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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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木虫 (小有名气)
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bxzc123: 金币+20, 翻译EPI+1, ★★★很有帮助 2014-11-25 22:11:57
bxzc123: 金币+20, 翻译EPI+1, ★★★很有帮助 2014-11-25 22:11:57
| 一种来自海洋细菌Alcanivorax的新型的glyA基因被克隆,并在大肠杆菌BL21(DE3)上表达。重组的glyA 编码了一个由418个氨基酸组成的多肽,被命名为 AdSHMT, 它显示出与来自Shewanella藻的SHMT最高70%的相似。在SDS-PAGE分析中纯化的酶为45 kDa的单链 (多肽)。结果发现,在50◦C和pH7.0时,AdSHMT表现出最大的活性。AdSHMT against dl-threo-3-phenylserine (这里的一个专业知识点,我不懂。应该是一生化反应)的 Km, Vmax, 和 Kcat 数值分别为0097摩尔/升,3255摩尔/分钟/毫克和2451/秒。更重要的是反相高效液相色谱法检测表明,在35 ◦ C时(AdSHMT)催化glycine为l-serine反应中当glycine的初始浓度为0.399 M时在第22小时AdSHMT取得了88.37%的转化率。从Alcanivorax SP到AdSHMT的分子转化率是目前在工业生产中应用的从E. coli到EcSHMT的转化率的1.26倍。因此,AdSHMT因其高的酶转化率可能在工业上得到应用。 |

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