解淀粉芽孢杆菌β1314葡聚糖酶的高效表达-efficient expression of β 1314 glucanase from bacillus amyloliquefaciens.docxVIP

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解淀粉芽孢杆菌β1314葡聚糖酶的高效表达-efficient expression of β 1314 glucanase from bacillus amyloliquefaciens.docx

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解淀粉芽孢杆菌β1314葡聚糖酶的高效表达-efficient expression of β 1314 glucanase from bacillus amyloliquefaciens

。斗，3-1，4-g1ucanase is an important industria1 enzyme. It has been widely used in industryandagriculture ， especiallyinbrewingandfeedindus町. Presently，。-1，3斗，4唱lucanase has mainly been produced by microbe 扭曲e country and abroad. Theresearches about ?-l ，3斗，4-g1ucanase have been focused on constructing recombinant strains and improving the expression level and enz归ne properties. 丁he purpose of this research is ω improve the expression level and properties of ?斗，3-1，4唰glucanase with the methods of molecular biology and gene engineeri吨， and recombinant enzymes were characteriz时，respectively. The main studies and results in this research are as fol1ows:A DNA fragment containing ?-l ，3-1，4也lucanase structure gene bglA with its own signal peptide sequences was obtained by PCR from genomic DNA of Baci/lus αmyloliqu价ciens CICIM B4801. The recombinant plasmid pUB Q-bglA，which contains promoter PQ，w出 constructed by inserting the amplified fra伊nent into the cloning vector pUB 110 directly. The recombinant plasmid pUB-PQ 翩 bglA was transformed into Bacillus amyloliqu呐ciens (CICIMB480 1) by electroporation. The bglA w，部overexpressed in the recombinant strain and themaximum enzyme activity wω，303 U/mL，which was higher 11.84 times than the originalstrain.ηle highest extracellular engzym activity of ?斗，3-1，4唱lucanase reached up to 2023 U/mL with fermentator，which improved 6.67 times compared to flask fermentation forrecombinant strain.翩ηle characterization researchs show that the optimal pH and tempera阳re of the enzyme wω6.5 and 550C，respectively. The isolate B. amyloliquφciens CICIM B4801 (pUB Q-bglA) showed high stability of ?-l ，3-1，4-g1ucanase activity at the pH and temperature of 5.8-6.6 and 40-550C，respectively. The ?斗，川，4 glucan邸e activity was+improved more or less by the addition of some ions such ωCa2飞 K飞 Zn2and Lt，and wassignificant inhibited by the addition of Mn2 ，Cu2飞 Fe2and EDTA.4，EPEWm-m曹-Ea呻HHm 4‘·--」山mm3A…bA++Keywords: 阳，川，4唱lucana