本文選題：�；撬� + L-半胱亞磺酸脫羧酶��； 參考：《微生物學(xué)報(bào)》2017年08期

【摘要】：【目的】完成一個(gè)來(lái)源于堿性污染土壤宏基因組文庫(kù)的新的L-半胱亞磺酸脫羧酶基因undec1A的鑒定,研究其酶學(xué)性質(zhì)并利用非理性設(shè)計(jì)技術(shù)對(duì)其進(jìn)行分子改造�！痉椒ā恳詐 ETBlue-2為表達(dá)載體構(gòu)建包含undec1A基因的重組表達(dá)質(zhì)粒,轉(zhuǎn)化至宿主細(xì)胞E.coli Tuner(DE3)p Lac?中構(gòu)建重組表達(dá)克隆,采用鎳親和層析完成了酶蛋白的分離純化,完成其生化特征研究,利用連續(xù)易錯(cuò)PCR技術(shù)對(duì)Undec1A蛋白進(jìn)行分子改造�！窘Y(jié)果】生物信息學(xué)分析結(jié)果揭示Undec1A蛋白與已知的L-半胱亞磺酸脫羧酶存在類(lèi)似的輔酶結(jié)合位點(diǎn)和底物識(shí)別催化基序等。分子對(duì)接結(jié)果顯示氨基酸殘基Val237、Asp239、Asp266、Ile267、Ala268和Lys298等決定了與底物分子L-半胱亞磺酸的識(shí)別和結(jié)合催化。以L(fǎng)-半胱亞磺酸作為底物,重組Undec1A蛋白的最適作用p H為7.0,最適作用溫度為35°C;分子動(dòng)力學(xué)常數(shù)K_m為(1.557±0.015)mmol/L,V_(max)為(49.07±3.19)μmol/(L·min),k_(cat)為(45.80±1.32)/min。利用連續(xù)易錯(cuò)PCR技術(shù)完成了親本酶的分子改造,分離篩選到了一個(gè)酶活力更高的突變酶Undec1A-1180。在優(yōu)化條件下,Undec1A-1180的比活力較親本酶提高了約5.62倍�！窘Y(jié)論】本研究為構(gòu)建�；撬岬纳锖铣晒に囂峁┝死碚搮⒖�,因而具有重要的實(shí)踐意義。
[Abstract]:[objective] to complete the identification of a new L- cysteinesulfonic acid decarboxylase gene undec1A from a macro genomic library of alkaline contaminated soil. The enzyme properties were studied and modified by irrational design. [methods] Recombinant expression plasmid containing undec1A gene was constructed by using p ETBlue-2 as expression vector and transformed into host cell E.coli Tuner(DE3)p? The enzyme protein was isolated and purified by nickel affinity chromatography, and its biochemical characteristics were studied. Continuous error-prone PCR technique was used to modify Undec1A protein. [results] Bioinformatics analysis revealed that Undec1A protein had similar coenzyme binding sites and substrate recognition catalytic motifs with known L- cysteinesulfonic acid decarboxylase. The results of molecular docking showed that the amino acid residues Val237Asp239Asp266Ala268 and Lys298 determined the recognition and binding catalysis with the substrate L- cysteinesulfonic acid (L- cysteinesulfonic acid). Using L- cysteine sulfonic acid as substrate, the optimal action of recombinant Undec1A protein was 7.0 and the optimum temperature was 35 擄C, and the molecular dynamics constant K _ m was 1.557 鹵0.015 mmol-1 路min ~ (-1) (n = 49.07 鹵3.19) 渭 mol/(L 路min ~ (-1) 路min ~ (-1) ~ (-1) 路min ~ (-1) ~ (-1) ~ (-1) 路min ~ (-1) ~ (-1) 路min ~ (-1). The molecular transformation of parental enzyme was carried out by continuous error-prone PCR, and a mutant enzyme Undec1A-1180 with higher enzyme activity was isolated and screened. Under the optimized conditions, the specific activity of Undec1A-1180 was 5.62 times higher than that of the parent enzyme. [conclusion] this study provides a theoretical reference for the construction of taurine biosynthesis process, so it has important practical significance.
【作者單位】： 亞熱帶農(nóng)業(yè)生物資源保護(hù)與利用國(guó)家重點(diǎn)實(shí)驗(yàn)室廣西大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(21262003) 2017年度廣西高等教育創(chuàng)優(yōu)計(jì)劃教學(xué)相關(guān)項(xiàng)目-優(yōu)勢(shì)特色專(zhuān)業(yè)項(xiàng)目(優(yōu)質(zhì)本科專(zhuān)業(yè))~~
【分類(lèi)號(hào)】：Q55;Q78

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