本文選題：Sortase + A介導(dǎo)��； 參考：《江南大學(xué)》2017年碩士論文

【摘要】：來(lái)自于近平滑假絲酵母(Candida parapsilosis CCTCC M203011)中的短鏈脫氫酶(S)-羰基還原酶II(SCRII)在輔酶NADPH的參與下,能夠特異性催化2-羥基苯乙酮合成(S)-苯基乙二醇,但催化效率較低、熱穩(wěn)定性不高。本研究利用sortase A(SrtA)的轉(zhuǎn)肽作用,通過在SCRII的C末端引入SrtA識(shí)別序列,在SrtA的介導(dǎo)下構(gòu)建功能和穩(wěn)定性均加強(qiáng)的SCRII寡聚體。以2-羥基苯乙酮作為底物,NADPH作為輔酶,SCRII寡聚體為催化劑高效制備(S)-苯基乙二醇。隨后,將SrtA介導(dǎo)的寡聚化擴(kuò)展至其它8種羰基還原酶,建立了增強(qiáng)羰基還原酶穩(wěn)定性,提高酶催化效率的平臺(tái)技術(shù)。主要研究?jī)?nèi)容包括:(1)從金黃色葡萄球菌Staphylococcus aureus ST451的基因組中釣取SrtA基因核心序列并構(gòu)建pET21a-srt A質(zhì)粒,實(shí)現(xiàn)了SrtA在大腸桿菌Escherichia coli BL21(DE3)中的高效表達(dá);通過設(shè)計(jì)引物在SCRII的C末端引入GGGGSLPETGG序列,構(gòu)建pET28a-scr II-mtf質(zhì)粒,誘導(dǎo)表達(dá)結(jié)果表明加入GGGGSLPETGG標(biāo)簽對(duì)酶的表達(dá)沒有影響;優(yōu)化了SrtA介導(dǎo)SCRII-mtf連接的條件,當(dāng)SrtA的酶濃度為1 mg·m L-1的情況下,Ca2+的最佳濃度為10 mmol·L-1,連接反應(yīng)的最佳溫度為25℃,連接時(shí)間為36 h時(shí),幾乎所有的SCRII-mtf都被消耗,此時(shí)連接產(chǎn)物產(chǎn)量最高,且連接產(chǎn)物成分主要為二聚體和三聚體。(2)測(cè)定了SCRII寡聚體的酶學(xué)性質(zhì),在35℃,pH 6.0的條件下,SCRII-mtf較SCRII酶活有略微提高,而SrtA介導(dǎo)產(chǎn)生的SCRII寡聚體對(duì)2-羥基苯乙酮的比活達(dá)到了38.5 U·mg-1,與SCRII相比提高了6倍;SCRII寡聚體在50℃時(shí)相對(duì)酶活最高,SCRII寡聚體和SCRII在50℃放置1 h,活性分別維持在90%以上和50%左右,表明SCRII寡聚化后,溫度穩(wěn)定性顯著提高;SCRII、SCRII-mtf和SCRII寡聚體對(duì)于pH的依賴性非常相似,但SCRII寡聚體的pH穩(wěn)定性明顯高于其它兩種酶;與SCRII相比,SCRII寡聚體的Km值降低了3.3倍,說明經(jīng)過寡聚化,SCRII寡聚體與底物2-羥基苯乙酮的親和力有所增加。(3)確定了SCRII寡聚體生物轉(zhuǎn)化(S)-苯基乙二醇的最適反應(yīng)條件,從整體上來(lái)看,SCRII寡聚體在35℃,pH 6.0,5g·L-1 2-羥基苯乙酮的條件下,2 h內(nèi)SCRII寡聚體轉(zhuǎn)化生成(S)-苯基乙二醇的產(chǎn)率高達(dá)90%以上;在3 h內(nèi)生成(S)-苯基乙二醇的產(chǎn)率和光學(xué)純度均達(dá)到了100%。(4)選取了另外8種氧化還原酶,利用SrtA構(gòu)建氧化還原酶寡聚體,8種寡聚體在SrtA的介導(dǎo)下均出現(xiàn)不同程度的寡聚化,圓二色譜顯示寡聚體的Tm值較原始酶升高了5℃-12℃,寡聚體酶活力較單體提高了5-11倍,生物轉(zhuǎn)化反應(yīng)6 h后,對(duì)應(yīng)手性產(chǎn)物的產(chǎn)率提高了25%-285%。
[Abstract]:The short chain dehydrogenase (SCRII) from Candida parapsilosis CCTCC M203011), with the participation of coenzyme NADPH, can specifically catalyze the synthesis of thio-phenylglycol from 2-hydroxyacetophenone, but the catalytic efficiency is low and the thermal stability is not high. The aim of this study was to construct a SCRII oligomer with enhanced function and stability by introducing SrtA recognition sequence into the C terminal of SCRII by using the transpeptide of sortase An SrtA. Using 2-hydroxyacetophenone as substrate, NADPH was used as coenzyme SCRII oligomer as catalyst. Subsequently, the SrtA mediated oligomerization was extended to the other eight carbonyl reductase, and the platform technology was established to enhance the stability of the carbonyl reductase and improve the catalytic efficiency of the enzyme. The main research contents include: 1) the core sequence of SrtA gene was isolated from the genome of Staphylococcus aureus Staphylococcus aureus ST451 and the plasmid pET21a-srt A was constructed. The high expression of SrtA in E. coli Escherichia coli BL21DDE3) was achieved. The pET28a-scr II-mtf plasmid was constructed by introducing GGGGSLPETGG sequence into the C terminal of SCRII by designing primers. The induced expression results showed that the addition of GGGGSLPETGG tag had no effect on the expression of the enzyme, and the conditions of SrtA mediated SCRII-mtf ligation were optimized. When the enzyme concentration of SrtA was 1 mg mL ~ (-1), the optimal concentration of Ca ~ (2 2) was 10 mmol L ~ (-1), the optimal reaction temperature was 25 鈩，

本文編號(hào)：1903441