密码子优化的吡喃糖氧化酶基因在毕赤酵母中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2021-0148

摘要/Abstract

摘要：

吡喃糖氧化酶（pyranose oxidase,PROD,简称P2O）在木质素降解、碳水化合物的生物转化合成中具有重要作用,还应用于生物燃料电池、生物传感器以及临床诊断分析中。本实验室进行了吡喃糖氧化酶在毕赤酵母中的高效表达及酶学性质研究,为以后吡喃糖氧化酶的工业化高效生产提供理论依据。利用生物技术方法,基于毕赤酵母密码子偏好性优化吡喃糖氧化酶基因密码子。通过基因外源表达技术构建吡喃糖氧化酶重组毕赤酵母GS115菌株以实现吡喃糖氧化酶的高效表达,并对重组吡喃糖氧化酶的酶学性质进行研究。优化高产重组菌株的发酵条件,在10 L发酵罐中进行扩大培养。结果显示,吡喃糖氧化酶密码子优化后,其在毕赤酵母中实现了高效表达。在10 L发酵罐经过132 h诱导表达后,酶活达220 U/mL。酶学性质研究发现：重组吡喃糖氧化酶最适作用温度为55℃,在不超过60℃条件下热稳定性良好。该酶作用的最适pH为6.5,在pH 5-9条件下,其相对酶活高于50%。P2O在较广的pH范围内均表现出较高的稳定性,尤其是对碱性条件有较强的耐性。金属离子Cu²⁺ 对酶活的抑制作用较大。底物特异性检测结果显示,该重组酶的最适底物为D-葡萄糖。该研究成功构建了密码子优化的吡喃糖氧化酶的重组表达质粒pPIC9K-P2O,并在毕赤酵母中实现了高效表达。

关键词: 吡喃糖氧化酶, 密码子优化, 毕赤酵母, 异源表达

Abstract:

Pyranose oxidase（P2O）plays an important role in lignin degradation and carbohydrate biosynthesis. P2O is also used in biological fuel cell,biosensors and clinical diagnostic analysis. The expression of pyranose oxidase in Pichia pastoris and enzymatic characters were carried out in our laboratory,which will provide a theoretical basis for the industrial and efficient production of pyranose oxidase in the future. Based on the codon preference of P. pastoris,the codon of pyranose oxidase gene was optimized by biotechnology. The recombinant P. pastoris GS115 was constructed via gene exogenous expression technology for achieving the efficient expression of P2O. The enzymatic properties of the recombinant P2O were studied. After optimizing the fermentation conditions of high-yielding recombinant strains,large-scale culture was conducted in 10 L fermenter. As results,P2O was highly expressed in P. pastoris after codon optimization. After 132 h induction culture in 10 L fermenter,enzyme activity reached 220 U/mL. The optimal temperature of recombinant pyranose oxidase was 55℃,and its thermal stability was good under the condition of no more than 60℃. The optimal pH for this enzyme was 6.5. In the range of pH 5-9,the relative enzyme activity was higher than 50%. P2O showed high stability in a wide range of pH,especially in alkaline conditions. Cu²⁺ presented a greater inhibition on enzyme activity. Assay of substrate specificity showed that the optimal substrate for the recombinant enzyme was D-glucose. In conclusion,the codon-optimized recombinant expression plasmid pPIC9K-P2O is successfully constructed in this study and highly expressed in P. pastoris GS115.

Key words: pyranose oxidase, codon optimization, Pichia pastoris, heterogeneous expression

王玥, 高庆华, 董聪, 罗同阳, 王庆庆. 密码子优化的吡喃糖氧化酶基因在毕赤酵母中的表达[J]. 生物技术通报, 2022, 38(4): 269-277.

WANG Yue, GAO Qing-hua, DONG Cong, LUO Tong-yang, WANG Qing-qing. Expression of Pyranose Oxidase with Optimized Codon in Pichia pastoris[J]. Biotechnology Bulletin, 2022, 38(4): 269-277.

图/表 11

图1 密码子优化后的基因序列方框内序列分别为 EcoR I 和 Not I的酶切位点;划线序列为6个组氨酸标签

Fig. 1 Gene sequence after the optimization of codons The boxed sequences indicate restriction sites EcoR Iand Not I. The underlined sequences show 6×His-tag

图2 pPIC9K-P2O质粒双酶切验证 M：DNA marker;1：pPIC9K-P2O质粒经EcoR I和Not I双酶切;2：pPIC9K-P2O质粒

Fig. 2 Verification of pPIC9K-P2O digested by double enzymes M：DNA marker. 1：Digestion of pPIC9K-P2O by EcoR I and Not I.2：pPIC9K-P2O plasmid

图3 转化子的获得

Fig. 3 Acquirement of transformants

图4 筛选P2O高表达量的重组菌株

Fig. 4 Screening of recombinant P. pastoris with high-yield P2O

图5 重组蛋白的SDS-PAGE结果 M：蛋白 marker;1：对照发酵上清液;2-8：阳性转化子发酵上清液

Fig. 5 SDS-PAGE result of recombinant protein M：Protein marker. 1：Supernatant of negative control. 2-8：Supernatant of positive transformants

图6 重组蛋白纯化结果 M：蛋白 marker;1-8：重组蛋白纯化

Fig. 6 Purification results of recombinant protein M：Protein marker. 1-8：Purification of recombinant protein

图7 10 L 发酵罐高密度发酵生产 P2O A：P2O酶活生长曲线;B：SDS-PAGE 结果;M：蛋白 marker;1-12：0、12、24、36、48、60、72、84、96、108、120、132 h后的重组蛋白积累量

Fig. 7 Production of P2O by high-density fermentation in 10 L bioreactor A：Growth curve of P2O activity. B：SDS-PAGE result. M：Protein marker. 1-12：Accumulation of recombinant protein after 0,12,24,36,48,60,72,84,96,108,120 and 132 h

图8 最适pH及pH稳定性 A：30℃不同pH条件下P2O相对酶活;B：30℃不同pH条件下保温24 h后P2O相对酶活;缓冲液分别是100 mmol/L 柠檬酸-柠檬酸钠（■,黑线）,磷酸钾（●,红线）,Tris-HCl（▲,蓝线）,甘氨酸-NaOH（▼,粉线）

Fig. 8 Optimal pH and pH stability A：Relative activity at different pH conditions at 30℃. B：The relative enzyme activity of P2O kept in the same temperature for 24 h at different pH conditions at 30℃. The used buffers were 100 mmol/L sodium citrate（■,black line）,potassium phosphate（●,red line）,Tris-HCl（▲,blue line）and glycine-NaOH（▼,pink line）

图9 最适温度及温度稳定性 A：在pH 6.5,不同温度下P2O相对酶活;B：在pH 6.5,不同温度下保温30 min后P2O相对酶活

Fig. 9 Optimal temperature and temperature stability A：Relative activity of P2O at different temperatures at pH 6.5. B：The relative enzyme activity of P2O kept in the different temperatures for 30 min at pH 6.5

图10 不同金属离子对P2O的影响

Fig. 10 Effects of metal ions on the activity of P2O

图11 P2O的底物特异性

Fig. 11 Substrate specificity of P2O

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