Expression of α-Cyclodextrin Glycosyltransferase Gene of Gebacillius sp. CHB1 in Bacillus subtilis
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摘要:
目的 构建分泌表达α-环糊精葡萄糖基转移酶（α-CGTase）的重组枯草芽胞杆菌，实现α-CGTase的安全高效表达。 方法 通过PCR法扩增嗜热地芽胞杆菌α-CGTase基因，运用EcoR Ⅰ/Xho Ⅰ分别对α-CGTase基因及pBES进行双酶切，然后将该基因片段插入到大肠杆菌-枯草芽胞杆菌穿梭载体pBES中，再电转化法转化枯草芽胞杆菌RIK1285；对重组枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT发酵条件进行探索。 结果 （1）利用发酵培养基分泌表达α-CGTase，重组枯草芽胞杆菌工程菌B.subtilis RIK1285/pBE-CGT发酵上清液产酶达到2.9 U·mL-1。（2）TB为最适发酵培养基（配方：甘油0.5%，蛋白胨1.2%，酵母粉2.4%，K2HPO4 1.64%，KH2PO4 0.23%）；在初始pH 6.5，温度为37℃下，摇瓶发酵培养24 h后，α-环糊精酶的环化活性达到5.3 U·mL-1，是野生菌株嗜热地芽胞杆菌（0.66 U·mL-1）的8倍。 结论 成功构建了枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT工程菌，并确定其最适发酵培养基和培养条件。 -
关键词:
- 枯草杆菌工程菌 /
- α-环糊精葡萄糖基转移酶 /
- 表达 /
- 优化
Abstract:Objective To construct a vector for expressing α-cyclodextrin glycosyltransferase (α-CGTase) gene in Bacillus subtilis. Method The α-CGTase gene from Gebacillius sp. CHB1 was amplified by PCR. The EcoR Ⅰ-digested pBES and Xho Ⅰ-digested α-CGT gene were connected and transformed into B. subtilis RIK1285. Subsequently, fermentation of the recombinant B. subtilis RIK1285/pBE-CGT was optimized. Result (1) The α-CGTase gene was expressed in a fermentation medium to show an enzymatic activity of 2.9 U·mL-1 by B. subtilis RIK1285/pBE-CGT. (2) Medium TB with the formula of 0.5% glycerol, 1.2% peptone, 2.4% yeast extract, 1.64% K2HPO4, and 0.23% KH2PO4 was found to be optimal for the fermentation. After fermentation in TB at 37℃ for 24h, the α-CGTase activity reached 5.3 U·mL-1, which was 8-fold of what the wild Gebacillius sp. CHB1 could generate. Conclusion The engineered B. subtilis RIK1285/pBE-CGT was successfully obtained and the fermentation process optimized. -
图 1 重组表达载体pBE-CGT单/双酶切验证
注：1为1 kb DNA标准分子量；2为重组载体单酶切；3为重组载体双酶切。
Figure 1. Verification of pBE-CGT obtained by single and double digestion
Note:1.DL 1 kb DNA Marker; 2.Single digestion; 3.Double digestion.
图 2 重组芽胞杆菌B. subtilis RIK1285/pBE-CGT菌落PCR验证
注：1为1 kb DNA标准分子量；2~6为CGT克隆子PCR。
Figure 2. PCR validation on B. subtilis RIK1285/pBE-CGT colonies
Note:1.DL 1 kb DNA Marker; 2-6.Colony PCR of CGT。
图 3 初始工程菌发酵环糊精酶活力和生长曲线
Figure 3. CGTase activity and cell growth in shaking-flask fermentation
图 4 优化后工程菌发酵环糊精酶活力和生长曲线
Figure 4. CGTase activity and cell growth in shaking-flask fermentation after optimization
表 1 不同温度对菌体生长和产酶的影响
Table 1. Effects of temperature on growth and enzyme production of bacteria
温度
Temperature/℃细胞密度
OD600环糊精酶活性
CGTase activity/(U·mL-1)25 6.9 1.6±0.11a 30 7.3 2.1±0.16b 37 8.3 2.9±0.14c 40 9.5 1.9±0.15b 注：同列数据后不同小写字母表示不同处理间差异显著(P＜0.05)。表 2~3同。
Note:Values are the means±SD(n=3).Means with different letters within the same column are signi cantly different (P＜0.05).The same as Table 2-3.
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表 2 不同pH对菌体生长和产酶的影响
Table 2. Effects of pH on growth and enzyme production of bacteria
酸碱度
pH细胞密度
OD600环糊精酶活性
CGTase activity/(U·mL-1)5.5 7.3 1.9±0.13a 6.0 7.9 2.7±0.17b 6.5 8.1 3.3±0.19c 7.0 8.3 3.0±0.15c 7.5 8.4 2.7±0.15b 8.0 8.1 2.1±0.12a
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表 3 不同培养基对产环糊精酶的影响
Table 3. Effects of culture medium on growth and enzyme production of bacteria
初始培养基
Initial medium细胞密度
OD600环糊精酶活性
CGTase activity/(U·mL-1)LB 8.3 2.9±0.17a TSB 9.2 3.5±0.16b M9 10.5 4.1±0.13c TB 12.3 5.3±0.21d
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