耐冷菌Pseudomonas sp.W7产低温蛋白酶水解牛骨蛋白研究
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摘要
我国骨资源丰富，但是在骨资源利用上存在极大的浪费，酶解骨蛋白生产高质量的蛋白胨，是一条提高畜骨资源利用的有效途径。蛋白胨是蛋白质经水解之后形成的由眎、胨、多肽和氨基酸等组成的混合物，主要用于各种微生物的培养提供N源。
本研究采用低温蛋白酶水解牛骨蛋白，利用单因素实验研究在不同温度，pH，酶的添加量，反应时间对牛骨蛋白水解的影响，并在此基础上，筛选出3个对酶解效果影响显著的因素：温度、时间、pH；进一步利用响应面法对筛选出的因素进行3因素3水平优化，获得低温蛋白酶水解牛骨蛋白的最优条件为：温度38℃，时间77min，pH7.1，酶的添加量150U/mL。优化后，测得牛骨蛋白水解产物的氨态氮含量达到4.55%。
为了进一步考察低温蛋白酶水解牛骨蛋白过程中各因素之间的相互作用，本文设计了水解动力学实验，根据实验数据及公式推导得到低温蛋白酶水解牛骨蛋白的动力学方程计算得到米氏常数Km为0.7g/L，Km较小，表明酶与底物的亲和度比较大；最大反应速率Vmax为0.541mol/L.min，Vmax较大，表明酶解反应能够很快到达反应终点；测得反应活化能Ea为4.012KJ/mol，活化能较低，说明低温蛋白酶对温度比较敏感，高温容易使酶失去活性。酶解动力学实验结果为日后发酵生产提供参数依据，采用低温蛋白酶，水解温度一般低于中温酶，同时所用的水解时间很短。
目前蛋白胨产品主要以氨氮、总氮含量作为质量检测的指标，而蛋白胨中多肽片段大小及分布显著影响微生物对蛋白胨的利用。本文对牛骨蛋白水解产物进行SDS-PAGE凝胶电泳检测，结果表明水解液中的肽段呈连续性分布，在200KDa-6.5KDa之间均有分布，随着时间的延长，大分子的肽段逐渐被水解成小分子的肽段。为了进一步确定水解产物的肽段分布，本实验选取Sephadex G-100、Sephadex G-50，Sephadex G-25三种分子量范围不同的凝胶层析，测得牛骨蛋白水解物中多肽分布为：45KDa-150KDa的多肽占牛骨蛋白胨总量的14.83%，11.7KDa-45KDa的多肽30.37%，1KDa-11.7KDa的多肽34.16%，小于1KDa的多肽10.74%。氨基酸含量检测结果表明：牛骨蛋白水解后游离氨基酸总量高于水解前游离氨基酸的60.21%，其中必需氨基酸占总量的26.72%，半必需氨基酸占总量的5.02%，鲜味氨基酸占46.3%，苦味氨基酸占34.57%。
与商品蛋白胨（国产）和胰蛋白胨（进口）对比结果显示，牛骨蛋白胨的氨态氮含量高，但是葡聚糖层析结果显示牛骨蛋白水解液中小分子多肽含量低于商品蛋白胨和胰蛋白胨，氨基酸分析结果表明牛骨蛋白水解后游离氨基酸含量均明显高于商品蛋白胨和胰蛋白胨。这一点也解释了为什么低温蛋白酶水解牛骨蛋白产物检测氨态氮含量高，而小分子多肽片段分布少。
Bone resources are very rich in China, but there is a great waste in the utilization ofbone resources. Enzymatic hydrolysis of bone protein is an effective way to improve theutilization of animal bone resources, which produces high quality proteinpeptone.Peptone is a mixture of nitrogen source composed by the proteose, peptone,polypeptide, amino acid that formed after enzyme hydrolysis. It can provide the Nsource for microbial growth.
In this study, low temperature protease was used to hydrolyze bovine protein.Thesingle factor experiments were considered according to pH, temperature, enzymeaddition and reaction time to optimize the condition for bovine-bone peptone hydrolysis.Based on the results of signle factor optimization,3important factors were selectedwhich affect ammonia nitrogen significantly, pH，temperature and reaction time.Theoptimum hydrolysis conditon was obtained by the response surface methodology (RSM)with temperature38℃, pH7.1, reaction time77min,enzyme addition150U/mL. Theammonia nitrogen content reached to4.55%,.
To further investigate the interaction of factors in the process of the the bovine bonehydrolysis by low-temperature protease, The hydrolytic kinetic experiment wasdesigned, the dynamics equation of protease hydrolysis based on experimental dataand formula:dh=aexp(-bh)=38.8194e01.17h The Michaelis constantKm was0.7g/L, indicating that the affinity of the enzyme and substrate was relativelylarge. The maximum reaction rate Vmax was0.541mol/L.min, which meant the speedof hydrolysis reaction was fast. Activation energy Ea4.012KJ/mol, lower activationenergy showed that low-temperature protease was more sensitive to temperature. Hightemperature makes the enzyme lost activity easily.The results of enzymatic kineticsexperiment can provide parameters for the fermentation in the future, the hydrolysis temperature of low-temperature protease is generally lower than the temperature ofmedium-temperature enzyme, while the hydrolysis time is very short.
The quality of peptone products normally is test as the content of ammonia nitrogenand total nitrogen according to factory standard. The size and distribution of the peptidefragment as nitrogen source have a great influence on microorganism growth andfermentation. SDS-PAGE gel electrophoresis was used to primarily determine thedistribution of peptide fragments,the results showed that the hydrolysis of peptide hadcontinuous distribution from the200KD to6.5KDa.With the extension of hydrolysistime, peptides of large molecular were hydrolyzed into low molecule peptides or freeamino acids. To further determine the distribution of peptide, we selected three types ofgel chromatography with different MW ranges, Sephadex G-100, Sephadex G-50, andSephadex G-25. The results indicated that peptide of45KDa to150KDa accounted for14.83%of the total amount of cattle bone peptone,11.7KDa to45KDa accounted for30.37%,1KDa to11.7KDa accounted for34.16%and smaller than1KDa accounted for10.74%of the total protein. The content of amino acid is an important standard todetermine the peptone quality. According to amino acid autoanalyzer,the total contentof free amino acids in bovine-bone peptone after hydrolysis is60.21%higher than thatbefore hydrolysis,among which the essential amino acids accounted for26.72%, halfessential amino acids accounted for5.02%. Flavor amino acid accounted for46.3%,bitter taste amino acids accounted for34.57%.
Compared with commodity peptone(domestic) and tryptone（import）, bovine-bonepeptone we obtained showed higher content of ammonia nitrogen, but Gelchromatography showed that commodity peptone and tryptone peptone contented morepeptide of smaller molecular. By amino acid analysis, this conflict was proved to bedue to a large number of bone protein broken down into free amino acids in thehydrolysis. The total amino acids and free amino acids in bovine bone protein weresignificantly higher than those in commodity peptone and tryptone.
本研究采用低温蛋白酶水解牛骨蛋白，利用单因素实验研究在不同温度，pH，酶的添加量，反应时间对牛骨蛋白水解的影响，并在此基础上，筛选出3个对酶解效果影响显著的因素：温度、时间、pH；进一步利用响应面法对筛选出的因素进行3因素3水平优化，获得低温蛋白酶水解牛骨蛋白的最优条件为：温度38℃，时间77min，pH7.1，酶的添加量150U/mL。优化后，测得牛骨蛋白水解产物的氨态氮含量达到4.55%。
为了进一步考察低温蛋白酶水解牛骨蛋白过程中各因素之间的相互作用，本文设计了水解动力学实验，根据实验数据及公式推导得到低温蛋白酶水解牛骨蛋白的动力学方程计算得到米氏常数Km为0.7g/L，Km较小，表明酶与底物的亲和度比较大；最大反应速率Vmax为0.541mol/L.min，Vmax较大，表明酶解反应能够很快到达反应终点；测得反应活化能Ea为4.012KJ/mol，活化能较低，说明低温蛋白酶对温度比较敏感，高温容易使酶失去活性。酶解动力学实验结果为日后发酵生产提供参数依据，采用低温蛋白酶，水解温度一般低于中温酶，同时所用的水解时间很短。
目前蛋白胨产品主要以氨氮、总氮含量作为质量检测的指标，而蛋白胨中多肽片段大小及分布显著影响微生物对蛋白胨的利用。本文对牛骨蛋白水解产物进行SDS-PAGE凝胶电泳检测，结果表明水解液中的肽段呈连续性分布，在200KDa-6.5KDa之间均有分布，随着时间的延长，大分子的肽段逐渐被水解成小分子的肽段。为了进一步确定水解产物的肽段分布，本实验选取Sephadex G-100、Sephadex G-50，Sephadex G-25三种分子量范围不同的凝胶层析，测得牛骨蛋白水解物中多肽分布为：45KDa-150KDa的多肽占牛骨蛋白胨总量的14.83%，11.7KDa-45KDa的多肽30.37%，1KDa-11.7KDa的多肽34.16%，小于1KDa的多肽10.74%。氨基酸含量检测结果表明：牛骨蛋白水解后游离氨基酸总量高于水解前游离氨基酸的60.21%，其中必需氨基酸占总量的26.72%，半必需氨基酸占总量的5.02%，鲜味氨基酸占46.3%，苦味氨基酸占34.57%。
与商品蛋白胨（国产）和胰蛋白胨（进口）对比结果显示，牛骨蛋白胨的氨态氮含量高，但是葡聚糖层析结果显示牛骨蛋白水解液中小分子多肽含量低于商品蛋白胨和胰蛋白胨，氨基酸分析结果表明牛骨蛋白水解后游离氨基酸含量均明显高于商品蛋白胨和胰蛋白胨。这一点也解释了为什么低温蛋白酶水解牛骨蛋白产物检测氨态氮含量高，而小分子多肽片段分布少。
Bone resources are very rich in China, but there is a great waste in the utilization ofbone resources. Enzymatic hydrolysis of bone protein is an effective way to improve theutilization of animal bone resources, which produces high quality proteinpeptone.Peptone is a mixture of nitrogen source composed by the proteose, peptone,polypeptide, amino acid that formed after enzyme hydrolysis. It can provide the Nsource for microbial growth.
In this study, low temperature protease was used to hydrolyze bovine protein.Thesingle factor experiments were considered according to pH, temperature, enzymeaddition and reaction time to optimize the condition for bovine-bone peptone hydrolysis.Based on the results of signle factor optimization,3important factors were selectedwhich affect ammonia nitrogen significantly, pH，temperature and reaction time.Theoptimum hydrolysis conditon was obtained by the response surface methodology (RSM)with temperature38℃, pH7.1, reaction time77min,enzyme addition150U/mL. Theammonia nitrogen content reached to4.55%,.
To further investigate the interaction of factors in the process of the the bovine bonehydrolysis by low-temperature protease, The hydrolytic kinetic experiment wasdesigned, the dynamics equation of protease hydrolysis based on experimental dataand formula:dh=aexp(-bh)=38.8194e01.17h The Michaelis constantKm was0.7g/L, indicating that the affinity of the enzyme and substrate was relativelylarge. The maximum reaction rate Vmax was0.541mol/L.min, which meant the speedof hydrolysis reaction was fast. Activation energy Ea4.012KJ/mol, lower activationenergy showed that low-temperature protease was more sensitive to temperature. Hightemperature makes the enzyme lost activity easily.The results of enzymatic kineticsexperiment can provide parameters for the fermentation in the future, the hydrolysis temperature of low-temperature protease is generally lower than the temperature ofmedium-temperature enzyme, while the hydrolysis time is very short.
The quality of peptone products normally is test as the content of ammonia nitrogenand total nitrogen according to factory standard. The size and distribution of the peptidefragment as nitrogen source have a great influence on microorganism growth andfermentation. SDS-PAGE gel electrophoresis was used to primarily determine thedistribution of peptide fragments,the results showed that the hydrolysis of peptide hadcontinuous distribution from the200KD to6.5KDa.With the extension of hydrolysistime, peptides of large molecular were hydrolyzed into low molecule peptides or freeamino acids. To further determine the distribution of peptide, we selected three types ofgel chromatography with different MW ranges, Sephadex G-100, Sephadex G-50, andSephadex G-25. The results indicated that peptide of45KDa to150KDa accounted for14.83%of the total amount of cattle bone peptone,11.7KDa to45KDa accounted for30.37%,1KDa to11.7KDa accounted for34.16%and smaller than1KDa accounted for10.74%of the total protein. The content of amino acid is an important standard todetermine the peptone quality. According to amino acid autoanalyzer,the total contentof free amino acids in bovine-bone peptone after hydrolysis is60.21%higher than thatbefore hydrolysis,among which the essential amino acids accounted for26.72%, halfessential amino acids accounted for5.02%. Flavor amino acid accounted for46.3%,bitter taste amino acids accounted for34.57%.
Compared with commodity peptone(domestic) and tryptone（import）, bovine-bonepeptone we obtained showed higher content of ammonia nitrogen, but Gelchromatography showed that commodity peptone and tryptone peptone contented morepeptide of smaller molecular. By amino acid analysis, this conflict was proved to bedue to a large number of bone protein broken down into free amino acids in thehydrolysis. The total amino acids and free amino acids in bovine bone protein weresignificantly higher than those in commodity peptone and tryptone.
引文
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