重组巴斯德毕赤酵母发酵生产几丁质酶的研究
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摘要
几丁质广泛分布于动物、微生物体内,是自然界中含量仅次于纤维素的生物多聚物,年生物合成量达100亿吨。作为自然界中唯一大量合成的碱性多糖,其具有很多不同于其他多糖的特性,并显现出独特的应用价值,是糖生物学开发应用研究的一个热点。
几丁质酶(EC3.2.1.14)能将几丁质β-1,4糖苷键水解,最终分解为N-乙酰氨基葡萄糖(GlcNAc)。在作物抗真菌病害、人类治疗真菌病、几丁质深加工等领域前景看好。因此,几丁质酶的批量生产势在必行。本研究的内容即是采用一株Mut+表型的重组巴斯德毕赤酵母生产水稻碱性几丁质酶,对发酵过程进行优化,并对其理化性质进行分析。
试验在摇瓶水平对巴斯德毕赤酵母表达几丁质酶的诱导条件进行了研究,得到的结果为:培养基以有机培养基BMMY为宜;离心与否对外源蛋白表达影响不大;添加0.5%的(NH4)2SO4、0.05%的油酸、添加1.0%的甲醇混合0.1%的甘油、0.67%的YNB、1.5%的酵母抽提物、pH6.0、每12 h补加一次甲醇、诱导108 h,重组几丁质酶表达量最高,提高甲醇浓度稳定性有利于蛋白表达量的提高;而添加氧载体抑制外源蛋白表达;表面活性剂诱导杂蛋白分泌;降低诱导温度不利于重组几丁质酶的表达。可以此作为发酵罐培养流加成分的参考。
在7 L发酵罐进行了放大试验,发现该菌株利用甲醇能力较强,完全能够以溶氧振荡为指针指示甲醇流加,通过蠕动泵连续流加甲醇诱导,外源蛋白表达量峰值比摇瓶诱导提前了近24 h,在84 h蛋白表达量达462.41 mg/L,单位得率提高了近2.7倍;几丁质酶酶活达到77.61 U/mL。
为进一步提高几丁质酶的表达量,在诱导阶段进行了甘油-甲醇混合碳源流加策略和甘油-甲醇交替流加策略。甘油-甲醇混合流加过程中甘油的限制性流加改善了菌体生长状况,菌体密度达到一个更高的水平,酵母生理活性的提高使诱导期得以延长,带动甲醇进一步诱导蛋白表达,表达量达到了495.33 mg/L,几丁质酶酶活达到88.31 U/mL;甘油-甲醇碳源交替流加策略打破了毕赤酵母细胞内AOX1的平衡,延缓了酵母细胞内AOX1饱和的时间,外源蛋白表达速率下降的趋势得以缓解,在120 h外源蛋白最高表达量达到516.45 mg/L,几丁质酶酶活达到99.3 U/mL。
对发酵上清液进行了硫酸铵分级沉淀和Sephadex G-100柱层析,得到纯化的几丁质酶。发现该几丁质酶对热不敏感;在酸性pH范围内酶活性较高,并且表现为双峰型,在碱性pH中酶活性降低,印证巴斯德毕赤酵母正确表达了水稻碱性几丁质酶。该几丁质酶对病原真菌(Rhizopus stolonifer,Botrytis cinerea)扩展初期表现出抑制作用,但后期抑制效果不明显。
Chitin is aβ-1,4-linked,insoluble linear polymer of N-acetylglucosamine (GlcNAc)and is the second most abundant organic compound on our planet following cellulose, biosynthesize 10 billion per year. And it’s distributed in animal, microbe. As the exclusive alk polysaccharide of the nature, there are some special characteristics of chitin. It is became the focal point of the exploratory development for its particular function.
Chitinase(EC3.2.1.14) is a glycosyl hydrolase that catalyzes the hydrolytic degradation of chitin. It has good prospects in territories as follow, for higher plants is a part of defense mechanism against fungal pathogens, cure disease of human inducing by fungal, deep processing of chitin, and so on. So it’s imperative to produce chitinase in large-scale. In this study a recombinant strain of Pichia pastoris with a phenotype of Mut+ was used to produce alk rice chitinase, which was secreted into the culture. The aim of the present work was to enhance the expression level of chitinase expressed by the recombinant Pichia pastoris, and analyze the biochemistry character.
In shake-flask, the conditions of the induction phase were investigated. The optimal medium was BMMY. The concentration of (NH4)2SO4 was 0.5%, oleic acid was 0.05%,YNB was 0.67%,yeast extract was 1.5%,1.0% methanol mixed-feed with 0.1% glycerol. The optimal pH condition was 6.0. Add methanol to final concentration of 1.0% every 12 hours to maintain induction without centrifuging after the growth phase. The highest productivity of chitinase was obtained when inducing for 108 hours. And it was propitious to enhance production by enhanced the stability of methanol concentration. But the oxygen vector, surface active agent, lower the induction temperature were not propitious to production. The above data could be used to direct the fed-batch fermentation.
By 7 L fermentor, different methanol feeding modes were evaluated. For the strong ability to utilize methanol, the methanol feed rate could be directed by the dissolve oxygen vibration. Continue feed methanol through peristaltic pump, the highest production level of recombinant chitinase obtained earlier nearly 24 hours than the shake-flask, and high to 462.41 mg/L, was 2.7 folds than shake-flask. And the chitinase activity was 77.61 U/mL.
In order to further enhance the recombinant chitinase expression level, the glycerol-methanol mixed-feed mode and the feeding mode of two carbon sources alternatively were evaluated. The feed of glycerol during glycerol-methanol mixed feed phase, improved the growth environment for cell, and the biomass became more. The improved activity of cell’s physiology, prolong the induction phase, to spur the methanol induced more recombinant chitinase, the expression level was high to 495.33 mg/L and the chitinase activity was 88.31 U/mL. When feeding of two carbon sources alternatively, the blance of AOX1 was broken. And the prolong of saturation time of AOX1 activity, staved the decline rate of heterologous protein production rate, and at the 120 hours the highest level was 516.45 mg/L,99.3 U/mL.
The fast purification of fermentation broth was conducted with (NH4)2SO4 and Sephadex G-100, and got pure chitinase. The pure chitinase was thermostable, keep higher activity in acid environment, and the profile showed the double summit in wide pH limit. In alk environment the activity became lower, proved the recombinant chitnase was alk. The pathogen(Rhizopus stolonifer, Botrytis cinerea) could be inhibited by the enzyme in a certain extent.
几丁质酶(EC3.2.1.14)能将几丁质β-1,4糖苷键水解,最终分解为N-乙酰氨基葡萄糖(GlcNAc)。在作物抗真菌病害、人类治疗真菌病、几丁质深加工等领域前景看好。因此,几丁质酶的批量生产势在必行。本研究的内容即是采用一株Mut+表型的重组巴斯德毕赤酵母生产水稻碱性几丁质酶,对发酵过程进行优化,并对其理化性质进行分析。
试验在摇瓶水平对巴斯德毕赤酵母表达几丁质酶的诱导条件进行了研究,得到的结果为:培养基以有机培养基BMMY为宜;离心与否对外源蛋白表达影响不大;添加0.5%的(NH4)2SO4、0.05%的油酸、添加1.0%的甲醇混合0.1%的甘油、0.67%的YNB、1.5%的酵母抽提物、pH6.0、每12 h补加一次甲醇、诱导108 h,重组几丁质酶表达量最高,提高甲醇浓度稳定性有利于蛋白表达量的提高;而添加氧载体抑制外源蛋白表达;表面活性剂诱导杂蛋白分泌;降低诱导温度不利于重组几丁质酶的表达。可以此作为发酵罐培养流加成分的参考。
在7 L发酵罐进行了放大试验,发现该菌株利用甲醇能力较强,完全能够以溶氧振荡为指针指示甲醇流加,通过蠕动泵连续流加甲醇诱导,外源蛋白表达量峰值比摇瓶诱导提前了近24 h,在84 h蛋白表达量达462.41 mg/L,单位得率提高了近2.7倍;几丁质酶酶活达到77.61 U/mL。
为进一步提高几丁质酶的表达量,在诱导阶段进行了甘油-甲醇混合碳源流加策略和甘油-甲醇交替流加策略。甘油-甲醇混合流加过程中甘油的限制性流加改善了菌体生长状况,菌体密度达到一个更高的水平,酵母生理活性的提高使诱导期得以延长,带动甲醇进一步诱导蛋白表达,表达量达到了495.33 mg/L,几丁质酶酶活达到88.31 U/mL;甘油-甲醇碳源交替流加策略打破了毕赤酵母细胞内AOX1的平衡,延缓了酵母细胞内AOX1饱和的时间,外源蛋白表达速率下降的趋势得以缓解,在120 h外源蛋白最高表达量达到516.45 mg/L,几丁质酶酶活达到99.3 U/mL。
对发酵上清液进行了硫酸铵分级沉淀和Sephadex G-100柱层析,得到纯化的几丁质酶。发现该几丁质酶对热不敏感;在酸性pH范围内酶活性较高,并且表现为双峰型,在碱性pH中酶活性降低,印证巴斯德毕赤酵母正确表达了水稻碱性几丁质酶。该几丁质酶对病原真菌(Rhizopus stolonifer,Botrytis cinerea)扩展初期表现出抑制作用,但后期抑制效果不明显。
Chitin is aβ-1,4-linked,insoluble linear polymer of N-acetylglucosamine (GlcNAc)and is the second most abundant organic compound on our planet following cellulose, biosynthesize 10 billion per year. And it’s distributed in animal, microbe. As the exclusive alk polysaccharide of the nature, there are some special characteristics of chitin. It is became the focal point of the exploratory development for its particular function.
Chitinase(EC3.2.1.14) is a glycosyl hydrolase that catalyzes the hydrolytic degradation of chitin. It has good prospects in territories as follow, for higher plants is a part of defense mechanism against fungal pathogens, cure disease of human inducing by fungal, deep processing of chitin, and so on. So it’s imperative to produce chitinase in large-scale. In this study a recombinant strain of Pichia pastoris with a phenotype of Mut+ was used to produce alk rice chitinase, which was secreted into the culture. The aim of the present work was to enhance the expression level of chitinase expressed by the recombinant Pichia pastoris, and analyze the biochemistry character.
In shake-flask, the conditions of the induction phase were investigated. The optimal medium was BMMY. The concentration of (NH4)2SO4 was 0.5%, oleic acid was 0.05%,YNB was 0.67%,yeast extract was 1.5%,1.0% methanol mixed-feed with 0.1% glycerol. The optimal pH condition was 6.0. Add methanol to final concentration of 1.0% every 12 hours to maintain induction without centrifuging after the growth phase. The highest productivity of chitinase was obtained when inducing for 108 hours. And it was propitious to enhance production by enhanced the stability of methanol concentration. But the oxygen vector, surface active agent, lower the induction temperature were not propitious to production. The above data could be used to direct the fed-batch fermentation.
By 7 L fermentor, different methanol feeding modes were evaluated. For the strong ability to utilize methanol, the methanol feed rate could be directed by the dissolve oxygen vibration. Continue feed methanol through peristaltic pump, the highest production level of recombinant chitinase obtained earlier nearly 24 hours than the shake-flask, and high to 462.41 mg/L, was 2.7 folds than shake-flask. And the chitinase activity was 77.61 U/mL.
In order to further enhance the recombinant chitinase expression level, the glycerol-methanol mixed-feed mode and the feeding mode of two carbon sources alternatively were evaluated. The feed of glycerol during glycerol-methanol mixed feed phase, improved the growth environment for cell, and the biomass became more. The improved activity of cell’s physiology, prolong the induction phase, to spur the methanol induced more recombinant chitinase, the expression level was high to 495.33 mg/L and the chitinase activity was 88.31 U/mL. When feeding of two carbon sources alternatively, the blance of AOX1 was broken. And the prolong of saturation time of AOX1 activity, staved the decline rate of heterologous protein production rate, and at the 120 hours the highest level was 516.45 mg/L,99.3 U/mL.
The fast purification of fermentation broth was conducted with (NH4)2SO4 and Sephadex G-100, and got pure chitinase. The pure chitinase was thermostable, keep higher activity in acid environment, and the profile showed the double summit in wide pH limit. In alk environment the activity became lower, proved the recombinant chitnase was alk. The pathogen(Rhizopus stolonifer, Botrytis cinerea) could be inhibited by the enzyme in a certain extent.
引文
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