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| 腐皮镰孢聚丁二酸丁二酯解聚酶基因优化及在毕赤酵母中的表达 |
| Gene Optimization of Polybutylene Succinate Depolymerase from Fusarium solani and Its Expression in Pichia pastoris |
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| DOI：doi:10.3969/j.issn.1005-7021.2018.04.002 |
| 中文关键词: 腐皮镰孢 聚丁二酸丁二酯 解聚酶 毕赤酵母 |
| 英文关键词: Fusarium solani polybutylene succinate depolymerase Pichia pastoris |
| 基金项目:国家自然科学基金项目（31570097） |
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| 中文摘要: |
| 聚丁二酸丁二酯是一种可替代传统塑料的生物降解塑料，有助于解决白色污染问题，但其在自然界中往往降解缓慢，获取高效降解微生物或解聚酶是使其快速有效降解的关键。通过构建重组毕赤酵母表达体系以实现腐皮镰孢聚丁二酸丁二酯解聚酶（FSC）的高效表达。根据毕赤酵母偏好密码子优化FSC的基因密码子，并导入毕赤酵母X33中实现其重组表达。进一步通过单因素实验优化了菌株的产酶条件。结果显示，优化后的FSC基因序列中的157 个碱基发生改变，G+C含量由59.6%降低到48.3%，序列同源性为77.34%；构建的重组表达载体pPICZα-FSC转入毕赤酵母X33，结合抗性平板初筛、SDS-PAGE和Western bolt 验证，以及摇瓶发酵酶活力测定获得了1株具有较高产酶能力的重组菌株L1；进一步确定其摇瓶发酵培养条件：培养基起始pH 6.0，摇床转速220 r/min，甲醇补加量1%，接种量8%，培养时间72 h，培养温度30 ℃，此优化条件下菌株发酵液酶活力可达110 U/mL。 |
| 英文摘要: |
| Polybutylene succinate is a biodegradable plastics that could substitute traditional plastic and conducive to solve white pollution problems. However, polybutylene succinate degrades slowly in nature, searching high efficient degrading microorganisms or depolymerases is the key to the fast and efficient degradation of polybutylene succinate. This study was to construct a recombinant Pichia pastoris expression system to realize the efficient expression of polybutylene succinate depolymerase from Fusarium solani (FSC). According to the encoded codon in P. pastoris to optimize FSC gene codon and transducted into P. pastoris X33 to realize its recombinant expression. The enzyme producing conditions of the strain was optimized further through single factor experiments. The results showed that 157 bases in the optimized FSC gene sequence had changed, the G+C content reduced from 59.6% to 48.3%, and the homology was at 77.34%; the constructed recombinant expression vector pPICZα-FSC had transferred into P. pastoris X33, combined with initial screening on resistant plate, a recombinant strain L1 with higher enzyme producing capability was obtained tested and verified with SDS PAGE and Western blot as well as with shaking flash determination for fermentation enzyme activity; later the shaking flash fermentation culture conditions were further confirmed as follow: the starting pH was at 6.0, the shaker rotation was at 220 r/min, the addition of methanol was 1%, inoculation at 8%, culture for 72 hours in 30 ℃, and under these conditions the enzyme activity of fermentation broth could reach up to 110 U/mL. |
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