甘露聚糖酶是一类将甘露聚糖降解为短链甘露寡糖及甘露糖的水解酶。为了开发耐酸耐高温的工程酶,从棘孢木霉(Trichodema asperellum)ND-1中克隆甘露聚糖酶基因TaMan5A并在毕赤酵母(Komagataella phaffii)X-33中成功表达。结果显示,该酶的蛋白质分子质量约为67 kDa。最适pH值和温度分别为4.0和65 ℃,在pH 2.0～6.0具有较强的稳定性,孵育1 h后仍具有80%的活性,且在20～65 ℃时,残留酶活力均能达到90%以上。1 mmol/L Co2+、脲和5 mmol/L SDS、Zn2+、Fe2+、Mn2+、Ni2+对TaMan5A的活力都有不同程度的激活作用。由于玉米秸秆富含纤维素与半纤维素,因此进一步确定了TaMan5A水解秸秆的最佳组合条件(玉米秸秆用量0.5 g,TaMan5A用量0.5 g,pH 4.0,水解2 d),并得到玉米秸秆用量和TaMan5A用量对玉米秸秆降解效率影响最大。该研究首次从棘孢木霉ND-1中克隆出TaMan5A基因,实验结果表明,TaMan5A是一种嗜酸、耐高温、热稳定性强的酶,具有较大的应用前景,值得进一步研究。
Mannanases are enzymes capable of degrading mannans into shorter mannooligosaccharides and mannose. In pursuit of developing acid-resistant and thermotolerant engineering enzymes, this experiment successfully cloned the mannanase gene TaMan5A from Trichoderma asperellum ND-1 and expressed it in Komagataella phaffii X-33. The recombinant TaMan5A exhibited a molecular weight of 67 kDa approximately. The optimum pH and temperature were determined to be 4.0 and 65 ℃, respectively. TaMan5A displayed robust stability within the pH range of 2.0 to 6.0, retaining 80% activity after incubation for 1 h. Furthermore, the TaMan5A retained over 90% activity between 20 ℃ and 65 ℃. 1 mmol/L Co2+, urea, and 5 mmol/L SDS, Zn2+, Fe2+, Mn2+, and Ni2+ all exhibited varying degrees of activation on the activity of TaMan5A. Due to the high content of cellulose and hemicellulose in corn stalks, the optimal conditions for the enzymatic hydrolysis of mannooligosaccharides in the stalks were further determined: corn stalk 0.5 g, TaMan5A 0.5 g, pH 4.0, and 2 d. It was found that the dosage of corn stalks and TaMan5A had the greatest impact on the degradation efficiency of corn stalks. Notably, the study innovatively cloned the TaMan5A gene from T. asperellum ND-1 for the first time. Our findings indicate that TaMan5A is an acidophilic enzyme with high thermal stability, offering promising applications and warranting further investigation.
[1] SUN Y C, ZHOU X H, ZHANG W, et al.Enhanced β-mannanase production by Bacillus licheniformis by optimizing carbon source and feeding regimes[J].Preparative Biochemistry & Biotechnology, 2022, 52(7):845-853.
[2] 雒翠梅, 王旭洁, 母军, 等.外源酸催化对木材半纤维素热降解规律的影响[J].北京林业大学学报, 2022, 44(4):147-156.
LUO C M, WANG X J, MU J, et al.Effects of exogenous acid catalysis on the thermal degradation law of wood hemicellulose[J].Journal of Beijing Forestry University, 2022, 44(4):147-156.
[3] 张瀚文, 陈正军, 张晨雨, 等.木质纤维素预处理技术研究现状与展望[J].化工矿物与加工, 2023:1-15.
ZHANG H W, CHEN Z J, ZHANG Y C, et al.Research status and prospects of wood cellulose pretreatment techniques[J].Chemical Engineering & Mineral Processing, 2023, 1-15.
[4] WEIMER P J.Degradation of cellulose and hemicellulose by ruminal microorganisms[J].Microorganisms, 2022, 10(12):2345.
[5] BEHERA S, DEV M J, SINGHAL R S.Cross-linked β-mannanase aggregates:Preparation, characterization, and application for producing partially hydrolyzed guar gum[J].Applied Biochemistry and Biotechnology, 2022, 194(5):1981-2004.
[6] LIAO H P, LI S X, ZHENG H P, et al.A new acidophilic thermostable endo-1, 4-β-mannanase from Penicillium oxalicum GZ-2:Cloning, characterization and functional expression in Pichia pastoris[J].BMC Biotechnology, 2014, 14:90.
[7] 徐智鹏, 周迪, 陈敬帮, 等.甘露聚糖酶在发酵棕榈粕中的应用及仿生消化研究[J].中国饲料, 2023(5):168-171.
XU Z P, ZHOU D, CHEN J B, et al.Application of mannanase in fermented palm meal and study on biomimetic digestion[J].China Feed, 2023(5):168-171.
[8] ANGURAL S, JASSAL S, WARMOOTA R, et al.An integrated approach for pulp biobleaching:Application of cocktail of enzymes[J].Environmental Science and Pollution Research, 2023, 30(19):57155-57163.
[9] 王烁, 季海蕊, 曹慧莹, 等.干酪乳杆菌产β-甘露聚糖酶发酵条件优化及在果汁澄清中的应用[J].中国酿造, 2022, 41(11):144-149.
WANG S, JI H R, CAO H Y, et al.Optimization of fermentation conditions and application in fruit juice clarification of β-mannanase from Lactobacillus casei[J].China Brewing, 2022, 41(11):144-149.
[10] PIAO M Y, LI A, DU H S, et al.A review of additives use in straw composting[J].Environmental Science and Pollution Research International, 2023, 30(20):57253-57270.
[11] 杜亚彬, 赵红梅, 张学磊, 等.基于WRF-CMAQ评估秸秆禁烧对区域空气质量的影响[J].中国环境科学, 2022, 42(12):5578-5588.
DU Y B, ZHAO H M, ZHANG X L, et al.Evaluation of the impacts of straw burning ban on air quality based on WRF-CMAQ[J].China Environmental Science, 2022, 42(12):5578-5588.
[12] 蒋斌, 陈多宏, 张涛, 等.华南水稻秸秆焚烧期碳质气溶胶组分特征及源贡献评估[J].生态环境学报, 2022, 31(12):2358-2366.
JIANG B, CHEN D H, ZHANG T, et al.Characteristics and sources of carbonaceous aerosols during the crop straw burning seasons in southern China[J].Ecology and Environmental Sciences, 2022, 31(12):2358-2366.
[13] 刘慧琳, 莫招育, 覃纹, 等.广西秸秆露天焚烧源排放清单及时空分布研究[J].环境污染与防治, 2022, 44(5):631-638.
LIU H L, MO Z Y, QIN W, et al.Emission inventory and the spatio-temporal distribution of pollutant from open field straw burning in Guangxi[J].Environmental Pollution & Control, 2022, 44(5):631-638.
[14] ÖZÇELIK A T, Y¹LMAZ S, INAN M.Pichia pastoris promoters[J].Methods in Molecular Biology, 2019, 1923:97-112.
[15] ZHAO W, ZHENG J, ZHOU H B.A thermotolerant and cold-active mannan endo-1, 4-β-mannosidase from Aspergillus niger CBS 513.88:Constitutive overexpression and high-density fermentation in Pichia pastoris[J].Bioresource Technology, 2011, 102(16):7538-7547.
[16] ZHENG F Z, SONG L N, BASIT A, et al.An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose[J].Carbohydrate Polymers, 2020, 237:116121.
[17] 和子涵, 穆广亚, 贾一然, 等.椰果内生枯草芽孢杆菌YZ-21甘露聚糖酶基因的克隆、表达及酶学性质研究[J].饲料工业, 2023, 44(15):71-78.
HE Z H, MU G Y, JIA Y R, et al.Cloning, expression and enzymatic properties of mannanase gene from Bacillus subtilis YZ-21 in coconut[J].Feed Industry, 2023, 44(15):71-78.
[18] 田庚, 高伟强, 陈晓波, 等.地衣芽孢杆菌KD-1β-甘露聚糖酶定点突变提高酶活性及稳定性[J].生物技术通报, 2021, 37(10):100-109.
TIAN G, GAO W Q, CHEN X B, et al.Directed mutagenesis of β-mannanase gene from Bacillus licheniformis KD-1 for improving enzyme activity and stability[J].Biotechnology Bulletin, 2021, 37(10):100-109.
[19] 邵丽杰, 寇巍, 曹焱鑫, 等.纤维素酶、半纤维素酶降解膨化玉米秸秆工艺优化[J].环境工程学报, 2014, 8(10):4373-4378.
SHAO L J, KOU W, CAO Y X, et al.Optimization of degradation of expanded corn straw by cellulase and hemicellulase[J].Chinese Journal of Environmental Engineering, 2014, 8(10):4373-4378.
[20] 李珊珊, 刘德海, 刁文涛, 等.黑曲霉β-甘露聚糖酶ManA耐热突变体的筛选[J].中国酿造, 2022, 41(3):62-68.
LI S S, LIU D H, DIAO W T, et al.Screening of thermostable mutants of β-mannanase ManA from Aspergillus niger[J].China Brewing, 2022, 41(3):62-68.
