Study On The Heterologous High Level Expression Of Poly (Vinyl Alcohol) Dehydrogenase From Sphingopyxis Sp.113P3

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.