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A novel glyA gene from the marine bacterium Alcanivorax sp. was cloned and expressed in Escherichia coli BL21(DE3). The recombinant glyA encodes a polypeptide of 418 amino acids, which was designated as AdSHMT that shows the highest identity (70%) with a SHMT from Shewanella algae. The purified enzyme showed a single band at about 45 kDa by SDS-PAGE analysis. It was found that AdSHMT exhibited the maximal activity at 50 ◦ C and pH 7.0. The K m , V max , and K cat values of AdSHMT against dl-threo-3-phenylserine were calculated to be 0.097 mol/L, 3.255 ?mol/min/mg and 2.451/s, respectively. More importantly, RP-HPLC detection showed that the AdSHMT achieved an 88.37% molecular conversion rate in catalyzing glycine to l-serine, with the final concentration of l-serine being 353.15 mM in the reaction at 35 ◦ C and 22nd hour when the initial concentration of the substrate (glycine) was 0.399 M. The molecular conversion rate of the AdSHMT from the Alcanivorax sp. was 1.26-fold that of the EcSHMT from the E. coli,which is currently applied in industrial production. Therefore, AdSHMT has the potential for industrial applications due to its high enzymatic conversion rate. |
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- 翻譯EPI: 11
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- 金幣: 3605.5
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- 注冊(cè): 2014-10-27
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- 專業(yè): 高分子合成化學(xué)
【答案】應(yīng)助回帖
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bxzc123: 金幣+20, 翻譯EPI+1, ★★★很有幫助 2014-11-25 22:11:57
bxzc123: 金幣+20, 翻譯EPI+1, ★★★很有幫助 2014-11-25 22:11:57
| 一種來自海洋細(xì)菌Alcanivorax的新型的glyA基因被克隆，并在大腸桿菌BL21（DE3）上表達(dá)。重組的glyA 編碼了一個(gè)由418個(gè)氨基酸組成的多肽，被命名為 AdSHMT， 它顯示出與來自Shewanella藻的SHMT最高70％的相似。在SDS-PAGE分析中純化的酶為45 kDa的單鏈 (多肽)。結(jié)果發(fā)現(xiàn)，在50◦C和pH7.0時(shí)，AdSHMT表現(xiàn)出最大的活性。AdSHMT against dl-threo-3-phenylserine (這里的一個(gè)專業(yè)知識(shí)點(diǎn)，我不懂。應(yīng)該是一生化反應(yīng))的 Km, Vmax, 和 Kcat 數(shù)值分別為0097摩爾/升，3255摩爾/分鐘/毫克和2451/秒。更重要的是反相高效液相色譜法檢測(cè)表明，在35 ◦ C時(shí)（AdSHMT）催化glycine為l-serine反應(yīng)中當(dāng)glycine的初始濃度為0.399 M時(shí)在第22小時(shí)AdSHMT取得了88.37%的轉(zhuǎn)化率。從Alcanivorax SP到AdSHMT的分子轉(zhuǎn)化率是目前在工業(yè)生產(chǎn)中應(yīng)用的從E. coli到EcSHMT的轉(zhuǎn)化率的1.26倍。因此，AdSHMT因其高的酶轉(zhuǎn)化率可能在工業(yè)上得到應(yīng)用。 |
2樓2014-11-25 10:06:15
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