundβ-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest.ResultsA β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes β-1,3-glucan. Insoluble β-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.ConclusionsThis study provides new insight into the impacts of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families.

Nocardiopsis species as potential sources of diverse and novel extracellular enzymes

“…Wild-type and recombinant strains over-expressing native and chimeric β-1,3-glucanases generally find applications in the biocontrol of fungi, in the production of yeast extract and in the clarification of wines (Cheng et al 2013). The β-1,3-glucanases derived from Nocardiopsis were unusual since they acted on insoluble glucans.…”

Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

“…b-1,3-Glucanosyltransferase from Aspergillus fumigatus, a member of GH 72, catalyzes the hydrolysis of b-1,3-glucan and transglycosylation reaction to produce water-insoluble b-1,3-glucans from oligosaccharides (Hartland et al, 1996). Some endo-1,3-b-glucanases possess carbohydrate-binding domains or immunoglobulin like domains, which enhance the ability of the enzymes to bind waterinsoluble substrate (van Bueren et al, 2005;Cheng et al, 2013). Glycosylphosphatidylinositol-anchored endo-1,3-b-glucanase in the ascomycete fungus Ustilago esculenta is localized in the plasma membrane and functions to modify the inner cell wall structure (de Groot et al, 2003;Nakajima et al, 2012).…”

Autolysis of Pichia pastoris induced by cold

“…The glucan-net is formed by long chains of β-1,3-glucan with chain branches attached through β-1,6 linkages (Orlean 2012 ). Endo-β-1,3-glucanases can degrade the glucan-net of S. cerevisiae cell wall (Baladrón et al 2002 ; Ferrer et al 1996 ; Tanaka and Phaff 1965 ) and they have been used to make the yeast cell wall fragile in certain production processes (Cheng et al 2013 ). Moreover, eng from P. pastoris encodes for a secreted putative endo-β-1,3-glucanase (ENG; accession no.…”