壳聚糖酶(EC 3.2.1.132,chitosanase,Csn)能够催化壳聚糖中β-1,4-糖苷键断裂生成功能性壳寡糖。该研究从茂原链霉菌(Streptomyces mobaraensis)DSM40587中鉴定了一种新型Csn基因smCsn46(GenBank 登录号:UBI35095.1),并将其在谷氨酰胺转氨酶缺陷型S.mobaraensis菌株(smY2022Δtg)中表达。smCsn46基因包含一个882 bp的开放阅读框,编码分子质量为32 kDa的蛋白质。序列比对显示,smCsn46与来自Streptomyces sp.N174、Streptomyces coelicolor A3(2)、Bacillus mojavensis SY1和Bacillus subtilis 168的GH46家族Csn的氨基酸序列一致性分别为73%、73.9%、49.3%和48.4%。利用谷氨酰胺转氨酶的启动子(PsmY2022)、信号肽和终止子在smY2022Δtg中表达smCsn46,胞外酶活力达到5.52 U/mL。酶学性质分析表明,smCsn46的最适温度和最适pH值分别为50 ℃和5.5。该酶在50 ℃下表现出良好的热稳定性和pH稳定性。值得注意的是,smCsn46对脱乙酰度(degree of deacetylation,DDA)在85%~95%的壳聚糖均具有催化活性,且活性随着DDA的降低而增加。催化95% DDA壳聚糖时,smCsn46主要产物为壳四糖。该研究不仅丰富了Csn的资源,还为食品级Csn的工业化生产提供了新的思路和方法。
Chitosanase (EC 3.2.1.132, Csn) catalyzes the hydrolysis of β-1,4-glycosidic bonds in chitosan to generate functional chitooligosaccharides.Here, a novel Csn gene smCsn46 (GenBank:UBI35095.1) from Streptomyces mobaraensis DSM40587 was heterologously expressed in a transglutaminase-deficient S.mobaraensis strain (smY2022Δtg).The 882 bp ORF encodes a 32 kDa protein, sharing 73.0%, 73.9%, 49.3%, and 48.4% amino acid sequence similarity with GH46 family Csn from Streptomyces sp.N174, Streptomyces coelicolor A3(2), Bacillus mojavensis SY1, and Bacillus subtilis 168, respectively.Using the endogenous transglutaminase promoter (PsmY2022) and secretion elements, extracellular activity of smCsn46 reached 5.52 U/mL.The enzyme exhibited optimal activity at 50 ℃ and pH 5.5 with stability under these conditions.Notably, the enzyme displayed substrate specificity toward chitosan substrates with 85%-95% degree of deacetylation (DDA), showing enhanced catalytic efficiency as DDA decreased.In the case of the catalysis of 95% DDA chitosan, the main product was chitotetrose.This work expands chitosanase genetic resources and provides a food-grade Streptomyces expression platform for industrial chitooligosaccharide production.
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