Altammar Khadijah A, Ling Jonathan Guyang, Al-Bajalan Hussein M, Chin Iuan-Sheau, Mackeen Mukram Mohamed, Mahadi Nor Muhammad, Murad Abdul Munir Abdul, Bakar Farah Diba Abu
Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
Int J Biol Macromol. 2022 Dec 1;222(Pt B):2353-2367. doi: 10.1016/j.ijbiomac.2022.10.022. Epub 2022 Oct 6.
Cutinases are hydrolytic enzymes secreted by phytopathogens to degrade cutin, the main polymeric component of plant cuticles. The multifaceted functionality of cutinases has allowed for their exploitation for catalytic reactions beyond their natural purpose. To diversify and expand the cutinase enzyme class, we identified five cutinase homologs from the saprotroph Aspergillus niger. One of these cutinases, AnCUT3, was over-expressed in Pichia pastoris and its biophysicochemical properties characterized. The purified recombinant AnCUT3 possessed an optimum temperature of 25 °C, an optimum pH of 5, and was stable at temperatures up to 50 °C (1 h incubation, melting point of 45.6 °C) and in a wide pH range. Kinetic studies of AnCUT3 using pNP ester substrates showed the highest catalytic efficiency, k/K of 859 mM s toward p-nitrophenyl decanoate (C10). Although its calculated molecular mass is 27 kDa, AnCUT3 was expressed as two glycosylated proteins of molecular weights 24 and 50 kDa. Glycan profiling detected the presence of atypical paucimannose N-glycans (≤ManGlcNAc) from recombinant AnCUT3, suggesting protein-dependent glycan processing of AnCUT3 in P. pastoris. AnCUT3 was also able to degrade and modify the surface of polycaprolactone and polyethylene terephthalate. Taken together, these features poise AnCUT3 as a potential biocatalyst for industrial applications.
角质酶是植物病原体分泌的水解酶，用于降解角质，角质是植物角质层的主要聚合物成分。角质酶的多方面功能使其可用于超出其自然功能的催化反应。为了使角质酶类别多样化并扩大其范围，我们从腐生菌黑曲霉中鉴定出了5种角质酶同源物。其中一种角质酶AnCUT3在毕赤酵母中过表达，并对其生物物理化学性质进行了表征。纯化后的重组AnCUT3的最适温度为25℃，最适pH为5，在高达50℃的温度下（孵育1小时，熔点为45.6℃）以及在较宽的pH范围内均稳定。使用对硝基苯酯底物对AnCUT3进行的动力学研究表明，其对癸酸对硝基苯酯（C10）的催化效率最高，k/K为859 mM s。尽管AnCUT3的计算分子量为27 kDa，但它表达为两种分子量分别为24 kDa和50 kDa的糖基化蛋白。聚糖谱分析检测到重组AnCUT3中存在非典型的寡甘露糖N-聚糖（≤ManGlcNAc），这表明毕赤酵母中AnCUT3的聚糖加工依赖于蛋白质。AnCUT3还能够降解和修饰聚己内酯和聚对苯二甲酸乙二酯的表面。综上所述，这些特性使AnCUT3有望成为工业应用中的潜在生物催化剂。
