Characterizations And Funtional Analysis Of β-1,4-glucanase From Gibberella Zeae Expressed In Pichia Pastosis

Gibberella zeae, known by its anamorph Fusarium graminearum, is a plant pathogen. They can cause the most devastating disease, such as head blight on wheat,barley and ear rot on corn. In infected kernels of head blight on wheat and ear rot on corn, the pathogen can produce two mycotoxins. They can cause vomiting, liver damage, and reproductive defects in livestock, and are harmful to human. During the penetrating progress of hyphae, pathogen will produce cell wall degrading enzymes,and β-glucanase is one of them. It is believed that β-glucanase is a cell wall modifying enzymes, it can degrade β-glucan and render other polysaccharide components in cell walls more susceptible to hydrolysis. It has been gained wide public attention.Endo-β-1, 4-glucanase(EC3.2.1.4) is a pivotal enzyme which is involved in cellulose hydrolyzation. It is able to break the β-1, 4-glycosidic bonds within cellulose chain into low molecular weight glucose polymers. This kind of enzyme have already formed a large family in prokaryotes and eukaryotes, displaying different properties. In this study, a putative endoglucanase based on NCBI database(Genbank ESU17701)from Gibberella zeae was re-synthesized according to the yeast bias codon through the PTDS method. Then it was secretively expressed in Pichia pastoris by electroporation.After the identification of the activity and the SDS-PAGE electrophoresis, the positive recombinant β-1, 4-glucanase yeast strains were obtained. Then the positive strains were screened out to expand and induced expressed. The related enzymatic characteristics and kinetic parameters were measured using the pure enzyme purified by Ni2+-NTA agarose affinity column. The results showed that the molecular weight of Gzegl AI was 27.17 k Da. The protein quantitative is 12.37 g/m L and Gzegl AI’s isoelectric point is 7.75. The optimal temperature and p H of Gzegl AI is 40 o C and 4.5,respectively. When the enzyme Gzegl AI was treated at 4o C, 24 h, it showed higher relative activity under acidic p H, especially in p H 4.0-6.5, more than 80 %. There is a bad thermostability when treating the enzyme under different temperatures in different time. It could keep 30 % of the initial enzyme activity when treated at 50 o C with half an hour. When the temperature reached 60 o C and higher, the enzyme activity fell sharply to zero completely within a short time. This experiment also explored influences of different metal ions on enzyme Gzegl AI at low and high(1 m M and 10 m M) concentrations. The study found that a low concentration of Mn2+, a high concentration of Mn2+ and Cu2+ had inhibitory effect on enzyme activity, and the inhibition of Cu2+ is stronger. However, the high concentration of Al3+ could drastically promote the enzyme reaction. The substrate specificity on different cellulose materials such as carboxylmethyl cellulose sodium salt(CMC-Na), absorbent cotton, filter paper and cellulose microcrystalline(< 6%) showed that Gzegl AI have a strong specificity on CMC-Na. The Km value is 73.92 g/L and the Vm value is 0.16 g /(L·min). The influences of two kinds of protein digestive enzymes, pepsin and trypsin on Gzegl AI were also studied. Pepsin had a certain degree of inhibition on the enzyme activity, on the contrary, trypsin had no effect. Using the 4h7 m.1.A three-dimensional structure in the database PDB as a template, the three-dimensional structure of Gzegl AI was made by homology modeling methods on the website SWISS-MODEL. The Gzegl AI was composed by 246 residues and had a primary sequence identity of 58.72% with4h7 m.1.A(Th EG3). It’s analysed that Gzegl AI is one member of the glycosyl hydrolase family 12 and the three catalytic residues Asp120, Glu137, and Glu221,were strictly conserved in the CHF12.In view of the current lack of related reports about β-1, 4-glucanase from Gibberella zeae, the experiment studied enzymatic properties and functions of β-1,4-glucanase expressed by Pichia pastoris. They could provide fundamental basis for subsequent discussion in the pathogenic process of β-1, 4-glucanase in Gibberella zeae and also be an alternative potential applications in the efficent construction of β-1,4-glucanase engineering strain and the commercialized production.