Overexpression And Characterization Of Bacillus Amyloliquefaciens β-1,3-1,4-Glucanase

β-1,3-1,4-glucanase is an important industrial enzyme. It has been widely used in industry and agriculture, especially in brewing and feed industry. Presently,β-1,3-1,4-glucanase has mainly been produced by microbe in the country and abroad. The researches aboutβ-1,3-1,4-glucanase have been focused on constructing recombinant strains and improving the expression level and enzyme properties. The purpose of this research is to improve the expression level and properties ofβ-1,3-1,4-glucanase with the methods of molecular biology and gene engineering, and recombinant enzymes were characterized, respectively. The main studies and results in this research are as follows:A DNA fragment containingβ-1,3-1,4-glucanase structure gene bglA with its own signal peptide sequences was obtained by PCR from genomic DNA of Bacillus amyloliquefaciens CICIM B4801. The recombinant plasmid pUB-PQ-bglA, which contains promoter PQ, was constructed by inserting the amplified fragment into the cloning vector pUB110 directly. The recombinant plasmid pUB-PQ-bglA was transformed into Bacillus amyloliquefaciens (CICIM B4801) by electroporation. The bglA was overexpressed in the recombinant strain and the maximum enzyme activity was 303 U/mL, which was higher 11.84 times than the original strain. The highest extracellular engzym activity ofβ-1,3-1,4-glucanase reached up to 2023 U/mL with fermentator, which improved 6.67 times compared to flask fermentation for recombinant strain.The characterization researchs show that the optimal pH and temperature of the enzyme was 6.5 and 55°C, respectively. The isolate B. amyloliquefaciens CICIM B4801 (pUB-PQ-bglA) showed high stability ofβ-1,3-1,4-glucanase activity at the pH and temperature of 5.8-6.6 and 40-55°C, respectively. Theβ-1,3-1,4-glucanase activity was improved more or less by the addition of some ions such as Ca²⁺, K⁺, Zn²⁺ and Li⁺, and was significant inhibited by the addition of Mn²⁺, Cu²⁺, Fe²⁺ and EDTA.