蹄甲角蛋白酶高产菌株的诱变选育及发酵条件的优化
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摘要
动物蹄甲蕴含了大量潜在的有用蛋白质和氨基酸,我国蹄甲角蛋白资源丰富,若合理利用则可作为优质饲料蛋白和药物。但是因为其结构稳定,极难降解,找到合适的方法是研究降解角蛋白的关键。传统物理、化学降解法存在众多缺陷,诸如效率低、耗能高、污染大,而采用生物降解法效果显著,既利用了蛋白资源,也利于环境保护,推动了角蛋白及角蛋白酶的在各领域中的广泛应用,以获得较高的经济价值和社会价值。
本课题主要针对动物蹄甲蛋白的资源化利用,从菌株的诱变选育入手,对产角蛋白酶的培养基成分和培养条件进行优化,并且研究角蛋白酶的性质,取得的主要研究结果如下:
1.对一株产角蛋白酶菌株N5进行形态特征观察、生理生化测定、16SrDNA序列分析,初步鉴定该菌为黄杆菌属(Chryseobacterium sp.)菌株。
2.对N5菌株进行连续紫外诱变,筛选得到高产突变株U3-22,利用考马斯亮蓝法测定U3-22菌株的角蛋白酶发酵活力达69.85U/mL,比出发菌株的25.43U/mL提高了2.75倍。
3.采用单因素试验和四因素三水平的正交试验L9(3~4),对黄杆菌U3-22菌株产角蛋白酶的培养基组成进行了优化,确定了U3-22菌株的最佳培养基组成为:可溶性淀粉1.5%、辅助氮源酵母浸出物1.5%、磷酸氢二钾0.05%、蹄甲粉0.4%;最佳培养条件为:温度37℃、接种量3%、装液量50mL、最适pH值8.5,在此培养条件下,诱变菌株U3-22的发酵产酶活力提高到160.34U/mL,是原来69.85U/mL的2.3倍。
4.从U3-22菌株的发酵液提取粗角蛋白酶,分析其酶学性质。该角蛋白酶最适反应温度是70℃,最适作用pH是7.5;K~+、Mg~(2+)、Na_2SO_3对该角蛋白酶有较明显的促进作用,而Mn~(2+)、Zn~(2+)的抑制作用较为明显;突变株U3-22产生的角蛋白酶对蹄甲粉具有很强的分解能力,且具有较好的热稳定性和pH稳定性。结果表明,U3-22产生的角蛋白酶是一种新型的耐高温、耐酸碱角蛋白酶,在动物蹄甲、羽毛等废弃资源的利用中有巨大的应用前景。
There are plenty of resources on hoof keratin in our country. Livestock and poultry hoof accumulate an abundance of potential protein and amino acids, which could be used for the source of excellent feedstuff protein. It is the key to find and appropriate way to degrade keratin, as the keratin is very difficult to be decomposed due to the steady configuration. Different from conventional methods such as physical or chemical degradation, biodegradation holds outstanding effect and no defects like low efficiency, high expend and excessive contamination, which not only makes use of the protein source but also helps to protect environment. As the applications of keratin and keratinase to all kinds of industry emerged more, those are considered to be provided with more values of economic and social, and with brilliant prospect.
This study is mainly focused on the utilization of keratin, starting from mutagenesis-screening optimal strain; the characterization of crude enzyme and the fermentation process have been researched. The results completed are reported as follows:
1. The strain N5 was identified as Chryseobacterium sp. based on morphology observation,physicochemical characteristics, 16SrDNA sequence analysis .
2. The high-yield hoof keratinase strain U3-22 was obtained by UV mutagenesis on the original strain N5. The hoof keratinase production reached 69.85U/mL using method of coomassie brilliant blue colorimetric, which was 2.75 times as higher as that of the original strain with 25.43U/mL.
3. Effects of medium components on keratinase production were carried out by orthogonal experimental design of the four factors, the optimal fermentation medium was determined as follows: starch soluble 1.5%, Yeast extract powder 1.5%, K2HPO4, 0.05%, hoof 0.4%; The best fermentation conditions were: the optimum cultural temperature 37℃, inoculating amount 3%, medium volume 50mL in 300mL flask, optimal pH 8.5. Under these conditions, the keratinase activity was 160.34U/mL, which was 2.3 times of the previous.
4. The crude keratinase had been extracted from the fermentation liquid of U3-22 strain, and the enzymatic characteristics were analyzed. The optimum reaction temperature and pH were 70℃and 7.5 respectively. The enzyme activity could be clearly activated by K~+, Mg~(2+)and Na_2SO_3, and inhibited by Mn~(2+) and Zn~(2+). The keratinase could degrade the hoof effectively. These results suggest that the keratinase from the mutant strain U3-22 is novel, thermal stable and pH-tolerant and it can be applied in waste disposal of hoof and feather.
本课题主要针对动物蹄甲蛋白的资源化利用,从菌株的诱变选育入手,对产角蛋白酶的培养基成分和培养条件进行优化,并且研究角蛋白酶的性质,取得的主要研究结果如下:
1.对一株产角蛋白酶菌株N5进行形态特征观察、生理生化测定、16SrDNA序列分析,初步鉴定该菌为黄杆菌属(Chryseobacterium sp.)菌株。
2.对N5菌株进行连续紫外诱变,筛选得到高产突变株U3-22,利用考马斯亮蓝法测定U3-22菌株的角蛋白酶发酵活力达69.85U/mL,比出发菌株的25.43U/mL提高了2.75倍。
3.采用单因素试验和四因素三水平的正交试验L9(3~4),对黄杆菌U3-22菌株产角蛋白酶的培养基组成进行了优化,确定了U3-22菌株的最佳培养基组成为:可溶性淀粉1.5%、辅助氮源酵母浸出物1.5%、磷酸氢二钾0.05%、蹄甲粉0.4%;最佳培养条件为:温度37℃、接种量3%、装液量50mL、最适pH值8.5,在此培养条件下,诱变菌株U3-22的发酵产酶活力提高到160.34U/mL,是原来69.85U/mL的2.3倍。
4.从U3-22菌株的发酵液提取粗角蛋白酶,分析其酶学性质。该角蛋白酶最适反应温度是70℃,最适作用pH是7.5;K~+、Mg~(2+)、Na_2SO_3对该角蛋白酶有较明显的促进作用,而Mn~(2+)、Zn~(2+)的抑制作用较为明显;突变株U3-22产生的角蛋白酶对蹄甲粉具有很强的分解能力,且具有较好的热稳定性和pH稳定性。结果表明,U3-22产生的角蛋白酶是一种新型的耐高温、耐酸碱角蛋白酶,在动物蹄甲、羽毛等废弃资源的利用中有巨大的应用前景。
There are plenty of resources on hoof keratin in our country. Livestock and poultry hoof accumulate an abundance of potential protein and amino acids, which could be used for the source of excellent feedstuff protein. It is the key to find and appropriate way to degrade keratin, as the keratin is very difficult to be decomposed due to the steady configuration. Different from conventional methods such as physical or chemical degradation, biodegradation holds outstanding effect and no defects like low efficiency, high expend and excessive contamination, which not only makes use of the protein source but also helps to protect environment. As the applications of keratin and keratinase to all kinds of industry emerged more, those are considered to be provided with more values of economic and social, and with brilliant prospect.
This study is mainly focused on the utilization of keratin, starting from mutagenesis-screening optimal strain; the characterization of crude enzyme and the fermentation process have been researched. The results completed are reported as follows:
1. The strain N5 was identified as Chryseobacterium sp. based on morphology observation,physicochemical characteristics, 16SrDNA sequence analysis .
2. The high-yield hoof keratinase strain U3-22 was obtained by UV mutagenesis on the original strain N5. The hoof keratinase production reached 69.85U/mL using method of coomassie brilliant blue colorimetric, which was 2.75 times as higher as that of the original strain with 25.43U/mL.
3. Effects of medium components on keratinase production were carried out by orthogonal experimental design of the four factors, the optimal fermentation medium was determined as follows: starch soluble 1.5%, Yeast extract powder 1.5%, K2HPO4, 0.05%, hoof 0.4%; The best fermentation conditions were: the optimum cultural temperature 37℃, inoculating amount 3%, medium volume 50mL in 300mL flask, optimal pH 8.5. Under these conditions, the keratinase activity was 160.34U/mL, which was 2.3 times of the previous.
4. The crude keratinase had been extracted from the fermentation liquid of U3-22 strain, and the enzymatic characteristics were analyzed. The optimum reaction temperature and pH were 70℃and 7.5 respectively. The enzyme activity could be clearly activated by K~+, Mg~(2+)and Na_2SO_3, and inhibited by Mn~(2+) and Zn~(2+). The keratinase could degrade the hoof effectively. These results suggest that the keratinase from the mutant strain U3-22 is novel, thermal stable and pH-tolerant and it can be applied in waste disposal of hoof and feather.
引文
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