Abstract
BackgroundEndo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. Penicillium oxalicum is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-β-mannanase gene from Penicillium oxalicum.ResultsA gene encoding an acidophilic thermostable endo-1,4-β-mannanase (E.C. 3.2.1.78) from Penicillium oxalicum GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in Pichia pastoris GS115. A high enzyme activity (84.4 U mL−1) was detected in the culture supernatant. The recombinant endo-1,4-β-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg−1 using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80°C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60°C at pH 4.0. The Km and Vmax values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL−1 and 1425.5 μmol min−1 mg−1, 2.1 mg mL−1 and 154.8 μmol min−1 mg−1, and 2.3 mg mL−1 and 18.9 μmol min−1 mg−1, respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe3+ and Hg2+. Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose.ConclusionOur study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-β-mannanase in Pichia pastoris is suitable for various biotechnology applications.
Highlights
Endo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries
Mannan polysaccharides are the major components of softwood hemicellulose and can be classified into linear mannan, glucomannan, galactomannan, and galactoglucomannan [1]
Cloning and sequence analysis of the endo-1,4-β-mannanase gene from P. oxalicum GZ-2 A 772-bp gene fragment was amplified from the gDNA of strain GZ-2 using the degenerate primers manA-df and manA-dr
Summary
Endo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. The most important is endo-1,4-β-mannanase (EC 3.2.1.78), which can catalyze random internal hydrolysis of β-1,4-mannosidic linkages in the main structure of β-1,4-mannans, glucomannans and galactomannans by releasing small manno-oligosaccharides [2] This type of enzyme has shown a wide range of potential uses in industrial applications, including food/feed, pharmaceutical, pulp/paper, gas well stimulation [3], as well as for second generation biofuels [4,5]. The breakdown of lignocellulose to fermentable sugars by enzymatic hydrolysis is one of the decisive bottlenecks, due to the recalcitrance of the plant cell wall and the high cost of enzymes Based on this application, these enzymes have recently received much attention for industrial use, with an everincreasing demand for renewable bioenergy utilization
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