【作者】 贾东旭；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 聚乙烯醇（PVA）主要以1,3-二醇键的化学结构存在，是一种难于被降解的高分子化合物。PVA具有很多优良性能，在工业上得到广泛应用。然而，大量PVA废水排放到环境，导致严重的污染，因此，实现PVA的生物降解具有重要的现实意义。PVA脱氢酶（PVADH）催化PVA降解的第一步反应，本论文以Sphingopyxis sp.113P3PVADH为研究对象，详细研究该酶基因在大肠杆菌（E. coli）和毕赤酵母（P. pastoris）中的表达情况，并对该酶的应用进行初步研究，具体研究内容如下：（1）Sphingopyxis sp.113P3聚乙烯醇脱氢酶基因（PVADH）的人工合成及在大肠杆菌（E. coli）中的融合表达与包涵体复性。以Sphingopyxis sp.113P3PVADH的氨基酸序列为模板，按P. pastoris密码子偏好性并替换掉E. coli稀有密码子，合成1965bp的基因序列。聚合酶链式反应（PCR）扩增1887bp的成熟酶基因（mPVADH），并插入质粒pET32a（+），转化E. coli BL21（DE3）。以20oC、0.05mM异丙基-β-D-硫代半乳糖苷（IPTG）、2%葡萄糖条件，诱导12h，目的蛋白仍以包涵体存在。通过包涵体复溶、稀释复性、重组肠激酶（rEK）切割、透析去除rEK和硫氧还蛋白（TrxA），纯化得到约67kDa的mPVADH蛋白，酶活和比酶活分别为60U/mL和109U/mg。（2）成熟PVADH在毕赤酵母（P. pastoris）中的高效表达、纯化和酶学性质研究。将mPVADH基因插入载体pPIC9K并电转化P. pastoris GS115，高拷贝转化子在摇瓶和3L发酵罐最高酶活分别为56和902U/mL，是首次在真核表达系统中成功表达PVADH。但是，表达的目的蛋白分子量小于预期，N-端氨基酸测序表明截短蛋白缺少mPVADH的1-81氨基酸。纯化的截短酶最适反应温度和pH分别为41-53oC和pH7.0-8.0；Ca²⁺对酶活有促进作用；催化最适底物（PVA1799）的米氏常数（Km）和最大反应速率（Vmax）分别是1.87mg/mL和34.9nmol/（min·mg）。（3）成熟PVADH在P. pastoris中截短表达分析。P. pastoris GS115/pPIC9K/mPVADH摇瓶诱导时，分别添加1%酪蛋白氨基酸、5g/L六偏磷酸钠；或将质粒pPIC9K/mPVADH转化P. pastoris SMD1168，表达的目的蛋白是截短的，说明不是胞外蛋白酶将目的蛋白降解。反转录PCR（RT-PCR）表明mPVADH转录时的mRNA是全长的，说明转录正确。分别缺失mPVADH截断位点前的ATG59、ATG64、ATG67和ATG76；或缺失mPVADH蛋白N端的1-21、22-51和52-81氨基酸，各突变转化子表达的目的蛋白仍然是截短的。同时，将绿色荧光蛋白（EGFP）与mPVADH融合，所得突变转化子表达的2段外源蛋白的分子量分别与EGFP和截短PVADH相当，说明融合蛋白翻译完整，但胞内蛋白酶将其错误切割。为了确定胞内蛋白酶对mPVADH的识别位点，首先针对蛋白酶Kex2的识别位点设计突变；然后针对其它蛋白酶，将mPVADH截断位点附近的氨基酸突变为（GlyGlyGlyGlySer）2，或缺失截断位点-5至+5的氨基酸序列，突变转化子表达的都是截短蛋白，说明不是Kex2错切mPVADH，该蛋白酶识别mPVADH的位点可能在截断位点后某区域，还有待于进一步研究。（4）截短PVADH（tPVADH）在P. pastoris中的过量表达与相关机制分析。将1644bp的tPVADH基因插入pPIC9K多克隆位点并转化P. pastoris GS115，转化子在摇瓶最高酶活为546U/mL，与表达mPVADH基因相比，酶活提高了10倍。荧光定量PCR（qRT-PCR）表明表达mPVADH的转录水平是表达tPVADH的1.9倍，但表达tPVADH基因时，胞外目的蛋白含量更多的原因可能是胞内蛋白酶对mPVADH错切影响了目的蛋白的分泌效率。在3L发酵罐上，分别优化了28和22oC诱导时的甲醇浓度，所得最高酶活分别是5515和8464U/mL；后者是文献报道的最高水平。低温诱导时，tPVADH的产量和生产强度大幅度提高归结于高细胞存活率、低胞外蛋白酶活力、高AOX酶活和高tPVADH转录水平。（5）tPVADH降解PVA1799的应用效果评价。红外光谱（IR）分析表明氧化型PVA（oxiPVA）的羰基在1735cm-1出现新吸收峰；¹H-NMR表明在3.8ppm处出现新吸收峰，该峰对应的官能团是oxiPVA二酮基之间的α-次甲基基团，说明tPVADH的功能是催化PVA分子上相邻羟基生成β-二酮基。凝胶过滤色谱（GPC）检测发现，PVA1799和oxiPVA的粘度分子量（Mv）分别是154786和71564，而OPH水解产物中大分子物质已消失，说明oxiPVA能自降解，OPH能加快oxiPVA的降解。最后，tPVADH和OPH联合处理40g/L的PVA溶液，其粘度下降约15%，说明tPVADH和OPH联合应用对PVA有一定降解效果。更多还原

【Abstract】 Poly（vinyl alcohol）（PVA） was a high molecular compound which was difficult to bedegraded, its basic structure was composed mainly of head-to-tail1,3-diol units. PVA hadmany perfect performances, which made its wide application in industry. However, a largeamount of waste water containing PVA were poured into rivers, leding to serious waterpollution. Thus, it was more significant to realize PVA biodegradion. Poly（vinyl alcohol）dehydrogenase （PVADH） took responsibility for catalyzing the first reaction of PVAbiodegradation. In this study, we investigated the heterologous expression of PVADH fromSphingopyxis sp.113P3in Escherichia coli （E. coli） and Pichia pastoris （P. pastoris） system,and some application researches were also done. The major results were summarized asfollows:（1） Synthesis of Sphingopyxis sp.113P3PVADH gene, fusion expression in E. coli andrenaturation of inclusion bodies.Using the amine acid subsequence of Sphingopyxis sp.113P3PVADH as template, a1965bp fragment was synthesized based on the codon bias of P. pastoris and taking place ofrare codons of E.coli. The1887bp gene without native signal peptide （mPVADH） wasamplified by polymerase chain reaction （PCR） and cloned into pET32a（+）. Under thecondition of induction at20oC,0.05mM isopropyl β-D-1-thiogalactopyranoside （IPTG）,2%glucose and induction of12h, the fusion protein of thioredoxin （TrxA）-PVADH wasexpressed mainly in the form of inclusion bodies in E.coli BL21（DE3）. Inclusion bodies weredissolved in buffer, followed by dilution renaturation, cleaving fusion protein by recombinantenterokinase （rEK）, removing rEK and TrxA by dialysis, the active mPVADH were obtained.The enzyme activity and specific activity were60U/mL and109U/mg protein, respectively.（2） Expression, purification, and enzymatic characterization of mature PVADH in P.pastoris using mPVADH gene.The mPVADH gene was amplified and inserted into pPIC9K. The recombinant plasmidwas linearized and transformed into P. pastoris GS115, a positive transformant of P. pastorisGS115/pPIC9K/mPVADH was seleted for expression. Its maxmun activity reached55and902U/mL in a shake flask and3L bioreactor. This study was the first acquired in theeukaryon expression system of P. pastoris. Surprisingly, the molecular weight （Mw） of targetprotein was apparent smaller than anticipation, and its N-terminal amine acids matched themPVADH starting from the82thamino acid. The optimum temperature and pH ranged for thepurified enzyme were41-53oC and7.0-8.0, respectively. Ca²⁺had stimulating effect on theactivity of PVADH. The enzyme had a Michaelis constant （Km） of1.89mg/mL and amaximum reaction rate （Vmax） of34.9nmol/（min） toward optimal substrate of PVA1799.（3） Truncation analysis of expression in P. pastoris using mPVADH gene.At shake flask level, adding1%casamino acid or5g/L sodium hexameta phosphate intoBMMY medium when induction of P. pastoris GS115/pPIC9K/mPVADH proceeded,SDS-PAGE results showed the target proteins were truncated. The expression level of intactPVADH was also not improved by transforming the recombinant plasmid pPIC9K/mPVADH into proteinase A-deficient SMD1168, indicating expression truncation was not incorrectcleavage by extracellular proteolytic. Reverse transcription polymerase chain reaction（RT-PCR） results showed that the mRNA of P. pastoris GS115/pPIC9K/mPVADH duringtranscription was intact, so there was no problem with transcription. Subsequently, a series ofdeletion mutations were carried out as follows: deleting ATG59, ATG64, ATG67and ATG76before the truncation site of mPVADH, respectively; deleting1-21,22-51and52-81amineacids at the N-terminal of mPVADH, respectively. The enzyme expressed by the mutatedtransformants were also truncated. Furthermore, fusion protein of EGFP-mPVADH wasexpressed in P. pastoris, the target protein was cleaved into two parts, which matched the Mwof truncated PVADH and EGFP. These results indicated that the translation was correct. Inorder to determine the recognition site of intracellular protease, a few mutations were donearming at the recognition site of Kex2. Referring to other intracellular protease, mutationswere proceeded as follows: changing amine acids near the truncation site to（GlyGlyGlyGlySer）2, or direct deleting amine acids of-5to+5areas. However, the targetproteins expressed by all the transformants were also truncated. In conclusion, someintracellular protease incorrectly cleaved mPVADH, the recognition site of this protease （notKex2） probably exited in the amine acids sequence of truncated PVADH, and relativeresearches should be done continuously to determine the cleaving mechanism.（4） Overproduction of a truncated PVADH （tPVADH） in recombinant P. pastoris usingtPVADH gene and related mechanism analysis.The1644bp tPVADH gene was amplified and inserted into pPIC9K, followed bylinearrization and transformation into P. pastoris GS115, a positive transformant of P.pastoris GS115/pPIC9K/tPVADH was picked out. The maximal tPVADH activity reached546U/mL in shake flask, which was nearly10times that of the mPVADH under the sameconditions （55U/mL）. Fluorescence quantitative PCR （qRT-PCR） showed that thetranscription level of mPVADH was1.9times higher than that of tPVADH in shake flask.However, there was less interest protein in the fermentation broth expressed by mPVADH.The most probable reason for the differences might be incorrect cleaving mPVADH by somepretease, the expression of tPVADH could overcome this incorrect cleaving with a higheryield of enzyme in fermentation broth, although its transcriptional level was lower comparedwith that of mPVADH. In3L bioreactor, the induction concentrations of methanol wereoptimized at28and22oC, and the highest activity reached5515and8464U/mL. Thesignificant improvement of tPVADH production at low induction are attribute to the highercell viability, lower extracellular proteases activity, and higher alcohol oxidase （AOX）activity at induction phase.（5） Effect evaluation of cleaving PVA by PVADH and OPH.Infrared spectrum （IR） spectra of the carboxylate groups in oxidized PVA （oxiPVA）showed a new absorption at1735cm-1, which was not observed for PVA1799.¹H-NMRresult showed that there was a new absorption peak at3.8ppm for oxiPVA, whichcorresponded to internal α-methylene protons of β-diketone. The molecular weight ofviscosity （Mv） of PVA1799and oxiPVA were154786and71564, respectively. Thehydrolyzates by OPH were nearly the low Mv substance. The results above illustrated that oxiPVA was not stable, while OPH could entirely degrade β-diketone structure of oxiPVA.Eventually, the PVA solution of40g/L was handled by PVADH and OPH, and the viscosityof PVA solution decreased approximately15%after reaction of24h. It was stated that addingtPVADH and OPH had a certain effect on PVA degradation.更多还原