青贮饲料中产纤维素酶细菌的筛选、发酵和产酶条件优化及酶学性质初探
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摘要
本研究分离筛选了一株产纤维素酶的芽抱杆菌ws-6,优化了该菌株的产酶发酵条件,初步研究了酶学性质,并进一步优化了菌株的发酵条件,结果分述如下:
菌株分离筛选及鉴定从乌苏窖藏饲料中分离筛选出6个产CMCase的菌株,以代号ws-6的菌株产酶水平最高,通过形态学、生理生化指标及16S rDNA序列比对,鉴定为地衣芽孢杆菌(Bacillus licheniformis)。菌株最适生长pH值为5.0～7.0,最适生长温度35℃,产CMCase活力达2.55U/mL。
菌株产酶条件及酶学性质经碳、氮源及微量元素的筛选实验和正交实验分析,最适合菌株ws-6产CMCase的发酵培养基组成为(g/L):葡萄糖15g、CMC-Na 5g、酵母膏15g、KH_2PO_40.5g、NaCl 5g。发酵产酶的最适初始pH值为6.5,最适培养温度为35℃,最适装液量30mL(250mL三角瓶)。对菌株ws-6的酶学性质进行初步研究表明:酶反应的最适pH值为5.0,最适温度为50℃,最适底物浓度为0.6%,最适反应时间为20min。
菌株发酵条件优化经碳、氮源及微量元素的筛选实验和正交实验分析,确定ws-6菌株发酵培养基的最佳组分为(g/L):蔗糖60g、蛋白胨30g、NaCl 30g、K2HPO4 2 g、MgSO_4 1 g、FeSO_4 0.02 g、MnSO4 0.01 g。对发酵条件进行了研究,发酵的最适初始pH值为5.5,最适培养温度为35℃,最适装液量为10mL(250mL三角瓶),通过优化培养基组分和培养条件后,菌株ws-6活菌数达到了6.58×10~(10) CFU·mL~(-1)。
This study has isolated a cellulose-producing Bacillus sp. ws-6, and optimized the fermental conditions and enzyme-producing conditions, and did a preliminary research on the enzyme property. The results are as follows:
Isolation and identification of strains 6 CMCase producing strains are isolated from silage in Wusu. Among them, the ws-6 strain has highest level of CMCase activity. Through the observation of the morphology, the physiological and biochemical identification and the 16S rDNA sequencing analysis, the strain was identified as Bacillus licheniformis. The optimum pH for the bacteria is 5.0 ~ 7.0, the optimal growth temperature is 35℃, its CMCase activity is up to 2.55 U/mL.
Enzyme Producing Conditions and Enzyme Properties of the Strain Through the screening and orthogonal design experiments of the carbon, nitrogen and trace elements, the most suitable components of medium for strain ws-6 to produce CMCase are found to be as follows (g / L): glucose 15 g, CMC-Na 5 g, yeast extract 15 g , KH_2PO_4 0.5g, NaCl 5g. The optimal initial pH for enzyme-producing fermentation is 6.5, the optimal temperature is 35℃when cultivating in 250 mL conical bottle with 30 mL medium.
The preliminary study on the enzyme properties of strain ws-6 indicate that the optimal pH for the enzyme is 5.0, the optimal temperature is 50℃, the optimal concentration of substrate is 0.6%, the optimal time for reaction is 20 min.
Optimization of fermental condition Through the screening and orthogonal design experiments of the carbon, nitrogen and trace elements, the most suitable components of medium for strain’s fermentation are found to be as follows (g / L): sucrose 60 g, peptone 30 g, NaCl 30 g, K_2HPO_4 2 g, MgSO4 1 g, FeSO4 0.02 g, MnSO4 0.01 g. The research on fermental condition indicate that the optimal initial pH of fermentation is 5.5, the optimal temperature is 35℃with the optimal volume of liquid medium is 10 mL in 250 mL conical bottle. The strain ws-6’s living barteria has reached 6.58×10~(10) CFU·mL(-1) after the optimization of the components of medium and cultivating condition.
菌株分离筛选及鉴定从乌苏窖藏饲料中分离筛选出6个产CMCase的菌株,以代号ws-6的菌株产酶水平最高,通过形态学、生理生化指标及16S rDNA序列比对,鉴定为地衣芽孢杆菌(Bacillus licheniformis)。菌株最适生长pH值为5.0～7.0,最适生长温度35℃,产CMCase活力达2.55U/mL。
菌株产酶条件及酶学性质经碳、氮源及微量元素的筛选实验和正交实验分析,最适合菌株ws-6产CMCase的发酵培养基组成为(g/L):葡萄糖15g、CMC-Na 5g、酵母膏15g、KH_2PO_40.5g、NaCl 5g。发酵产酶的最适初始pH值为6.5,最适培养温度为35℃,最适装液量30mL(250mL三角瓶)。对菌株ws-6的酶学性质进行初步研究表明:酶反应的最适pH值为5.0,最适温度为50℃,最适底物浓度为0.6%,最适反应时间为20min。
菌株发酵条件优化经碳、氮源及微量元素的筛选实验和正交实验分析,确定ws-6菌株发酵培养基的最佳组分为(g/L):蔗糖60g、蛋白胨30g、NaCl 30g、K2HPO4 2 g、MgSO_4 1 g、FeSO_4 0.02 g、MnSO4 0.01 g。对发酵条件进行了研究,发酵的最适初始pH值为5.5,最适培养温度为35℃,最适装液量为10mL(250mL三角瓶),通过优化培养基组分和培养条件后,菌株ws-6活菌数达到了6.58×10~(10) CFU·mL~(-1)。
This study has isolated a cellulose-producing Bacillus sp. ws-6, and optimized the fermental conditions and enzyme-producing conditions, and did a preliminary research on the enzyme property. The results are as follows:
Isolation and identification of strains 6 CMCase producing strains are isolated from silage in Wusu. Among them, the ws-6 strain has highest level of CMCase activity. Through the observation of the morphology, the physiological and biochemical identification and the 16S rDNA sequencing analysis, the strain was identified as Bacillus licheniformis. The optimum pH for the bacteria is 5.0 ~ 7.0, the optimal growth temperature is 35℃, its CMCase activity is up to 2.55 U/mL.
Enzyme Producing Conditions and Enzyme Properties of the Strain Through the screening and orthogonal design experiments of the carbon, nitrogen and trace elements, the most suitable components of medium for strain ws-6 to produce CMCase are found to be as follows (g / L): glucose 15 g, CMC-Na 5 g, yeast extract 15 g , KH_2PO_4 0.5g, NaCl 5g. The optimal initial pH for enzyme-producing fermentation is 6.5, the optimal temperature is 35℃when cultivating in 250 mL conical bottle with 30 mL medium.
The preliminary study on the enzyme properties of strain ws-6 indicate that the optimal pH for the enzyme is 5.0, the optimal temperature is 50℃, the optimal concentration of substrate is 0.6%, the optimal time for reaction is 20 min.
Optimization of fermental condition Through the screening and orthogonal design experiments of the carbon, nitrogen and trace elements, the most suitable components of medium for strain’s fermentation are found to be as follows (g / L): sucrose 60 g, peptone 30 g, NaCl 30 g, K_2HPO_4 2 g, MgSO4 1 g, FeSO4 0.02 g, MnSO4 0.01 g. The research on fermental condition indicate that the optimal initial pH of fermentation is 5.5, the optimal temperature is 35℃with the optimal volume of liquid medium is 10 mL in 250 mL conical bottle. The strain ws-6’s living barteria has reached 6.58×10~(10) CFU·mL(-1) after the optimization of the components of medium and cultivating condition.
引文
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