Serratia sp.SYBC H发酵产蛋白酶，酶学性质，应用及其基因克隆研究
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摘要
蛋白酶是一类水解肽键的酶,其广泛应用于食品、饲料、医药、化工等行业,因此研究其发酵产酶、酶学特性及应用具有一定的理论价值和潜在的商业价值。本论文以从腐烂蓝藻中分离的一株沙雷氏菌为蛋白酶生产菌株,研究了以下内容:采用三种统计方法进行Serratia sp. SYBC H发酵浮萍产蛋白酶的摇瓶优化。先采用单因素实验,对影响Serratia sp. SYBC H发酵浮萍生产蛋白酶的因素进行筛选;接着用正交试验筛选出影响产酶的主要因素;最后挑选出三个主要因素进行响应面水平优化以及考察这三个主要因素之间是否存在交互性。实验结果表明:主要因素小麦粉20 g/L、浮萍43.9 g/L、NaCl 0.08 mol/L对蛋白酶生产的贡献最大,同时发现主要因素浮萍和小麦粉之间存在交互作用。
通过5 L发酵罐放大发酵,对操作条件如pH控制、通风量和搅拌转速进行了研究。结果发现,发酵过程中控制pH,蛋白酶的生产高于发酵过程中不控制pH的对照,通风量和搅拌转速为6.0 vvm和800 r/min时,发酵液的蛋白酶活最高(1450.4 U/mL)。培养基中添加吐温80的主要作用是改善细胞膜的渗透性,促进蛋白酶向胞外排放。
采用三种统计方法对Serratia sp. SYBC H摇瓶发酵蓝藻生产蛋白酶进行优化。先采用单因素实验,对影响产酶的因素进行筛选,接着用正交试验筛选出主要因素;最后对主要因素进行响应面水平优化以及考察它们之间是否存在交互性。结果发现当主要因素蓝藻50 g/L、尿素0.05 mol/L和硫酸锌1.5 mmol/L时,蛋白酶活最高,达到704.7U/mL.另外,发现主要因素(蓝藻、尿素和硫酸锌)之间不存在交互作用。
发酵上清液中的粗酶通过硫酸铵分级盐析、阴离子交换和凝胶过滤三步分离纯化,得到电泳纯蛋白酶。经过SDS-PAGE电泳检验,发现该纯蛋白酶的分子量经估算大约为59 kDa。该酶的最佳反应pH为8.0左右,在pH 5-11范围内,蛋白酶活相对较高。该蛋白酶的最适反应温度为40°C左右,在30-80°C之间,酶活相对较高。该酶受Mn~(2+)和Na~+显著激活,而其它金属离子抑制酶活,尤其是5 mmol/L Co~(2+)可以完全抑制该丝氨酸蛋白酶。在60°C下锰离子可提高该酶的热稳定性。与常见的丝氨酸蛋白酶不同的是,该Serratia sp. SYBC H丝氨酸蛋白酶不受Ca~(2+)激活,且酶的热稳定性下降。此酶不仅在亲水性有机溶剂(50%, v/v)如丙酮、甲醇、二甲基亚砜和二甲基甲酰胺中稳定而且在疏水性有机溶剂如正己烷庚烷,辛烷中稳定。另外,该丝氨酸蛋白酶耐受非离子表面活性剂(20%,v/v)吐温80、(25%, v/v) Triton X-100和甘油,但对阴离子表面活性剂SDS (1%)却较敏感。此酶受PMSF显著抑制,表明该蛋白酶是丝氨酸蛋白酶。
Serratia sp. SYBC H蛋白酶有显著的去血污能力;对富含β型蛋白结构的羽毛粉也有很强的降解作用,水解上清液中的可溶性多肽均3-5倍地增长。然而,该酶对于富含α型蛋白结构的头发,羊毛即使在其最适反应温度40°C下作用24 h都不催化降解。
该酶可在多种有机溶剂如吡啶、二甲基亚砜、二甲基甲酰胺、甲醇和丙酮中催化蔗糖酯合成,该酶既可催化酰基供体乙酸酐也可催化酰基供体乙酸乙烯酯。该酶既可非定向催化,合成多种取代酯;也可定向催化,合成单一产物,如在溶剂吡啶与正己烷(1:1, v/v)中,该酶催化乙酸酐与蔗糖合成蔗糖八乙酸酯,在30°C下反应24 h左右,蔗糖八乙酸酯的产率可达90%。
该丝氨酸蛋白酶核酸序列长1,288 bp,编码429个氨基酸残基。序列中疏水性氨基酸残基比例(43.62%)大于亲水性氨基酸残基比例(28.64%)。尤其是位于成熟肽C端的活性中心Ser残基附近存在较多的疏水性氨基酸残基,推测这可能与蛋白酶的有机溶剂稳定性有关,从分子水平上论证了Serratia sp. SYBC H丝氨酸蛋白酶一级结构与有机溶剂稳定性功能之间的关系。
Proteases are a kind of enzymes which catalyze the hydrolysis of peptide bonds. They are widely used in food, feed, medicine, chemical industries and so on. They have theoretical and potential commercial values to study on proteases production, enzymatic properties and applications. The strain Serratia sp. SYBC H isolated from decayed Cyanobacteria in Taihu Lake (Jiangsu, China) was used as the protease producer. The contents in this thesis are as follows:
Three statistical methods were used for shaken flask optimization for the protease production by Serratia sp. SYBC H with duckweed as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Then, orthogonal design was applied to find the significant variables. Finally, response surface methodology (RSM) including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the three significant variables. The optimal level of the significant variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, and sodium chloride 0.08 mol/L, respectively. The interaction between duckweed and wheat flour was significant.
The operating conditions such as pH, aeration, stirring speed were studied in 5-liter fermentor, respectively. It is found that the yield of the protease was higher when pH value was controlled in fermentation process than that of control experiments with no pH control. The maximum protease activity (1450.4 U/mL) was observed from the ventilation rate at 6.0 vvm, and stirring speed at 800 r/min. The effect of Tween 80 supplemented in fermentation medium is to improve the permeability of Serratia sp. SYBC H cell membranes, and to promote the protease secretion into supernatant.
Three statistical methods were used for shaken flask optimization for proteases production by Serratia sp. SYBC H with cyanobacteria as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Orthogonal design was then applied to find the significant variables. Finally, response surface methodology (RSM) including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the significant variables. The optimal level of the significant variables for the maximum protease production was cyanobacteria 50.0 g/L, ZnSO4.7H2O 1.5 mmol/L and Urea 0.05 mol/L. The maximum protease activity reached 704.7 U/mL in the optimized medium. There was no interaction between the significant factors.
The crude protease from fermentation supernatant was purified through three steps involving (NH4)2SO4 salt precipitation, DEAE-cellulose anion-exchange chromatography, and gel filtration. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH ranges between 5.0 and 11.0, with the maximum activity at about pH 8.0. It is also fairly active over the temperature ranges between 30°C and 80°C, with the maximum activity at about 40°C. The protease activity was substantially stimulated by Mn~(2+) and Na+ (5mmol/L), respectively. Other metal ions inhibited the protease activity, especially, 5 mmol/L Co~(2+) can completely inhibit the protease activity. In addition, Mn~(2+) enhanced the thermostability of the protease significantly at 60°C. However, different from other serine proteases, the activity and stability of serine protease from Serratia sp. SYBC H were inhibited by Ca~(2+). The protease not only remained relatively high activity in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH, but also showed high stability in hydrophobic solvents such as hexane, heptane and octane. The protease retained relatively high initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. But, there was obvious reduction in protease activity in the presence of the anionic surfactant SDS at 1% (w/v) concentration. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease.
It is found that the protease from Serratia sp. SYBC H has significant capacity to remove blood. The protease efficiently catalyzed the degradation of the feather meal rich inβ-structure of proteins. In the hydrolytic supernatant, soluble polypeptides were 3-5 times that of control with the inactivated protease. However, the protease can not catalyzeα-rich proteins as hair and wool, even at its optimal temperature of 40°C for 24 h..
The protease can catalyze sucrose esters syntheses in a variety of organic solvents such as pyridine, DMSO, DMF, methanol, acetone. As far as acyl donors were concerned, acetic anhydride and vinyl acetate can be catalyzed by the protease from Serratia sp. SYBC H. The protease can catalyze the formation of sucrose esters with varying degrees of substitution (DS), with the performance of non-directed catalysis. It is also catalyze the synthesis of a single product, exhibiting directed catalysis. For example, in pyridine and hexane (1:1, v/v), the protease catalyzed the acylation of sucrose with acetic anhydride to form sucrose octaacetate. The yield of sucrose octaacetatecan can reach 90% under 30°C, 200 r/min and 24 h.
The serine protease from Serratia sp. SYBC H comprised of 1,288 bp encoding 429 amino acid residues. The proportion of the hydrophobic amino acids residues (43.62%) was higher than that of hydrophilic amino acids residues (28.64%) in the mature peptides of the serine protease from Serratia sp. SYBC H. Especially, the presence of high content of hydrophobic amino acid residues surrounding the catalytic residue Ser in C-terminal mature peptide may be related to the solvents stability of protease, which demonstrated the relationship between structure and function of the solvent-stable serine protease from Serratia sp. SYBC H.
通过5 L发酵罐放大发酵,对操作条件如pH控制、通风量和搅拌转速进行了研究。结果发现,发酵过程中控制pH,蛋白酶的生产高于发酵过程中不控制pH的对照,通风量和搅拌转速为6.0 vvm和800 r/min时,发酵液的蛋白酶活最高(1450.4 U/mL)。培养基中添加吐温80的主要作用是改善细胞膜的渗透性,促进蛋白酶向胞外排放。
采用三种统计方法对Serratia sp. SYBC H摇瓶发酵蓝藻生产蛋白酶进行优化。先采用单因素实验,对影响产酶的因素进行筛选,接着用正交试验筛选出主要因素;最后对主要因素进行响应面水平优化以及考察它们之间是否存在交互性。结果发现当主要因素蓝藻50 g/L、尿素0.05 mol/L和硫酸锌1.5 mmol/L时,蛋白酶活最高,达到704.7U/mL.另外,发现主要因素(蓝藻、尿素和硫酸锌)之间不存在交互作用。
发酵上清液中的粗酶通过硫酸铵分级盐析、阴离子交换和凝胶过滤三步分离纯化,得到电泳纯蛋白酶。经过SDS-PAGE电泳检验,发现该纯蛋白酶的分子量经估算大约为59 kDa。该酶的最佳反应pH为8.0左右,在pH 5-11范围内,蛋白酶活相对较高。该蛋白酶的最适反应温度为40°C左右,在30-80°C之间,酶活相对较高。该酶受Mn~(2+)和Na~+显著激活,而其它金属离子抑制酶活,尤其是5 mmol/L Co~(2+)可以完全抑制该丝氨酸蛋白酶。在60°C下锰离子可提高该酶的热稳定性。与常见的丝氨酸蛋白酶不同的是,该Serratia sp. SYBC H丝氨酸蛋白酶不受Ca~(2+)激活,且酶的热稳定性下降。此酶不仅在亲水性有机溶剂(50%, v/v)如丙酮、甲醇、二甲基亚砜和二甲基甲酰胺中稳定而且在疏水性有机溶剂如正己烷庚烷,辛烷中稳定。另外,该丝氨酸蛋白酶耐受非离子表面活性剂(20%,v/v)吐温80、(25%, v/v) Triton X-100和甘油,但对阴离子表面活性剂SDS (1%)却较敏感。此酶受PMSF显著抑制,表明该蛋白酶是丝氨酸蛋白酶。
Serratia sp. SYBC H蛋白酶有显著的去血污能力;对富含β型蛋白结构的羽毛粉也有很强的降解作用,水解上清液中的可溶性多肽均3-5倍地增长。然而,该酶对于富含α型蛋白结构的头发,羊毛即使在其最适反应温度40°C下作用24 h都不催化降解。
该酶可在多种有机溶剂如吡啶、二甲基亚砜、二甲基甲酰胺、甲醇和丙酮中催化蔗糖酯合成,该酶既可催化酰基供体乙酸酐也可催化酰基供体乙酸乙烯酯。该酶既可非定向催化,合成多种取代酯;也可定向催化,合成单一产物,如在溶剂吡啶与正己烷(1:1, v/v)中,该酶催化乙酸酐与蔗糖合成蔗糖八乙酸酯,在30°C下反应24 h左右,蔗糖八乙酸酯的产率可达90%。
该丝氨酸蛋白酶核酸序列长1,288 bp,编码429个氨基酸残基。序列中疏水性氨基酸残基比例(43.62%)大于亲水性氨基酸残基比例(28.64%)。尤其是位于成熟肽C端的活性中心Ser残基附近存在较多的疏水性氨基酸残基,推测这可能与蛋白酶的有机溶剂稳定性有关,从分子水平上论证了Serratia sp. SYBC H丝氨酸蛋白酶一级结构与有机溶剂稳定性功能之间的关系。
Proteases are a kind of enzymes which catalyze the hydrolysis of peptide bonds. They are widely used in food, feed, medicine, chemical industries and so on. They have theoretical and potential commercial values to study on proteases production, enzymatic properties and applications. The strain Serratia sp. SYBC H isolated from decayed Cyanobacteria in Taihu Lake (Jiangsu, China) was used as the protease producer. The contents in this thesis are as follows:
Three statistical methods were used for shaken flask optimization for the protease production by Serratia sp. SYBC H with duckweed as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Then, orthogonal design was applied to find the significant variables. Finally, response surface methodology (RSM) including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the three significant variables. The optimal level of the significant variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, and sodium chloride 0.08 mol/L, respectively. The interaction between duckweed and wheat flour was significant.
The operating conditions such as pH, aeration, stirring speed were studied in 5-liter fermentor, respectively. It is found that the yield of the protease was higher when pH value was controlled in fermentation process than that of control experiments with no pH control. The maximum protease activity (1450.4 U/mL) was observed from the ventilation rate at 6.0 vvm, and stirring speed at 800 r/min. The effect of Tween 80 supplemented in fermentation medium is to improve the permeability of Serratia sp. SYBC H cell membranes, and to promote the protease secretion into supernatant.
Three statistical methods were used for shaken flask optimization for proteases production by Serratia sp. SYBC H with cyanobacteria as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Orthogonal design was then applied to find the significant variables. Finally, response surface methodology (RSM) including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the significant variables. The optimal level of the significant variables for the maximum protease production was cyanobacteria 50.0 g/L, ZnSO4.7H2O 1.5 mmol/L and Urea 0.05 mol/L. The maximum protease activity reached 704.7 U/mL in the optimized medium. There was no interaction between the significant factors.
The crude protease from fermentation supernatant was purified through three steps involving (NH4)2SO4 salt precipitation, DEAE-cellulose anion-exchange chromatography, and gel filtration. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH ranges between 5.0 and 11.0, with the maximum activity at about pH 8.0. It is also fairly active over the temperature ranges between 30°C and 80°C, with the maximum activity at about 40°C. The protease activity was substantially stimulated by Mn~(2+) and Na+ (5mmol/L), respectively. Other metal ions inhibited the protease activity, especially, 5 mmol/L Co~(2+) can completely inhibit the protease activity. In addition, Mn~(2+) enhanced the thermostability of the protease significantly at 60°C. However, different from other serine proteases, the activity and stability of serine protease from Serratia sp. SYBC H were inhibited by Ca~(2+). The protease not only remained relatively high activity in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH, but also showed high stability in hydrophobic solvents such as hexane, heptane and octane. The protease retained relatively high initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. But, there was obvious reduction in protease activity in the presence of the anionic surfactant SDS at 1% (w/v) concentration. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease.
It is found that the protease from Serratia sp. SYBC H has significant capacity to remove blood. The protease efficiently catalyzed the degradation of the feather meal rich inβ-structure of proteins. In the hydrolytic supernatant, soluble polypeptides were 3-5 times that of control with the inactivated protease. However, the protease can not catalyzeα-rich proteins as hair and wool, even at its optimal temperature of 40°C for 24 h..
The protease can catalyze sucrose esters syntheses in a variety of organic solvents such as pyridine, DMSO, DMF, methanol, acetone. As far as acyl donors were concerned, acetic anhydride and vinyl acetate can be catalyzed by the protease from Serratia sp. SYBC H. The protease can catalyze the formation of sucrose esters with varying degrees of substitution (DS), with the performance of non-directed catalysis. It is also catalyze the synthesis of a single product, exhibiting directed catalysis. For example, in pyridine and hexane (1:1, v/v), the protease catalyzed the acylation of sucrose with acetic anhydride to form sucrose octaacetate. The yield of sucrose octaacetatecan can reach 90% under 30°C, 200 r/min and 24 h.
The serine protease from Serratia sp. SYBC H comprised of 1,288 bp encoding 429 amino acid residues. The proportion of the hydrophobic amino acids residues (43.62%) was higher than that of hydrophilic amino acids residues (28.64%) in the mature peptides of the serine protease from Serratia sp. SYBC H. Especially, the presence of high content of hydrophobic amino acid residues surrounding the catalytic residue Ser in C-terminal mature peptide may be related to the solvents stability of protease, which demonstrated the relationship between structure and function of the solvent-stable serine protease from Serratia sp. SYBC H.
引文
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