Nagarajan Dilli Rani, Krishnan Chandraraj
Department of Biotechnology, Indian Institute of Technology Madras, Chennai-600036, India.
Protein Expr Purif. 2010 Mar;70(1):122-8. doi: 10.1016/j.pep.2009.09.020. Epub 2009 Oct 6.
Bacillus subtilis KCC103 hyper-produces alpha-amylase and the synthesis is resistant to carbon catabolite repression. The strain efficiently produced alpha-amylase in low cost agro-biomass based medium rich in simple sugars without catabolite repression. Here, the catabolite repression resistant promoter (amyR4) of alpha-amylase was isolated from KCC103 and used to synthesize recombinant enzymes in B. subtilis. When the bgaB gene encoding beta-galactosidase of Bacillus stearothermophilus was cloned and expressed under the amyR4 promoter, high level of beta-galactosidase activity was found in Escherichia coli (28 U/ml)) and B. subtilis (19 U/ml). Further, the genes encoding endoxylanase (xynA) and carboxymethyl cellulase (bglC) from B. subtilis were cloned with signal peptides and expressed with CCR-resistant amyR4 promoter. In E. coli, the expression was intracellular with activities of cellulase and xylanase at 76 and 105IU/ml respectively. The expression was extracellular in B. subtilis with activities at 17 and 17 IU/ml of cellulase and xylanase respectively in LB medium. When recombinant B. subtilis was cultured in LB-glucose medium, the synthesis of recombinant enzymes was not subject to catabolite repression and the expression was observed throughout the growth. This is important as glucose in the medium can prevent sporulation of the Bacillus and prevent activation of the other scavenger pathways that leads to degradation of recombinant proteins. The catabolite derepressed promoter of alpha-amylase from B. subtilis KCC103 can be efficiently used for overexpression of various industrial enzymes.
枯草芽孢杆菌KCC103能过量产生α-淀粉酶，且其合成不受碳分解代谢物阻遏的影响。该菌株能在富含单糖且无分解代谢物阻遏的低成本农业生物质培养基中高效产生α-淀粉酶。在此，从KCC103中分离出α-淀粉酶的分解代谢物阻遏抗性启动子（amyR4），并用于在枯草芽孢杆菌中合成重组酶。当将嗜热脂肪芽孢杆菌编码β-半乳糖苷酶的bgaB基因在amyR4启动子下克隆并表达时，在大肠杆菌（28 U/ml）和枯草芽孢杆菌（19 U/ml）中均发现了高水平的β-半乳糖苷酶活性。此外，来自枯草芽孢杆菌的编码内切木聚糖酶（xynA）和羧甲基纤维素酶（bglC）的基因与信号肽一起被克隆，并在抗CCR的amyR4启动子下表达。在大肠杆菌中，表达是胞内的，纤维素酶和木聚糖酶的活性分别为76和105 IU/ml。在枯草芽孢杆菌中，表达是胞外的，在LB培养基中纤维素酶和木聚糖酶的活性分别为17和17 IU/ml。当重组枯草芽孢杆菌在LB-葡萄糖培养基中培养时，重组酶的合成不受分解代谢物阻遏的影响，并且在整个生长过程中都能观察到表达。这很重要，因为培养基中的葡萄糖可以防止芽孢杆菌形成芽孢，并防止导致重组蛋白降解的其他清除途径的激活。枯草芽孢杆菌KCC103的α-淀粉酶的分解代谢物去阻遏启动子可有效地用于各种工业酶的过表达。
