Molecular Cloning, Expression, and Enzyme Activity of Glucose Oxidase Gene from Soil Thermophilic Streptomyces

Mahsa Karimiabar; Hamed Ahari; Kumarss Amini

doi:10.30498/ijb.2021.272122.2979

Molecular Cloning, Expression, and Enzyme Activity of Glucose Oxidase Gene from Soil Thermophilic Streptomyces

Iran J Biotechnol. 2022 Jan 1;20(1):e2979. doi: 10.30498/ijb.2021.272122.2979. eCollection 2022 Jan.

Authors

Mahsa Karimiabar¹, Hamed Ahari¹, Kumarss Amini²

Affiliations

¹ Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
² Department of Microbiology, Faculty of Basic Science, Saveh Branch, Islamic Azad University, Saveh, Iran.

Abstract

Background: Glucose oxidase is an oxidoreductase that depletes oxygen in food processing and is used in biosensors, glucose diagnostic kits, food processing, cosmetics, and chemical industries. This enzyme is often isolated from fungi, such as Penicillium and Aspergillus Niger.

Objectives: The objective of this study was to clone and express a full-length GOX gene from soil thermophilic streptomyces for bioinformatic and enzyme activity evaluations.

Materials and methods: After collecting samples from the Gandom Beryan area of Kerman province, Iran, Streptomyces strains were identified with specific biochemical and molecular tests. Streptomyces strains with glucose oxidase gene were detected by PCR, and the amplified gene fragment was cloned into Escherichia coli host bacterium using TA cloning technique. The expression of the cloned GOX gene in the host bacterium was measured using real-time PCR, and the recombinant plasmids were sequenced. The enzymatic activity was measured in the extracts of E. coli cells carrying the plasmids.

Results: After screening the samples, 12 strains of Streptomyces were identified, 4 of which carried the GOX gene. The GOX open reading frame, obtained by PCR, was cloned into a vector and transformed into Escherichia coli origami to generate GOX-producing bacteria. Enzyme activity was confirmed and a phylogenetic tree showed the degree of kinship between Streptomyces species and other species, including Streptomyces SP MI02-7b. The expression levels of GOX genes mRNA were found to be approximately 4-fold higher in transformed E. coli than in soil thermophilic Streptomyces (P <0.001).

Conclusion: This study showed that natural thermostable streptomyces producing glucose oxidase enzyme could be found in Iran. The enzyme gene was successfully transformed into Escherichia coli generating a recombinant host with high yield capability that can be a major step towards the production of this enzyme from indigenous strains. It should be emphasized that the GOX enzyme produced by these strains is profitable due to high production levels correlated to the optimum condition in cheap culture media, short fermentation cycles, high expression capability, and ease of growth.

Keywords: Cloning; Expression; Glucose oxidase; Streptomyces.