Properties of the endogenous cellulase from Panesthia cribrata saussure and purification of major endo-β-1,4-glucanase components

Abstract

The cellulase of Panesthia cribrata consists of at least six endo-β-1,4-glucanase (EC 3.2.1.4) and two β-glucosidase (EC 3.2.1.21) components. The two major endo-β-1,4-glucanase components, named EG1 and EG2, comprise 13% of the soluble protein in the foregut and midgut contents. They were purified by a combination of gel chromatography and ion-exchange FPLC and had M r of 53,600 and 48,800, respectively. With carboxymethylcellulose (CMC) the K m and V max values for EG1 and 9.4 mg/ml and 22.2 mg reducing sugar/min/mg protein; the corresponding values for EG2 were 6.8 and 88.3l. Values for K m, V max and k cat were also calculated for activity against cellotetraose and cellopentaose. Neither component hydrolysed cellobiose or cellotriose. The relative activities of EG1 and EG2 against CMC and microcrystalline cellulose Sigmacell Type 20 were 5200:1 and 3500:1. It is proposed that the inefficiency of EG1 and EG2 is compensated for by their secretion in large amounts into the gut. Four minor endo-β-1,4-glucanase components, comprising between 4 and 10% of the endo-β-1,4-glucanase activity were also present but could not be separated from the β-glucosidase components and therefore were not characterised. There were two β-glucosidase components, named GD1 and GD2, which were partially purified and characterised. The K m for GD1, the major component, was 10.6 mM with p- nitrophenyl-β- D - glucopyranoside . K m values were also determined for both components with cellodextrins ranging from cellobiose to cellopentaose. GD1 was competitively inhibited by gluconon-δ-1, 5-lactone ( K i = 0.33 mM) but was unaffected by glucose at physiological concentrations. Neither component was active against crystalline cellulose or CMC.