Cloning And Expression Of Constitutive Endoglucanase Gene From Rhizopus Stolonifer And Enzymology Properties Of Recombinant Strains

Cellulase can be divided into the repression enzymes and the induction enzyme.The repression enzymes which is produce cellulose enzyme in the respective medium, these enzymes is generally lower expression and enzyme activity is very low.The induction enzyme is cellulose enzyme of vaccination in culture medium containing cellulose matrix, belongs to the cellulose enzyme induction type. Most of the cellulose is not soluble in nature, and how solid insoluble cellulose inter fungal cells to induce cellulose enzyme gene expression, early in the20th century, researchers have put forward the fungus can produce low levels of basic cellulase, under the condition of no induced can also produce,which is to identify the function of the basic cellulase extracellular cellulose substrates, combined with the substrate, and very low levels of hydrolysis, small molecules produced by the hydrolysis of soluble products, can enter the intracellular to induce efficient of cellulose enzyme gene expression, the guess turned out into a kind of model of filamentous fungi decompose cellulose. So this the type of research have great significance. This experiment for the cloning and expression of constitutive endoglucanase gene from rhizopus stolonifer and enzymology properties of recombinant strains, help us to understand the features of constitutive endoglucanase.In our Experiment we send the Rhizopus stolonifer in2%glucose as the sole carbon source culture medium, the composition of the enzyme is detected in the fermented liquid have constitutive endoglucanase enzyme activity, and to research the optimize conditions for enzyme.it is concluded that the optimal culture conditions for temperature30℃, table speed is180RPM, with fluid amount to60ml/250ml, add0.3%calcium carbonate. At this point the creeping type of rhizopus constitutive endoglucanase secretion in30h the maximum is.92IU.We have constructed the rhizopus stolonifer constitutive cDNA library, to screening positive clones,two gene were fonded, zegl size of1164bp, zeg2size of1251bp. Zeg1encoding387amino acids, the formulas for zegl C1814H2858N556O567S24, relative molecular weight of42297.4Da, theoretical isoelectric point9.42, belong to alkaline protein, speculated that half-life is more than10h. Hydrophilic coefficient (GRAVY) an average of0.459, in combination with ProtScale analysis result, the inference zegl for soluble protein. Zeg2size of1251bp encoding1251amino acids, the formulas for zeg2C1959H3046N594O607S24. relative molecular weight of45400.7Da, theoretical isoelectric point9.44, belong to alkaline protein that half-life is more than10h. Hydrophilic coefficient (GRAVY) an average of0.505, in combination with ProtScale analysis result, the inference zeg2for soluble protein. By comparing zegl and zeg2with86%similarity, a similar catalytic activity area. The CDD (conservative sequence database) analysis that the protein belongs to glycoside hydrolase family5, the PDB (protein structure database) analysis that the key catalytic residues in the fifth family, predict two protein147th of the glutamic acid (E) and199th of the tryptophan acid(W) were protein catalytic residues.Enzymology properties of recombinant strains Show that draw zegl and zeg2optimum reaction temperature is50℃, the optimum pH is5.0. Two enzymes in30-50℃enzyme stability is better, when temperature is greater than60℃soon lost enzyme activity, zegl than zeg2stability is good. Zeg1in pH between4.07.0enzyme activity is relatively stable, zeg2in pH between5.07.0enzyme activity is relatively stable. The stability of the pH can be seen more zegl more stable than zeg2. Study the effect of metal ions of recombinant enzyme activity, the results show that Mg2+、Ca2+、Zn2+to cut inside zegl glucohexaose glycosidase have obvious promoting effect, Ca2+to cut inside zeg2glucohexaose glycosidase have obvious promoting effect, Al3+、Mn2+cut inside of the two kinds of recombinant enzyme glucohexaose glycosidase has obvious inhibitory effect. Use CMC-SDS-PAGE electrophoresis accurately measure zegl and zeg2protein molecular weight55kD,58kD.