嗜热毛壳菌甘露聚糖酶Ct Man26的性质及其在甘露寡糖制备中的应用

Characterization of Mannanase CtMan26 from Chaetomium thermophilum and Its Application in Mannooligosaccharide Preparation

旨在实现嗜热毛壳菌(Chaetomium thermophilum)中的甘露聚糖酶Ct Man26在毕赤酵母(Pichia pastoris)中高效表达,探究重组酶Ct Man26的酶学性质及在甘露寡糖(mannooligosaccharides,MOS)制备中的潜力。甘露聚糖酶Ct Man26共含448个氨基酸,由碳水化合物结合模块CBM35、Linker及GH26家族的催化域模块组成,催化结构域上的Asp-395、Leu-396和CBM35辅助模块上的Leu-100受到多个疏水作用力,并可与Linker上氨基酸形成多个氢键。重组酶Ct Man26经P.pastoris表达后,存在N-糖基化修饰,蛋白表观分子质量约为55 kDa。重组酶Ct Man26的最适温度为55℃,最适pH值为5.0,50、60℃处理1 h后酶活力仍保持在90%以上,在pH 4.0~12.0范围表现出良好的稳定性。以魔芋甘露聚糖为底物时,重组酶Ct Man26的比活力为23.15 U/mg,米氏常数为4.75 mg/mL,最大反应速度为27.17μmol/(min·mg)。重组酶Ct Man26制备的MOS主要为甘露糖(14.2 mg/L)、甘露二糖(20.1 mg/L)、甘露三糖(6.1 mg/L)和甘露四糖(2.5 mg/L)。进一步研究发现,制备的MOS表现出较好的抗氧化能力,当样品质量浓度为2.4 mg/mL时,对1,1-二苯基-2-三硝基苯肼自由基清除率、2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基清除率、羟自由基清除率以及超氧阴离子自由基清除率分别为(90.80±0.65)%、(80.7±1.07)%、(69.10±1.10)%和(76.7±3.14)%。同时,MOS具有明显的益生元特性,能够抑制大肠杆菌(Escherichia coli)的生长,促进有益菌戊糖片球菌(Pediococcus pentosaceus)和植物乳杆菌(Lactobacillu plantarum)的生长,且随着MOS添加量增加及培养时间的延长越发明显。综上所述,重组甘露聚糖酶Ct Man26具有较好的酶学特性,在MOS的制备中具有潜在的应用价值。

The aim of this study was to achieve efficient expression of mannanase CtMan26 from Chaetomium thermophilum in Pichia pastoris and to investigate the enzymatic properties of CtMan26 and its potential in the preparation of mannooligosaccharides(MOS).The mannanase CtMan26 contained a total of 448 amino acids and consisted of the family 35 carbohydrate-binding module(CBM35),Linker and the catalytic domain module of the GH26 family.Asp-395 and Leu-396 in the catalytic domain and Leu-100 in the auxiliary module of CBM35 were subjected to multiple hydrophobic forces and could form multiple hydrogen bonds with amino acids in the Linker.Recombinant enzyme CtMan26 was expressed with N-glycosylation modification.CtMan26 showed an apparent molecular mass of about 55 kDa.The optimum temperature of the recombinant enzyme was 55℃,and the optimum pH was 5.0.The enzymatic activity was maintained at more than 90%of its original value after being incubated at 50 and 60℃for 1 hour,and it showed good stability at pH ranging from 4.0 to 12.0.The specific activity of CtMan26 was 23.15 U/mg,and its Km and Vmax values were 4.75 mg/mL and 27.17μmol/(min·mg),respectively,when konjac glucomannan was used as the substrate.MOS prepared with the recombinant enzyme mainly consisted of mannose(14.2 mg/L),mannose disaccharides(20.1 mg/L),mannose trisaccharides(6.1 mg/L)and mannose tetrasaccharides(2.5 mg/L).The MOS exhibited good antioxidant capacity,which scavenged(90.80±0.65)%of 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical,(80.7±1.07)%of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)cation radical,(69.10±1.10)%of hydroxyl radical and(76.7±3.14)%of superoxide anion radical at 2.4 mg/mL concentration.Meanwhile,the MOS had obvious prebiotic properties,which could inhibit the growth of Escherichia coli and promote the growth of the beneficial bacteria Pediococcus pentosaceus and Lactobacillu plantarum the effect being more pronounced with increasing concentration and incubation time.In conclusion,the recombinant mannanase CtMan26 has good enzyme properties and therefore has potential application in the preparation of MOS.