Expression of Beta-galactosidase Gene in Pichia pastoris Improved by Non-methanol Induced Episomal Expression Vector
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摘要: 半乳糖苷酶是一种重要的食品工业用酶,目前主要通过毕赤酵母甲醇诱导型表达系统进行生产,但甲醇的使用存在火灾、残留毒性等安全隐患,已逐渐成为食品工业用酶生产中备受关注的问题之一。为满足β-半乳糖苷酶安全生产的需要,本研究利用强组成型启动子PGCW14和源自酵母的自我复制序列PARS构建了一种新型非甲醇诱导游离型表达载体pGCW14ZαA-PARS,并且在此基础上分别构建了非甲醇诱导游离型重组表达菌株KM71/p GCW14ZαA-PARS-Aoβ-GAL,用以改善米曲霉Aspergillus Oryzae RIB40(ATCC42149)来源的β-半乳糖苷酶基因(Aoβ-GAL)在毕赤酵母中的表达。实验结果表明,该非甲醇诱导游离型表达载体在毕赤酵母中传代培养90代后仍保持83.22%的遗传稳定性,完全能够满足工业上大规模生产的需要。在10%流速补充碳源的条件下,非甲醇诱导游离表达菌株KM71/pGCW14ZαA-PARS-Aoβ-GAL经高密度发酵的最高酶活性和比活性分别为126.4 U/m L和26.2 U/mg,分别是传统甲醇诱导型表达菌株KM71/pPIC9k-Aoβ-GAL的1.94倍和3.12倍,且发酵周期缩短了36 h。可见,本研究构建的非甲醇诱导游离型表达载体可有效改善β-半乳糖苷酶在毕赤酵母中的表达,且在食品工业用酶的安全、高效工业化生产中具有一定的应用前景。
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关键词:
- 组成型启动子PGCW14 /
- 游离型表达载体 /
- 重组毕赤酵母 /
- β-半乳糖苷酶 /
- 高密度发酵
Abstract: galactosidase is an important enzyme for the food industry, and it is usually produced by Pichia pastoris methanol-induced expression system. However, the use of methanol has several safety hazards such as causing fire and residual toxicity, which has gradually become one of the most concerned issues in industrial production of food-grade enzymes. In order to meet the needs of safe production of β-galactosidase, this study constructed a novel methanol-free episomal expression vector, pGCW14 ZαA-PARS, using strong constitutive promoter PGCW14 and the yeast-derived self-replicating sequence PARS. Then the recombinant expression strain KM71/pGCW14 ZαA-PARS-Aoβ-GAL was constructed to improve the expression of the β-galactosidase gene（Aoβ-GAL）derived from Aspergillus oryzae RIB40（ATCC42149）. Results showed that the methanol-free episomal expression vector maintained 83.22% genetic stability after subculture in Pichia pastoris for 90 generations, which could fully meet the needs of industrial large-scale production. After high-density fermentation under a 10% flow rate fed-batch of carbon source, the highest enzyme activity and specific activity of the strain KM71/pGCW14 ZαA-PARS-Aoβ-GAL were 126.4 U/m L and 26.2 U/mg, which were 1.94 times and 3.12 times of those of the traditional methanol-inducible expression strain KM71/pPIC9 k-Aoβ-GAL, respectively. Besides, the fermentation cycle was shortened by 36 h. It indicated that the novel methanol-free episomal expression vector constructed in this study could effectively improve the expression of β-galactosidase in Pichia pastoris, and has great application prospects in the safe and efficient industrial production of food enzymes. -
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