内切β-1,4-葡聚糖酶是参与纤维素材料分解的关键糖苷水解酶(GH)。在这项研究中,为了发现一种新型的冷适应β-1,4-D-葡聚糖降解酶,编码来自南极地衣(Cladoniaborealis)相关细菌LichenicolacladonioiPAMC26568的细胞外β-1,4-葡聚糖酶(GluL)的基因,在大肠杆菌BL21中鉴定并重组表达。GluL基因(1044-bp)编码由单个催化GH8结构域组成的非模块化多肽,与来自高松葡糖醋杆菌(WP_182950054)的未表征蛋白具有55%的最高序列同一性。重组内切β-1,4-葡聚糖酶(rGluL:38.0kDa)在pH4.0和45°C下最有效地降解羧甲基纤维素钠(CMC),甚至在3°C时也显示出其最大降解活性的约23%。在浓度为0.1至0.5M的1mMMn2或NaCl存在下,rGluL的生物催化活性显着提高>1.3倍。而该酶被1mMHg2和Fe2以及5mMN-溴代琥珀酰亚胺和0.5%十二烷基硫酸钠大大下调。rGluL是一种真正的内切β-1,4-葡聚糖酶,可以优先分解由3至6个D-葡萄糖组成的D-纤维寡糖,CMC,和大麦β-葡聚糖,没有其他额外的糖苷水解酶活性。rGluL对大麦β-葡聚糖的比活性(15.1Umg-1)和kcat/Km值(6.35mg-1s-1mL)约高1.8倍和2.2倍,分别,与其对CMC的比活性(8.3Umg-1)和kcat/Km值(2.83mg-1s-1mL)相比。CMC的酶解,D-细胞四糖,D-纤维六糖主要产生D-纤维二糖,伴随着D-葡萄糖,D-细胞三糖,和D-细胞四糖。然而,rGluL对D-纤维戊糖的裂解导致仅产生D-纤维二糖和D-纤维三糖。本研究的发现暗示rGluL是一部小说,酸性,和冷适应的GH8内切-β-1,4-葡聚糖酶具有高比活性,可以在包括纺织和食品工艺在内的低温工艺中用作有前途的候选物。
Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic
GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted
GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.