Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany.
Food Microbiol. 2012 Apr;29(2):205-14. doi: 10.1016/j.fm.2011.07.008. Epub 2011 Jul 28.
Branched chain aminotransferases (IlvE/BcaT), specific for leucine, isoleucine, and valine initialize the formation of methyl-branched volatiles, which are strongly linked to the typical aroma of cured meat products. Lactobacillus sakei, one of the dominating lactic acid bacteria species for meat fermentations and commonly used as starter lacks this enzyme, whereas the presence of IlvE has been reported for Lactobacillus paracasei, a non-starter lactic acid bacterium occurring in meat products and with probiotic properties, in Staphylococcus carnosus, a catalase positive cocci also used as starter for meat products, and in Enterococcus faecalis belonging to the natural microbiota of meat and that may impact on the aroma of fermented meat products. The genes for branched-chain aminotransferases of these three bacterial species were used to complement the IlvE negative strain L. sakei TMW1.1322. For that purpose, ilvE genes were heterologously expressed in L. sakei TMW1.1322 under the control of the constitutive L. sakei promotor pldhL via replicative plasmids and chromosomal integration. To examine effective expression the constructs were transcriptionally coupled to mCherry, a red fluorescent protein. Aminotransferase activities and formation of volatile compounds were compared. Activities of L. sakei ArcT and AspD purified enzymes were also measured. Conversion of branched chain amino acids to the corresponding α-keto-acids was significantly increased in all transformants expressing the ilvE genes. The activity of IlvE obtained from L. paracasei was highest. Substrate specificities of IlvEs towards leucine, isoleucine and valine were similar. However, enhanced transaminase activities did not increase formation of the respective methyl-branched volatiles by recombinant L. sakei strains. This indicates that presence of ilvE cannot be the only bottleneck in aroma formation from amino acids. Amino acid or peptide uptake into the cell via specific transport systems and the conversion of α-keto acids to the corresponding aldehydes, alcohols and carboxylic acids must be considered as further limiting steps.
支链氨基酸转氨酶（IlvE/BcaT），特异性针对亮氨酸、异亮氨酸和缬氨酸，起始形成甲基支链挥发物，这与腌制肉产品的典型香气强烈相关。乳杆菌（Lactobacillus sakei）是肉发酵中占主导地位的乳酸菌之一，通常用作发酵剂，但缺乏这种酶，而 IlvE 已被报道存在于非发酵剂乳酸菌乳杆菌副干酪亚种（Lactobacillus paracasei）中，该菌存在于肉产品中，具有益生菌特性，也存在于肉发酵剂阳性球菌金黄色葡萄球菌（Staphylococcus carnosus）和粪肠球菌（Enterococcus faecalis）中，这些菌属于肉的天然菌群，可能会影响发酵肉产品的香气。这三种细菌的支链氨基酸转氨酶基因被用于补充缺乏 IlvE 的乳杆菌（Lactobacillus sakei）菌株 TMW1.1322。为此，ilvE 基因通过复制质粒和染色体整合，在组成型乳杆菌（Lactobacillus sakei）启动子 pldhL 的控制下在乳杆菌（Lactobacillus sakei）TMW1.1322 中异源表达。为了检查有效表达，构建物与红色荧光蛋白 mCherry 转录偶联。比较了转氨酶活性和挥发性化合物的形成。还测量了 L. sakei ArcT 和 AspD 纯化酶的转氨酶活性。在表达 ilvE 基因的所有转化体中，支链氨基酸转化为相应的α-酮酸的转化率显著增加。来自乳杆菌副干酪亚种的 IlvE 的活性最高。IlvE 对亮氨酸、异亮氨酸和缬氨酸的底物特异性相似。然而，重组乳杆菌（Lactobacillus sakei）菌株转氨酶活性的增强并没有增加相应的甲基支链挥发物的形成。这表明，ilvE 的存在不能成为氨基酸形成香气的唯一瓶颈。通过特定的转运系统将氨基酸或肽摄取到细胞内，以及将α-酮酸转化为相应的醛、醇和羧酸，必须被视为进一步的限制步骤。
