产纤维素酶细菌的分离、鉴定与酶学性质研究
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摘要
目的筛选并鉴定纤维素酶高产菌株,初步研究其所产纤维素酶的酶学性质,为纤维素资源的综合利用提供菌种资源。方法通过刚果红染色和酶活测定,从广州凉茶中药渣堆肥和黑龙江玉米地土壤中筛选出纤维素酶高产菌株,进行基于16S rDNA分析的分子鉴定,同时采用单因素试验方法确定菌株最佳培养时间,判断温度、pH值、金属离子、有机溶剂和盐浓度对所产纤维素酶活性及稳定性的影响。结果筛选获得3株纤维素酶高产菌株,其中Z-1鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus),Z-2和H-3鉴定为枯草芽孢杆菌(B.subtilis),其所产CMCase酶学活性分别为977.92 U/mL、1 089.79 U/mL和1 538.44 U/mL。所产纤维素酶最适反应温度为50℃,最适pH值范围为pH4～5,该纤维素酶可被Ag~+、Co~(2+)和Mg~(2+)离子激活,也可被一定浓度的Tris-100和甲醇激活。经浓度为1.5 mol/L的NaCl处理4 h后,其酶活力仍剩余56.34%以上。结论 B.methylotrophicus Z-1,B.subtilis Z-2以及B.subtilis H-3为纤维素酶高产菌株,其所产纤维素酶耐热、耐酸、耐盐,并可被部分金属离子和有机物质所激活,具有潜在的开发价值。
Objective To provide strain resources for the comprehensive utilization of cellulose resources by screening of cellulase-producing strains and studying of the enzymatic properties of cellulase. Methods CMC-Congo red staining and enzyme activity assay were used to screen the cellulase-producing strains from compost of traditional Chinese herbal tea residue in Guangzhou and soil of cornfields in Heilongjiang province. 16 S rDNA sequence analysis was used for gene identification of the strains. Single factor tests were used to determine influence of temperature,pH,metal ions,organic solvents and salt concentration on the cellulase activity and stability. Results 3 strains of Bacillus Z-1,Z-2 and H-3 were screened out,and their cellulase activities were 977.92 U/mL,1 089.79 U/mL,and 1 538.44 U/mL,respectively. The optimal reaction temperature was 50 ℃,and the optimal pH was pH 4.0～5.0. The cellulases were activated by Ag~+,Co~(2+),Mg~(2+),Tris-100,and methanol in a certain concentration range. The relative cellulase activity still reached to 56.34% under 1.5 mol/L sodium chloride solution treatment for 4 h. Conclusion The cellulases produced by B. methylotrophicus Z-1,B. subtilis Z-2,and B. subtilis H-3 are resistant to heat,acid and salt,and can be activated by some metal ions and organic substances,which exhibit the potential development value.
Objective To provide strain resources for the comprehensive utilization of cellulose resources by screening of cellulase-producing strains and studying of the enzymatic properties of cellulase. Methods CMC-Congo red staining and enzyme activity assay were used to screen the cellulase-producing strains from compost of traditional Chinese herbal tea residue in Guangzhou and soil of cornfields in Heilongjiang province. 16 S rDNA sequence analysis was used for gene identification of the strains. Single factor tests were used to determine influence of temperature,pH,metal ions,organic solvents and salt concentration on the cellulase activity and stability. Results 3 strains of Bacillus Z-1,Z-2 and H-3 were screened out,and their cellulase activities were 977.92 U/mL,1 089.79 U/mL,and 1 538.44 U/mL,respectively. The optimal reaction temperature was 50 ℃,and the optimal pH was pH 4.0～5.0. The cellulases were activated by Ag~+,Co~(2+),Mg~(2+),Tris-100,and methanol in a certain concentration range. The relative cellulase activity still reached to 56.34% under 1.5 mol/L sodium chloride solution treatment for 4 h. Conclusion The cellulases produced by B. methylotrophicus Z-1,B. subtilis Z-2,and B. subtilis H-3 are resistant to heat,acid and salt,and can be activated by some metal ions and organic substances,which exhibit the potential development value.
引文
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[6] 吴文韬,鞠美庭,刘金鹏,等. 一株纤维素降解菌的分离、鉴定及对玉米秸秆的降解特性[J]. 微生物学通报,2013,40(4):712-719.
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[10] 曾思泉,凌娟,林丽云,等. 1株红树林来源枝孢属真菌的分离鉴定及纤维素酶性质分析[J].微生物学杂志,2018,38(2):37-42.
[11] 李华,高丽. β-葡萄糖苷酶活性测定方法的研究进展[J]. 食品与生物技术学报,2007,26(2):107-114.
[12] 穆春雷,武晓森,李术娜,等. 低温产纤维素酶菌株的筛选、鉴定及纤维素酶学性质[J]. 微生物学通报,2013,40(7):1193-1201.
[13] 黄春凯,左小明,王红蕾,等.一株产纤维素酶菌株的分离、鉴定及产酶特性[J]. 微生物学通报,2015,42(4):646-653.
[14] 马旭光,张宗舟,蔺海明,等. 黑曲霉高产纤维素酶活突变株的筛选[J]. 中国酿造,2008,186:61-63.
[15] 孟建宇,冯福应,刘向红,等. 1株纤维素降解菌的筛选及产酶条件研究[J]. 微生物学杂志,2011,31(4):47-51.
[16] 周东兴,王广栋,邬欣慧,等.腐熟堆肥中纤维素降解菌筛选鉴定及酶学特性研究[J].东北农业大学学报,2018,49(5):60～68.
[17] 王春燕,刘四新,高阳,等. 红树林土壤纤维素酶产生菌筛选及产酶条件研究[J]. 食品与生物技术学报,2011,30(1):144-150.
[18] 张璐璐,卫旭彪,斯大勇,等. 产纤维素酶芽孢杆菌的筛选及鉴定[J]. 饲料工业,2016,37(18):45-50.
[19] 余祖华,丁轲,侯奎,等. 产纤维素酶地衣芽孢杆菌的诱变选育及其产酶条件优化[J]. 中国畜牧兽医,2016,43(04):1006-1011.
[20] YIN Lijung,LIN Hsinhung,JIANG Shanntzong. Bioproperties of potent nattokinase from Bacillus subtilis YJ1[J]. J Agric Food Chem,2010,58(9):5737-5742.
[21] 吴自祥,万学瑞,马亚茹,等. 产纤维素酶细菌的分离筛选与鉴定[J]. 草原与草坪,2017,37(2):7-11.
[2] 杨明霞,陈莉娜,刘雪平. 再生纤维素纤维的开发现状及发展趋势[J]. 防治科学与工程,2016,33(3):169-173.
[3] 邱春生,孙力平. 木质纤维素类农业废弃物生物转化资源化研究进展[J]. 安徽农业科学,2014,42(18):5928-5932.
[4] BAYER E A,LAMED R,HIMMEL M E. The potential of cellulases and cellulosomes for cellulosic waste management[J]. Curr Opin Biotechnol,2007,18(3):237-245.
[5] ANNAMALAI N,RAJESWARI M V,ELAYARAJA S,et al. Thermostable,haloalkaline cellulase from Bacillus halodurans,CAS 1 by conversion of lignocellulosic wastes[J]. Carbohydr Polym,2013,94(1):409-415.
[6] 吴文韬,鞠美庭,刘金鹏,等. 一株纤维素降解菌的分离、鉴定及对玉米秸秆的降解特性[J]. 微生物学通报,2013,40(4):712-719.
[7] 张景强,林鹿,孙勇,等. 纤维素结构与解结晶的研究进展[J].林产化学与工业,2008,28(06):109-114.
[8] 严鸿林,唐彩琰,SANGRILA S,等. 细菌产生的纤维素酶[J]. 国外畜牧学(猪与禽),2018,38(9):51-54.
[9] 李保深,高大文,张博. 一株产纤维素酶真菌的筛选、鉴定及降解效果[J]. 东北林业大学学报,2011,39(7):134-137.
[10] 曾思泉,凌娟,林丽云,等. 1株红树林来源枝孢属真菌的分离鉴定及纤维素酶性质分析[J].微生物学杂志,2018,38(2):37-42.
[11] 李华,高丽. β-葡萄糖苷酶活性测定方法的研究进展[J]. 食品与生物技术学报,2007,26(2):107-114.
[12] 穆春雷,武晓森,李术娜,等. 低温产纤维素酶菌株的筛选、鉴定及纤维素酶学性质[J]. 微生物学通报,2013,40(7):1193-1201.
[13] 黄春凯,左小明,王红蕾,等.一株产纤维素酶菌株的分离、鉴定及产酶特性[J]. 微生物学通报,2015,42(4):646-653.
[14] 马旭光,张宗舟,蔺海明,等. 黑曲霉高产纤维素酶活突变株的筛选[J]. 中国酿造,2008,186:61-63.
[15] 孟建宇,冯福应,刘向红,等. 1株纤维素降解菌的筛选及产酶条件研究[J]. 微生物学杂志,2011,31(4):47-51.
[16] 周东兴,王广栋,邬欣慧,等.腐熟堆肥中纤维素降解菌筛选鉴定及酶学特性研究[J].东北农业大学学报,2018,49(5):60～68.
[17] 王春燕,刘四新,高阳,等. 红树林土壤纤维素酶产生菌筛选及产酶条件研究[J]. 食品与生物技术学报,2011,30(1):144-150.
[18] 张璐璐,卫旭彪,斯大勇,等. 产纤维素酶芽孢杆菌的筛选及鉴定[J]. 饲料工业,2016,37(18):45-50.
[19] 余祖华,丁轲,侯奎,等. 产纤维素酶地衣芽孢杆菌的诱变选育及其产酶条件优化[J]. 中国畜牧兽医,2016,43(04):1006-1011.
[20] YIN Lijung,LIN Hsinhung,JIANG Shanntzong. Bioproperties of potent nattokinase from Bacillus subtilis YJ1[J]. J Agric Food Chem,2010,58(9):5737-5742.
[21] 吴自祥,万学瑞,马亚茹,等. 产纤维素酶细菌的分离筛选与鉴定[J]. 草原与草坪,2017,37(2):7-11.
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