Insight into the Substrate Specificity of Lactobacillus paracasei Aspartate Ammonia-Lyase
Insight into the Substrate Specificity of Lactobacillus paracasei Aspartate Ammonia-Lyase
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深入了解副干酪乳杆菌天冬氨酸氨裂解酶的底物特异性
DOI：
10.3390/fermentation9010049
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
L. Hsieh
中科院分区：
文献类型：
--
作者：
Yi;W. You;Yung;Jhih‐Ying Ciou;L. Hsieh
天冬氨酸解氨酶（AAL）催化天冬氨酸向富马酸和氨的可逆转化反应。本研究将副干酪乳杆菌LpAAL基因在大肠杆菌中进行了异源表达。除了重组His标记的LpAAL蛋白外，还使用麦芽糖结合蛋白（MBP）融合的LpAAL蛋白来增强其蛋白溶解性和表达水平。这两种重组蛋白显示出广泛的底物特异性，催化天冬氨酸，富马酸，苯丙氨酸，酪氨酸产生富马酸，天冬氨酸，反式肉桂酸，和p-香豆酸，分别。LpAAL蛋白对四种底物的最适反应pH和温度分别为8.0和40 °C。LpAAL蛋白对天冬氨酸、富马酸、苯丙氨酸和酪氨酸作为底物的Km值分别为5.7、8.5、4.4和1.2 mM。以天冬氨酸、富马酸、苯丙氨酸和酪氨酸为底物的LpAAL蛋白的kcat值分别为6.7、0.45、4.96和0.02 s-1。因此，天冬氨酸、富马酸、苯丙氨酸和酪氨酸是LpAAL酶的真正底物。
Aspartate ammonia-lyase (AAL) catalyzes the reversible conversion reactions of aspartate to fumaric acid and ammonia. In this work, Lactobacillus paracasei LpAAL gene was heterologously expressed in Escherichia coli. As well as a recombinant His-tagged LpAAL protein, a maltose-binding protein (MBP) fused LpAAL protein was used to enhance its protein solubility and expression level. Both recombinant proteins showed broad substrate specificity, catalyzing aspartic acid, fumaric acid, phenylalanine, and tyrosine to produce fumaric acid, aspartic acid, trans-cinnamic acid, and p-coumaric acid, respectively. The optimum reaction pH and temperature of LpAAL protein for four substrates were measured at 8.0 and 40 °C, respectively. The Km values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 5.7, 8.5, 4.4, and 1.2 mM, respectively. The kcat values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 6.7, 0.45, 4.96, and 0.02 s−1, respectively. Therefore, aspartic acid, fumaric acid, phenylalanine, and tyrosine are bona fide substrates for LpAAL enzyme.