底物结合域的融合增强角蛋白酶的催化能力并促进羽毛废料的酶促转化,底物结合域的融合增强角蛋白酶的催化能力并促进羽毛废料的酶促转化,Journal of Agricultural and Food Chemistry

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底物结合域的融合增强角蛋白酶的催化能力并促进羽毛废料的酶促转化,底物结合域的融合增强角蛋白酶的催化能力并促进羽毛废料的酶促转化
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2023-07-18 , DOI: 10.1021/acs.jafc.3c03064
Xiaomei Ji _{1,

2,

3} , Zheng Peng _{1,

2,

3} , Jie Song _{1,

2,

3} , Guoqiang Zhang _{1,

2,

3} , Juan Zhang _{1,

2,

3}

Affiliation

角蛋白酶在角蛋白水解中的独特作用引起了人们对羽毛废物回收的巨大兴趣。然而，由于羽毛角蛋白的高疏水性，角蛋白酶水解羽毛的催化能力通常较低。在这项研究中，我们旨在通过将底物结合域融合到酶中来提高角蛋白酶羽毛水解效率。我们筛选了几种碳水化合物结合模块（CBM）和连接肽。我们选择了最有希望的候选者来构建、克隆和表达融合角蛋白酶 KerZ1/CBM-L8，其羽毛水解效率为 7.8 × 10 –8 ^g /U。与 KerZ1 相比，KerZ1/CBM-L8 的羽毛水解效率提高了 2.71 倍，k _cat值提高了 179%，这意味着更高的催化效率，K _m和结合常数 ( K ) 值较低，这表明 KerZ1/CBM-L8-角蛋白结合亲和力较高。此外，使用等温滴定量热法测定的与底物的结合位点（ N ）的数量比 KerZ1 高 24.1 倍。因此，底物结合结构域的融合提高了角蛋白酶与疏水性底物的结合能力，从而提高了其羽毛水解效率。因此，使用融合角蛋白酶将显着提高羽毛废料的回收率。,角蛋白酶在角蛋白水解中的独特作用引起了人们对羽毛废物回收的巨大兴趣。然而，由于羽毛角蛋白的高疏水性，角蛋白酶水解羽毛的催化能力通常较低。在这项研究中，我们旨在通过将底物结合域融合到酶中来提高角蛋白酶羽毛水解效率。我们筛选了几种碳水化合物结合模块（CBM）和连接肽。我们选择了最有希望的候选者来构建、克隆和表达融合角蛋白酶 KerZ1/CBM-L8，其羽毛水解效率为 7.8 × 10 –8 ^g /U。与 KerZ1 相比，KerZ1/CBM-L8 的羽毛水解效率提高了 2.71 倍，k _cat值提高了 179%，这意味着更高的催化效率，K _m和结合常数 ( K ) 值较低，这表明 KerZ1/CBM-L8-角蛋白结合亲和力较高。此外，使用等温滴定量热法测定的与底物的结合位点（ N ）的数量比 KerZ1 高 24.1 倍。因此，底物结合结构域的融合提高了角蛋白酶与疏水性底物的结合能力，从而提高了其羽毛水解效率。因此，使用融合角蛋白酶将显着提高羽毛废料的回收率。

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Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10^–8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a k_cat value that is 179% higher, which translates to higher catalytic efficiency, and K_m and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8–keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.

更新日期：2023-07-18

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