Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (6): 299-309.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1215
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YANG Lu1,2(), YUAN Yuan2, FANG Zhi-kai2, LIN Ru2, JIANG Hong1,2(), ZHOU Jian1,2()
Received:
2023-12-25
Online:
2024-06-26
Published:
2024-05-15
Contact:
JIANG Hong, ZHOU Jian
E-mail:1950302732@qq.com;jianghong709@163.com;zjian503@163.com
YANG Lu, YUAN Yuan, FANG Zhi-kai, LIN Ru, JIANG Hong, ZHOU Jian. Identification of a Streptomyces Strain and Study on the Fermentation Process of Geldanamycin Production[J]. Biotechnology Bulletin, 2024, 40(6): 299-309.
培养基类型 Type of culture medium | 培养基配方 Component of culture medium |
---|---|
种子培养基Seed medium | 葡萄糖1.8%;麦芽糊精1.0%;黄豆饼粉2.25%;酵母粉0.5%;MgSO4·7H2O 0.1%;K2HPO4·3H2O 0.1%(pH 7.0) |
发酵培养基Fermentation medium | 淀粉4.0%;黄豆饼粉4.0%;氯化钴0.001%;豆油0.5%;碳酸钙1.0%(pH 7.2) |
胰蛋白胨-酵母膏琼脂ISP1 | 胰蛋白胨0.5%;酵母膏0.3%;琼脂2%(pH 7.0-7.4) |
酵母膏-麦芽膏琼脂ISP2 | 酵母膏0.4%;麦芽膏1%;葡萄糖0.4%;琼脂2%(pH 7.3) |
燕麦粉琼脂ISP3 | 燕麦粉2%;微盐0.1%;琼脂2%(pH 7.3) |
无机盐-淀粉琼脂ISP4 | 可溶性淀粉1%;K2HPO4·3H2O 0.1%;MgSO4·7H2O 0.1%;NaCl 0.1%;(NH4)2SO4 0.2%;CaCO3 0.2%;琼脂2% |
甘油-天冬酰胺琼脂ISP5 | L-天冬酰胺0.%;K2HPO4·3H2O 0.1%;甘油1%;微盐0.1%;琼脂2% |
蛋白胨酵母提取物铁琼脂ISP6 | Bacto蛋白胨铁琼脂3.6%;酵母膏0.1%(pH 7.0-7.2) |
酪氨酸琼脂ISP7 | 酵母膏0.1%;L-酪氨酸0.1%;NaCl 0.85%;琼脂2%(pH 7.5) |
高氏1号培养基G1 | 可溶性淀粉2%;KNO3 0.1%;K2HPO4·3H2O 0.05%;MgSO4·7H2O 0.05%;NaCl 0.05%;FeSO4 0.001%;琼脂2%(pH 7.2-7.4) |
马铃薯培养基PDA | 马铃薯20%;葡萄糖2%;琼脂2%(pH 6-6.5) |
蛋白胨-甘油培养基CM008 | 蛋白胨0.5%;牛肉提取物0.3%;甘油2%;琼脂1.5%(pH 7.0) |
葡萄糖-天门冬素培养基CM0011 | 葡萄糖1%;天门冬素0.05%;K2HPO4·3H2O 0.05%;琼脂1.5%(pH 7.2-7.4) |
查氏-蔗糖Charlery - sucrose | 蔗糖3%;K2HPO4·3H2O 0.1%;MgSO4·7H2O 0.05%;KCl 0.05%;NaNO3 0.2%;琼脂2%(pH 7.2-7.4) |
Table 1 Type and formula of medium
培养基类型 Type of culture medium | 培养基配方 Component of culture medium |
---|---|
种子培养基Seed medium | 葡萄糖1.8%;麦芽糊精1.0%;黄豆饼粉2.25%;酵母粉0.5%;MgSO4·7H2O 0.1%;K2HPO4·3H2O 0.1%(pH 7.0) |
发酵培养基Fermentation medium | 淀粉4.0%;黄豆饼粉4.0%;氯化钴0.001%;豆油0.5%;碳酸钙1.0%(pH 7.2) |
胰蛋白胨-酵母膏琼脂ISP1 | 胰蛋白胨0.5%;酵母膏0.3%;琼脂2%(pH 7.0-7.4) |
酵母膏-麦芽膏琼脂ISP2 | 酵母膏0.4%;麦芽膏1%;葡萄糖0.4%;琼脂2%(pH 7.3) |
燕麦粉琼脂ISP3 | 燕麦粉2%;微盐0.1%;琼脂2%(pH 7.3) |
无机盐-淀粉琼脂ISP4 | 可溶性淀粉1%;K2HPO4·3H2O 0.1%;MgSO4·7H2O 0.1%;NaCl 0.1%;(NH4)2SO4 0.2%;CaCO3 0.2%;琼脂2% |
甘油-天冬酰胺琼脂ISP5 | L-天冬酰胺0.%;K2HPO4·3H2O 0.1%;甘油1%;微盐0.1%;琼脂2% |
蛋白胨酵母提取物铁琼脂ISP6 | Bacto蛋白胨铁琼脂3.6%;酵母膏0.1%(pH 7.0-7.2) |
酪氨酸琼脂ISP7 | 酵母膏0.1%;L-酪氨酸0.1%;NaCl 0.85%;琼脂2%(pH 7.5) |
高氏1号培养基G1 | 可溶性淀粉2%;KNO3 0.1%;K2HPO4·3H2O 0.05%;MgSO4·7H2O 0.05%;NaCl 0.05%;FeSO4 0.001%;琼脂2%(pH 7.2-7.4) |
马铃薯培养基PDA | 马铃薯20%;葡萄糖2%;琼脂2%(pH 6-6.5) |
蛋白胨-甘油培养基CM008 | 蛋白胨0.5%;牛肉提取物0.3%;甘油2%;琼脂1.5%(pH 7.0) |
葡萄糖-天门冬素培养基CM0011 | 葡萄糖1%;天门冬素0.05%;K2HPO4·3H2O 0.05%;琼脂1.5%(pH 7.2-7.4) |
查氏-蔗糖Charlery - sucrose | 蔗糖3%;K2HPO4·3H2O 0.1%;MgSO4·7H2O 0.05%;KCl 0.05%;NaNO3 0.2%;琼脂2%(pH 7.2-7.4) |
水平 Level | A:葡萄糖Glucose/(g·L-1) | B:黄豆饼粉Soybean cake powder/(g·L-1) | C:硫酸铵 (NH4)2SO4/(g·L-1) |
---|---|---|---|
-1 | 80 | 15 | 2.5 |
0 | 100 | 20 | 3.0 |
1 | 120 | 25 | 3.5 |
Table 2 Optimized Box-Behnken test factors and levels via geldamycin
水平 Level | A:葡萄糖Glucose/(g·L-1) | B:黄豆饼粉Soybean cake powder/(g·L-1) | C:硫酸铵 (NH4)2SO4/(g·L-1) |
---|---|---|---|
-1 | 80 | 15 | 2.5 |
0 | 100 | 20 | 3.0 |
1 | 120 | 25 | 3.5 |
培养基 Medium | 生长状态 Growth state | 表面颜色 Surface color | 背面颜色 Back color | 气生菌丝 Aerial mycelium | 基质菌丝 Substrate mycelium | 产孢情况 Spore production situation | 可溶性色素 Soluble pigment | 质地 Texture |
---|---|---|---|---|---|---|---|---|
ISP1 | +++ | 绿白色 | 绿白色 | 浅绿灰色 | 绿白色 | 适中 | 无 | 圆形,波浪边缘,表面不光滑 |
ISP2 | ++++ | 绿灰色 | 中等黄褐色 | 绿灰色 | 绿白色 | 较厚 | 有 | 圆形,波浪边缘,表面不光滑,较多褶皱 |
ISP3 | ++++ | 绿灰色 | 浅绿灰色 | 绿灰色 | 绿白色 | 较厚 | 无 | 圆形,锯形边缘,表面不光滑 |
ISP4 | ++++ | 灰黄绿 | 黄灰色 | 灰黄绿 | 绿白色 | 较厚 | 有 | 圆形,锯形边缘,表面不光滑 |
ISP5 | ++++ | 浅绿灰色 | 绿白色 | 浅绿灰色 | 绿白色 | 较厚 | 无 | 圆形,锯形边缘,表面不光滑 |
ISP6 | +++ | 绿白色 | 深黄色 | 绿白色 | 绿白色 | 较薄 | 无 | 圆形,波浪边缘,表面较光滑 |
ISP7 | ++++ | 浅灰黄色 | 深黄褐色 | 浅灰黄色 | 浅灰黄色 | 适中 | 有 | 圆形,波浪边缘,表面光滑 |
G1 | ++++ | 灰黄绿 | 浅绿灰色 | 灰黄绿 | 淡黄绿色 | 较厚 | 有 | 圆形,波浪边缘,表面光滑 |
PDA | ++++ | 绿灰色 | 绿灰色 | 绿灰色 | 绿白色 | 较厚 | 有 | 圆形,波浪边缘,表面不光滑 |
CM008 | +++ | 绿白色 | 中等黄色 | 浅绿灰色 | 绿白色 | 适中 | 无 | 圆形,波浪边缘,表面不光滑,较多褶皱 |
CM0011 | ++++ | 灰黄棕色 | 绿白色 | 灰黄棕色 | 黄白色 | 较厚 | 无 | 圆形,波浪边缘,表面较光滑 |
查氏-蔗糖 Charlery - sucrose | ++ | 绿白色 | 淡绿色 | 绿白色 | 绿白色 | 较薄 | 无 | 圆形,波浪边缘,表面较光滑 |
Table 3 Culture characteristics of FIM18-0592
培养基 Medium | 生长状态 Growth state | 表面颜色 Surface color | 背面颜色 Back color | 气生菌丝 Aerial mycelium | 基质菌丝 Substrate mycelium | 产孢情况 Spore production situation | 可溶性色素 Soluble pigment | 质地 Texture |
---|---|---|---|---|---|---|---|---|
ISP1 | +++ | 绿白色 | 绿白色 | 浅绿灰色 | 绿白色 | 适中 | 无 | 圆形,波浪边缘,表面不光滑 |
ISP2 | ++++ | 绿灰色 | 中等黄褐色 | 绿灰色 | 绿白色 | 较厚 | 有 | 圆形,波浪边缘,表面不光滑,较多褶皱 |
ISP3 | ++++ | 绿灰色 | 浅绿灰色 | 绿灰色 | 绿白色 | 较厚 | 无 | 圆形,锯形边缘,表面不光滑 |
ISP4 | ++++ | 灰黄绿 | 黄灰色 | 灰黄绿 | 绿白色 | 较厚 | 有 | 圆形,锯形边缘,表面不光滑 |
ISP5 | ++++ | 浅绿灰色 | 绿白色 | 浅绿灰色 | 绿白色 | 较厚 | 无 | 圆形,锯形边缘,表面不光滑 |
ISP6 | +++ | 绿白色 | 深黄色 | 绿白色 | 绿白色 | 较薄 | 无 | 圆形,波浪边缘,表面较光滑 |
ISP7 | ++++ | 浅灰黄色 | 深黄褐色 | 浅灰黄色 | 浅灰黄色 | 适中 | 有 | 圆形,波浪边缘,表面光滑 |
G1 | ++++ | 灰黄绿 | 浅绿灰色 | 灰黄绿 | 淡黄绿色 | 较厚 | 有 | 圆形,波浪边缘,表面光滑 |
PDA | ++++ | 绿灰色 | 绿灰色 | 绿灰色 | 绿白色 | 较厚 | 有 | 圆形,波浪边缘,表面不光滑 |
CM008 | +++ | 绿白色 | 中等黄色 | 浅绿灰色 | 绿白色 | 适中 | 无 | 圆形,波浪边缘,表面不光滑,较多褶皱 |
CM0011 | ++++ | 灰黄棕色 | 绿白色 | 灰黄棕色 | 黄白色 | 较厚 | 无 | 圆形,波浪边缘,表面较光滑 |
查氏-蔗糖 Charlery - sucrose | ++ | 绿白色 | 淡绿色 | 绿白色 | 绿白色 | 较薄 | 无 | 圆形,波浪边缘,表面较光滑 |
分组Group | 葡萄糖Glucose/(g·L-1) | 黄豆饼粉Soybean cake powder/(g·L-1) | 硫酸铵(NH4)2SO4/(g·L-1) | 相对效价Relative titer/% |
---|---|---|---|---|
1 | 60 | 10 | 2.0 | 104 |
2 | 80 | 15 | 2.5 | 114 |
3 | 100 | 20 | 3.0 | 136 |
4 | 120 | 25 | 3.5 | 31 |
5 | 140 | 30 | 4.0 | 15 |
Table 4 Design and results of steepest-climbing test
分组Group | 葡萄糖Glucose/(g·L-1) | 黄豆饼粉Soybean cake powder/(g·L-1) | 硫酸铵(NH4)2SO4/(g·L-1) | 相对效价Relative titer/% |
---|---|---|---|---|
1 | 60 | 10 | 2.0 | 104 |
2 | 80 | 15 | 2.5 | 114 |
3 | 100 | 20 | 3.0 | 136 |
4 | 120 | 25 | 3.5 | 31 |
5 | 140 | 30 | 4.0 | 15 |
分组Group | A:葡萄糖Glucose/(g·L-1) | B:黄豆饼粉Soybean cake powder/(g·L-1) | C:硫酸铵(NH4)2SO4/(g·L-1) | 相对效价Relative titer(Y) |
---|---|---|---|---|
1 | 1 | 1 | 0 | 145.79±37.27 |
2 | 0 | 0 | 0 | 163.10±10.45 |
3 | -1 | -1 | 0 | 146.09±11.86 |
4 | 1 | 0 | -1 | 134.75±13.15 |
5 | 0 | 0 | 0 | 165.58±1.63 |
6 | 0 | 0 | 0 | 153.91±16.84 |
7 | 0 | 1 | 1 | 25.25±0.59 |
8 | 0 | 0 | 0 | 158.94±5.90 |
9 | -1 | 1 | 0 | 37.32±1.45 |
10 | 1 | -1 | 0 | 138.31±1.15 |
11 | 1 | 0 | 1 | 135.49±7.29 |
12 | -1 | 0 | -1 | 153.49±1.61 |
13 | 0 | 1 | -1 | 146.57±13.94 |
14 | 0 | -1 | -1 | 140.41±2.33 |
15 | -1 | 0 | 1 | 49.91±8.92 |
16 | 0 | -1 | 1 | 148.60±12.41 |
17 | 0 | 0 | 0 | 154.04±1.46 |
Table 5 Response surface design and test results
分组Group | A:葡萄糖Glucose/(g·L-1) | B:黄豆饼粉Soybean cake powder/(g·L-1) | C:硫酸铵(NH4)2SO4/(g·L-1) | 相对效价Relative titer(Y) |
---|---|---|---|---|
1 | 1 | 1 | 0 | 145.79±37.27 |
2 | 0 | 0 | 0 | 163.10±10.45 |
3 | -1 | -1 | 0 | 146.09±11.86 |
4 | 1 | 0 | -1 | 134.75±13.15 |
5 | 0 | 0 | 0 | 165.58±1.63 |
6 | 0 | 0 | 0 | 153.91±16.84 |
7 | 0 | 1 | 1 | 25.25±0.59 |
8 | 0 | 0 | 0 | 158.94±5.90 |
9 | -1 | 1 | 0 | 37.32±1.45 |
10 | 1 | -1 | 0 | 138.31±1.15 |
11 | 1 | 0 | 1 | 135.49±7.29 |
12 | -1 | 0 | -1 | 153.49±1.61 |
13 | 0 | 1 | -1 | 146.57±13.94 |
14 | 0 | -1 | -1 | 140.41±2.33 |
15 | -1 | 0 | 1 | 49.91±8.92 |
16 | 0 | -1 | 1 | 148.60±12.41 |
17 | 0 | 0 | 0 | 154.04±1.46 |
来源Source | 平方和Sum of squares | 自由度 Df | 均方Mean square | F | P |
---|---|---|---|---|---|
模型Model | 31 930.65 | 9 | 3 547.85 | 83.14 | < 0.000 1 |
A-葡萄糖 | 3 508.21 | 1 | 3 508.21 | 82.21 | < 0.000 1 |
B-黄豆饼粉 | 5 967.02 | 1 | 5 967.02 | 139.83 | < 0.000 1 |
C-硫酸铵 | 5 830.98 | 1 | 5 830.98 | 136.64 | < 0.000 1 |
AB | 3 378.32 | 1 | 3 378.32 | 79.17 | < 0.000 1 |
AC | 2 721.11 | 1 | 2 721.11 | 63.76 | < 0.000 1 |
BC | 4 193.53 | 1 | 4 193.53 | 98.27 | < 0.000 1 |
A2 | 1 604.43 | 1 | 1 604.43 | 37.60 | 0.000 5 |
B2 | 2 173.81 | 1 | 2 173.81 | 50.94 | 0.000 2 |
C2 | 1 890.01 | 1 | 1 890.01 | 44.29 | 0.000 3 |
失拟Lack of fit | 188.18 | 3 | 62.73 | 2.27 | 0.222 5 |
残差误差Residual error | 298.72 | 7 | 42.67 | R2=0.990 7 | |
纯误差Pure error | 110.54 | 4 | 27.64 | Adj R2=0.978 8 | |
合计 Total | 32 229.37 | 16 |
Table 5 Variance analysis and significance error of regression equation
来源Source | 平方和Sum of squares | 自由度 Df | 均方Mean square | F | P |
---|---|---|---|---|---|
模型Model | 31 930.65 | 9 | 3 547.85 | 83.14 | < 0.000 1 |
A-葡萄糖 | 3 508.21 | 1 | 3 508.21 | 82.21 | < 0.000 1 |
B-黄豆饼粉 | 5 967.02 | 1 | 5 967.02 | 139.83 | < 0.000 1 |
C-硫酸铵 | 5 830.98 | 1 | 5 830.98 | 136.64 | < 0.000 1 |
AB | 3 378.32 | 1 | 3 378.32 | 79.17 | < 0.000 1 |
AC | 2 721.11 | 1 | 2 721.11 | 63.76 | < 0.000 1 |
BC | 4 193.53 | 1 | 4 193.53 | 98.27 | < 0.000 1 |
A2 | 1 604.43 | 1 | 1 604.43 | 37.60 | 0.000 5 |
B2 | 2 173.81 | 1 | 2 173.81 | 50.94 | 0.000 2 |
C2 | 1 890.01 | 1 | 1 890.01 | 44.29 | 0.000 3 |
失拟Lack of fit | 188.18 | 3 | 62.73 | 2.27 | 0.222 5 |
残差误差Residual error | 298.72 | 7 | 42.67 | R2=0.990 7 | |
纯误差Pure error | 110.54 | 4 | 27.64 | Adj R2=0.978 8 | |
合计 Total | 32 229.37 | 16 |
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