Urease operon is highly conserved within the species and urease-negative strains are rare in nature. MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in , coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in determined a substitution of Asp with Asn in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp Asn substitution in UreE on the urease activity of , gene of the reference strain DSM 20617 () was replaced by gene of strain MIMO1 () to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp with Asn in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

中文翻译：

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将嗜热链球菌 DSM 20617T 的金属分子伴侣 UreE 中的 Asp29 替换为 Asn29 可增加脲酶活性并预期牛奶发酵过程中的尿素水解

脲酶操纵子在物种内高度保守，脲酶阴性菌株在自然界中很少见。 MIMO1 是从商业酸奶中分离出来的，之前被定性为脲酶阳性 Ni 依赖性菌株。除了编码镍 ABC 转运蛋白通透酶的突变之外，菌株 MIMO1 还显示出编码参与镍递送至脲酶催化位点的金属伴侣的基因突变。单碱基突变决定了金属伴侣中不参与催化活性的保守蛋白质区域中天冬氨酸被天冬氨酸取代。为了探讨UreE中Asp Asn取代对脲酶活性的影响，将参考菌株DSM 20617()基因替换为菌株MIMO1()基因，得到重组ES3。凝胶内脲酶活性检测表明，UreE 中 Asn 取代 Asp 导致酶复合物具有更高的稳定性。此外，与野生型相比，重组ES3显示出更高水平的脲酶活性，而基因表达水平没有任何可检测到的增加，从而突出了UreE不仅在Ni组装中而且在脲酶活性水平上的作用。在牛奶中生长期间，与野生型相比，重组 ES3 显示出预期的脲酶活性以及类似的牛奶发酵性能。通过比较脲酶阳性和脲酶阴性菌株/突变体获得的总体数据证实，脲酶活性强烈影响牛奶发酵过程，特别是同质乳酸发酵的产量。