A low-temperature active endo-β-1,4-mannanase from Bacillus subtilis TD7 and its gene expression in Escherichia coli

Abstract

A low-temperature active endo-β-1,4-mannanase (YBMan) from Bacillus subtilis TD7 was isolated, characterized and successfully expressed in Escherichia coli to enhance the yield of mannanase for a potential application as a gel-breaker in guar gum-based fracturing fluids in oilfields. YBMan showed good compatibility with a wide temperature range and retained about 70% relative activity at 20°C compared to its optimal temperature (65°C). This is the highest relative activity among reported low-temperature active mannanases against guar gum. The gene (1104 bp) of strain TD7 coding a protein with 367 amino acid residues was cloned and its expression generated two recombinant mannanases, TBMan-1 and TBMan-2. Compared to the wild type, the protein yield of TBMan-1 from a one-liter shake flask broth increased 5.6-fold, and the specific activity (crude enzyme) increased 6.4-fold. The total enzyme activity increased 35.8-fold with a total activity of approximately 79550 U. Moreover, TBMan-1 had at 20°C still about 80% relative activity. The enzyme was evaluated also for its application as gel-breaker and showed excellent ability for viscosity reduction with guar gum at 20°C. Low-temperature activity and high yield make the recombinant β-mannanase attractive for applications with guar-based hydraulic fracturing fluids and other biotechnological aspects.