Abstract：
This study reports an alternative strategy for the expression of a recombinant L-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl β-D-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of D-galactose into D-tagatose. L-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant L-AI aiming its high-scale production.
摘要：
这项研究报告了通过使用葡萄糖和甘油作为碳源并使用残余乳清乳糖作为诱导剂的自动诱导，从屎肠球菌DBFIQE36中表达重组L-AI的替代策略。还评估了市售乳糖和异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)作为比较酶表达水平的诱导物。酶提取物通过亲和层析纯化，characterized,并应用于将D-半乳糖生物转化为D-塔格糖。L-AI的催化活性为1.67±0.14、1.52±0.01和0.7±0.04U/mL，当使用商业乳糖表达时，来自乳清的乳糖，IPTG,分别。通过改变酶提取的方案可以获得更高的活性，例如,乳清酶提取物的催化活性为3.8U/mL。与用IPTG表达的酶相比,酶纯化后使用乳糖(商业或残余乳清)产生的酶提取物的比活性也更高。当使用4g/L的残余乳清乳糖进行11小时的酶表达时,获得了最好的结果。这些结果证明了针对重组L-AI的有效表达的替代和经济方案的效力,该重组L-AI旨在其大规模生产。
