Screening and identification of native cellulose-degrading bacteria from soil and cloning of endoglucanase enzyme gene

Marefat1, Arezu; Sadeghi, Leila; Dehghan, Gholam Reza

Screening and identification of native cellulose-degrading bacteria from soil and cloning of endoglucanase enzyme gene

Document Type : Original Research

Authors

Arezu Marefat1

Leila Sadeghi

Gholam reza Dehghan

Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran

Abstract

In recent years, biocatalysts have widespread application in industry because they can do chemical reactions with the lowest energy and highest efficiency. Bacterial enzymes are more useful in this field due to simple cloning and expression process in the manipulated host. By considering specific role of endoglucanase enzymes in cellulose hydrolyzing reactions, these types of enzymes are more applicable in related industries. The produced glucose through enzymatic hydrolysis could be used in different industries such as biofuel and ethanol production and in the food industry as sweetener. Therefore, cloning and production of Endoglucanase in manipulated hosts has been developed in recent years. This study was performed to isolate, screen and identify native endoglucanase -producing strains from soil around the roots of the maple tree. Isolated strains were identified using 16S rRNA gene sequencing. After identifying of the bacteria (Enterobacter hormaechei), Endoglucanase enzyme gene was amplified using degenerate primers at first and then by specific primers with restriction enzymes sequences. DNA fragment and plasmid vector were treated by specific restriction enzymes and then ligated to each other. Then recombinant plasmid transferred to the E. coli BL-21 as expression host and kinetic properties of recombinant enzyme were evaluated. Expression of the target protein was done by stimulating the Lac operon by using 1 mM of IPTG and the kinetic features of the recombinant enzyme such as Vmax and Km evaluated as 45 µmol/min and 1.4 mg/ml respectively. The optimum conditions for enzyme activity tend to be 37°C at a pH of 7.

Keywords

Cellulose

Carboxy methyl cellulose

Screening

Degenerated primers

Endoglucanase enzyme

Enterobacter hormaechei

Subjects

Industrial Biotechnology

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