Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon, South Korea.
Int J Biol Macromol. 2012 Jan 1;50(1):103-11. doi: 10.1016/j.ijbiomac.2011.10.003. Epub 2011 Oct 15.
A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser(10)-Asp(176)-His(179)), and a conserved sequence motif Ser(S)(10)-Gly(G)(77)-Asn(N)(79)-His(H)(179) with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and β-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.
从无害李斯特菌 CLIP 11262 中鉴定出一种新型寡聚水解酶（LI22），并对其进行了特性分析和固定化，以用于工业应用。LI22 的序列分析揭示了一个假定的催化三联体（Ser(10)-Asp(176)-His(179)），以及一个保守的序列基序 Ser(S)(10)-Gly(G)(77)-Asn(N)(79)-His(H)(179)，与 SGNH-水解酶超家族的其他成员具有中等相似性（<30%）。LI22 能够水解对硝基苯乙酸酯、α-和β-萘乙酸酯，而 S10A 突变体完全失去了活性。使用凝胶过滤、圆二色性（CD）、荧光、分子建模和凝胶过滤研究了 LI22 的结构特性。我们已经表明，在 30%TFE 或 50%乙醇溶液中孵育时，LI22 会转化为卷曲的淀粉样纤维。通过用硫酸铵沉淀酶并随后用戊二醛交联来制备 LI22 的交联酶聚集体。LI22-CLEA 具有更高的热稳定性和化学稳定性，以及在重复使用后的良好耐久性，突出了其在制药和化学工业中作为生物催化剂的潜在适用性。
