甲壳素,β-1,4-连接的N-乙酰葡糖胺(GlcNAc)的聚合物,可以通过各种几丁质酶降解为有价值的寡糖。在这项研究中,希里卡纳希瓦氏菌JW44的基因组,显示出显著的几丁质分解活性,进行了调查,以了解其甲壳素降解潜力。然后克隆了该菌株的几丁质酶基因SkChi65,表达,并纯化以表征其酶学性质和底物水解。基因组分析表明,在JW44中与几丁质利用相关的14个基因中,有6个属于糖苷水解酶(GH)家族,因为它们具有几丁质结合和催化的功能域。重组几丁质酶SkChi65,由1129个氨基酸组成,被鉴定为GH18家族的成员,并拥有两个具有典型基序[A/N]KWWT[N/S/Q]的几丁质结合域和一个具有基序DxxDxE的催化域,SxGG,YxR,和[E/D]xx[V/I]。SkChi65在37°C下最佳表达为139.95kDa的活性蛋白,其中1.0mM异丙基-β-d-硫代吡喃半乳糖苷诱导6小时。纯化的SkChi65在30-50°C和pH5.5-8.0的范围内显示出高稳定性,在40°C和pH7.0下最佳。动力学参数Km,Vmax,SkChi65对胶体几丁质的kcat为27.2μM,分别为299.2μMs-1和10,203s-1。除了胶体甲壳素,SkChi65对乙二醇壳聚糖和结晶几丁质显示出高活性。薄层色谱分析后,主要产品有N,N'-二乙酰几丁二糖,和以(GlcNAc)2-6作为底物的GlcNAc。总的来说,SkChi65可以表现出对不同底物的外切和内切几丁质酶活性,菌株JW44具有很高的工业应用潜力,具有优良的甲壳素生物转化能力。
Chitin, a polymer of β-1,4-linked N-acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of Shewanella khirikhana JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene SkChi65 from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 °C with 1.0 mM isopropyl-β-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30-50 °C and pH 5.5-8.0 with optima at 40 °C and pH 7.0. The kinetic parameters Km, Vmax, and kcat of SkChi65 towards colloidal chitin were 27.2 μM, 299.2 μMs-1, and 10,203 s-1, respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were N,N\'-diacetylchitobiose, and GlcNAc with (GlcNAc)2-6 used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion.