一株产纤维素酶嗜盐真菌的分离、鉴定及发酵条件优化
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摘要
纤维素酶是生物燃料产业的关键酶系。本文通过刚果红染色法从腌制一年的诺邓火腿上分离到一株具有纤维素酶活性的嗜盐真菌YFCC2018SY。以形态学结合分子系统学手段对其进行鉴定,用胞外酶活测定法探索其产酶规律,并通过响应面法优化其产酶条件。结果表明该菌株属于球孢枝孢菌,且能分泌滤纸酶、内切酶和β‐葡萄糖苷酶3种嗜盐纤维素酶。响应面分析得到最优发酵条件为:NaCl含量88.58g/L、装瓶量51.21mL、起始pH 7.72。通过优化,纤维素酶活力由113.3U/mL提高到302.8U/mL,提高了167%。上述结果可以为嗜盐纤维素酶开发利用提供参考。
Cellulases are the key enzymes used in the biofuel industry. A halophilic fungus cellulase-produced,designated as YFCC2018 SY, was isolated from Nuodeng ham salted for a year by using the Congo red dye test.Identification of the strain was based on morphology and phylogenetic analysis. Extracellular enzyme activity was monitored to study the pattern of cellulase production during liquid-state fermentation by the strain. Optimum conditions of cellulase production were obtained by the response surface methodology. Strain YFCC2018 SY was identified as Cladosporium sphaerospermum, and the strain could secrete three halophilic cellulases, FPase,CMCase and β-glucosidase. The optimal conditions of the maximal activity were indicated as follows: NaCl content, 88.58 g/L; liquid volume, 51.21 mL; initial pH value, 7.72. Under the conditions, cellulase activity was increased by 167%, ranging from 113.3 to 302.8 U/mL. Our results could provide a reference for development and utilization of halophilic cellulase.
Cellulases are the key enzymes used in the biofuel industry. A halophilic fungus cellulase-produced,designated as YFCC2018 SY, was isolated from Nuodeng ham salted for a year by using the Congo red dye test.Identification of the strain was based on morphology and phylogenetic analysis. Extracellular enzyme activity was monitored to study the pattern of cellulase production during liquid-state fermentation by the strain. Optimum conditions of cellulase production were obtained by the response surface methodology. Strain YFCC2018 SY was identified as Cladosporium sphaerospermum, and the strain could secrete three halophilic cellulases, FPase,CMCase and β-glucosidase. The optimal conditions of the maximal activity were indicated as follows: NaCl content, 88.58 g/L; liquid volume, 51.21 mL; initial pH value, 7.72. Under the conditions, cellulase activity was increased by 167%, ranging from 113.3 to 302.8 U/mL. Our results could provide a reference for development and utilization of halophilic cellulase.
引文
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Du JH,Huan P,Li LZ,An SQ,Jiang ZB,Ma XD,2018.Isolation and study of an extremely halophilic fungus.Journal of Hubei University(Natural Science),40(2):189-194(in Chinese)
Gong MF,Guan QL,Ou XF,Chen XM,Xie ZL,2017.Screening and production of cellulase of bacteria isolated from intestinal tract of Pomacea canaliculata.Journal of Biobased Materials&Bioenergy,11(4):278-281
Gunde-Cimerman N,Ramos J,Plemenita?A,2009.Halotolerant and halophilic fungi.Mycological Research,113(11):1231-1241
Gunny AAN,Arbain D,Jamal P,Gumba RE,2015.Improvement of halophilic cellulase production from locally isolated fungal strain.Saudi Journal of Biological Sciences,22(4):476-483
Guo J,2015.Study on the cellulose degradation and identification of three strains of halotolerant and halophilic bacteria isolated from Qinghai Lake.Chinese Qinghai Journal of Animal&Veterinary Sciences,45(5):23-26(in Chinese)
Kong FJ,Wang XJ,2017.Halophilic microoganisms in salt lakes on Tibet Plateau and their potential application.Science&Technology Review,35(12):27-31(in Chinese)
Korany AH,Ali AE,Essam TM,Megahed SA,2017.Optimization of cellulase production by Halobacillus sp.QLS 31 isolated from Lake Qarun,Egypt.Applied Biochemistry&Biotechnology,183(1):189-199
Liu JF,Xue DS,He KK,Chen HY,Yao SJ,2012.Screening of a cellulase producing halotolerant strain marine Aspergillu sp.and its fermentation conditions.Journal of Food Science&Biotechnology,31(7):711-718(in Chinese)
Liu S,Fan BQ,2012.Screening of a straw cellulose-degrading fungi QSH3-3 and study on its characteristics of cellulase production.Plant Nutrition and Fertilizer Science,18(1):218-226(in Chinese)
Mosier NS,Hall P,Ladisch CM,Ladisch MR,1999.Reaction kinetics,molecular action,and mechanisms of cellulolytic proteins.Advances in Biochemical Engineering/Biotechnology,65:23-40
Qin WN,Zhao K,Guo LZ,Lu WD,2016.Identification,process optimization of cellulase production of halophilic actinomycete DN-A4,and characterization of the enzymatic properties of its cellulase.Journal of Qingdao Agricultural University(Natural Science),33(1):27-34(in Chinese)
Wang Y,Han YF,Liang ZQ,2016.Screening of superior lignin-degrading thermotolerant fungal strains from Wuyi Mountains and assessment of their degradation effects on straw lignin.Mycosystema,35(10):1169-1177(in Chinese)
Wang Y,Han YF,Liang ZQ,2017a.Rice straw degradation and enzyme production of two Taifanglania strains.Mycosystema,36(5):598-603(in Chinese)
Wang Y,Han YF,Yu H,Liang ZQ,2017b.Characterization and optimal fermentation conditions of cold-adapted cellulase produced by Scopulariopsis hibernica.Mycosystema,36(8):1132-1140(in Chinese)
Wang Y,Wang YR,Luo Y,Chen WH,Han YF,Liang ZQ,2014.Acold-adapted cellulase-producing strain of Scopulariopsis hibernica.Journal of Mountain Agriculture and Biology,33(3):50-54(in Chinese)
White TJ,Bruns T,Lee S,Taylor JW,1990.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.In:Innis MA,Gelfand DH,Sninsky JJ,White TJ(eds.)PCR protocols:a guide to methods and applications.Academic Press,New York.315-322
Wu YF,Gu AX,Wang HK,2018.Isolation and identification of halophilic and halotolerant fungi from roots of Suaeda plants.Acta Agriculturae Zhejiangensis,30(4):649-655(in Chinese)
Yu HY,Li X,2015.Alkali-stable cellulase from a halophilic isolate,Gracilibacillus sp.SK1 and its application in lignocellulosic saccharification for ethanol production.Biomass&Bioenergy,81:19-25
Zalar P,de Hoog GS,Schroers HJ,Crous PW,Groenewald JZ,Gunde-Cimerman N,2007.Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum,with descriptions of seven new species from hypersaline environments.Studies in Mycology,58:157-183
Zhang B,Liu KH,Chen WQ,Yang FY,Ding XW,Deng BW,2016.Identification and fermentation conditions of a cellulase-producing fungus from Huama Salt Lake in northern Shaanxi.Journal of Northwest A&F University(Natural Science Edition),44(4):149-156(in Chinese)
Zhang HD,Zhang GM,Yao CX,Junaid M,Lu ZH,Zhang HJ,Ma YH,2015.Structural insight of a trimodular halophilic cellulase with a family 46 carbohydrate-binding module.PLoS One,10(11):e0142107
杜剑晖,还萍,李岭卓,安爽晴,江正兵,马向东,2018.一株极端嗜盐真菌的筛选及初步研究.湖北大学学报(自然科学版),40(2):189-194
郭璟,2015.分离自青海湖的三株耐盐菌及嗜盐菌的鉴定及纤维素降解能力的研究.青海畜牧兽医杂志,45(5):23-26
孔凡晶,王现洁,2017.青藏高原盐湖嗜盐微生物资源及应用前景.科技导报,35(12):27-31
刘杰凤,薛栋升,何可可,陈慧英,姚善泾,2012.一株耐盐纤维素酶海洋曲霉的筛选及产酶条件研究.食品与生物技术学报,31(7):711-718
刘爽,范丙全,2012.秸秆纤维素降解真菌QSH3-3的筛选及其特性研究.植物营养与肥料学报,18(1):218-226
秦雯娜,赵坤,郭立忠,卢伟东,2016.1株产纤维素酶嗜盐放线菌的鉴定、发酵优化及酶学性质的研究.青岛农业大学学报(自然科学版),33(1):27-34
王垚,韩燕峰,梁宗琦,2016.采自武夷山高温木质素降解菌优良菌株筛选及其木质素降解效果测定.菌物学报,35(10):1169-1177
王垚,韩燕峰,梁宗琦,2017a.两株戴氏霉对水稻秸秆的降解及产酶研究.菌物学报,36(5):598-603
王垚,韩燕峰,虞泓,梁宗琦,2017b.爱尔兰帚霉产低温纤维素酶的酶学性质和发酵工艺.菌物学报,36(8):1132-1140
王垚,王玉荣,罗韵,陈万浩,韩燕峰,梁宗琦,2014.一株在低温条件下产纤维素酶的草生帚霉.山地农业生物学报,33(3):50-54
吴曰福,顾爱星,王洪凯,2018.碱蓬根系嗜盐耐盐真菌的分离与鉴定.浙江农业学报,30(4):649-655
张波,刘开辉,陈文强,杨方玉,丁小维,邓百万,2016.陕北花马盐湖1株产纤维素酶真菌的鉴定及产酶条件研究.西北农林科技大学学报(自然科学版),44(4):149-156
Du JH,Huan P,Li LZ,An SQ,Jiang ZB,Ma XD,2018.Isolation and study of an extremely halophilic fungus.Journal of Hubei University(Natural Science),40(2):189-194(in Chinese)
Gong MF,Guan QL,Ou XF,Chen XM,Xie ZL,2017.Screening and production of cellulase of bacteria isolated from intestinal tract of Pomacea canaliculata.Journal of Biobased Materials&Bioenergy,11(4):278-281
Gunde-Cimerman N,Ramos J,Plemenita?A,2009.Halotolerant and halophilic fungi.Mycological Research,113(11):1231-1241
Gunny AAN,Arbain D,Jamal P,Gumba RE,2015.Improvement of halophilic cellulase production from locally isolated fungal strain.Saudi Journal of Biological Sciences,22(4):476-483
Guo J,2015.Study on the cellulose degradation and identification of three strains of halotolerant and halophilic bacteria isolated from Qinghai Lake.Chinese Qinghai Journal of Animal&Veterinary Sciences,45(5):23-26(in Chinese)
Kong FJ,Wang XJ,2017.Halophilic microoganisms in salt lakes on Tibet Plateau and their potential application.Science&Technology Review,35(12):27-31(in Chinese)
Korany AH,Ali AE,Essam TM,Megahed SA,2017.Optimization of cellulase production by Halobacillus sp.QLS 31 isolated from Lake Qarun,Egypt.Applied Biochemistry&Biotechnology,183(1):189-199
Liu JF,Xue DS,He KK,Chen HY,Yao SJ,2012.Screening of a cellulase producing halotolerant strain marine Aspergillu sp.and its fermentation conditions.Journal of Food Science&Biotechnology,31(7):711-718(in Chinese)
Liu S,Fan BQ,2012.Screening of a straw cellulose-degrading fungi QSH3-3 and study on its characteristics of cellulase production.Plant Nutrition and Fertilizer Science,18(1):218-226(in Chinese)
Mosier NS,Hall P,Ladisch CM,Ladisch MR,1999.Reaction kinetics,molecular action,and mechanisms of cellulolytic proteins.Advances in Biochemical Engineering/Biotechnology,65:23-40
Qin WN,Zhao K,Guo LZ,Lu WD,2016.Identification,process optimization of cellulase production of halophilic actinomycete DN-A4,and characterization of the enzymatic properties of its cellulase.Journal of Qingdao Agricultural University(Natural Science),33(1):27-34(in Chinese)
Wang Y,Han YF,Liang ZQ,2016.Screening of superior lignin-degrading thermotolerant fungal strains from Wuyi Mountains and assessment of their degradation effects on straw lignin.Mycosystema,35(10):1169-1177(in Chinese)
Wang Y,Han YF,Liang ZQ,2017a.Rice straw degradation and enzyme production of two Taifanglania strains.Mycosystema,36(5):598-603(in Chinese)
Wang Y,Han YF,Yu H,Liang ZQ,2017b.Characterization and optimal fermentation conditions of cold-adapted cellulase produced by Scopulariopsis hibernica.Mycosystema,36(8):1132-1140(in Chinese)
Wang Y,Wang YR,Luo Y,Chen WH,Han YF,Liang ZQ,2014.Acold-adapted cellulase-producing strain of Scopulariopsis hibernica.Journal of Mountain Agriculture and Biology,33(3):50-54(in Chinese)
White TJ,Bruns T,Lee S,Taylor JW,1990.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.In:Innis MA,Gelfand DH,Sninsky JJ,White TJ(eds.)PCR protocols:a guide to methods and applications.Academic Press,New York.315-322
Wu YF,Gu AX,Wang HK,2018.Isolation and identification of halophilic and halotolerant fungi from roots of Suaeda plants.Acta Agriculturae Zhejiangensis,30(4):649-655(in Chinese)
Yu HY,Li X,2015.Alkali-stable cellulase from a halophilic isolate,Gracilibacillus sp.SK1 and its application in lignocellulosic saccharification for ethanol production.Biomass&Bioenergy,81:19-25
Zalar P,de Hoog GS,Schroers HJ,Crous PW,Groenewald JZ,Gunde-Cimerman N,2007.Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum,with descriptions of seven new species from hypersaline environments.Studies in Mycology,58:157-183
Zhang B,Liu KH,Chen WQ,Yang FY,Ding XW,Deng BW,2016.Identification and fermentation conditions of a cellulase-producing fungus from Huama Salt Lake in northern Shaanxi.Journal of Northwest A&F University(Natural Science Edition),44(4):149-156(in Chinese)
Zhang HD,Zhang GM,Yao CX,Junaid M,Lu ZH,Zhang HJ,Ma YH,2015.Structural insight of a trimodular halophilic cellulase with a family 46 carbohydrate-binding module.PLoS One,10(11):e0142107
杜剑晖,还萍,李岭卓,安爽晴,江正兵,马向东,2018.一株极端嗜盐真菌的筛选及初步研究.湖北大学学报(自然科学版),40(2):189-194
郭璟,2015.分离自青海湖的三株耐盐菌及嗜盐菌的鉴定及纤维素降解能力的研究.青海畜牧兽医杂志,45(5):23-26
孔凡晶,王现洁,2017.青藏高原盐湖嗜盐微生物资源及应用前景.科技导报,35(12):27-31
刘杰凤,薛栋升,何可可,陈慧英,姚善泾,2012.一株耐盐纤维素酶海洋曲霉的筛选及产酶条件研究.食品与生物技术学报,31(7):711-718
刘爽,范丙全,2012.秸秆纤维素降解真菌QSH3-3的筛选及其特性研究.植物营养与肥料学报,18(1):218-226
秦雯娜,赵坤,郭立忠,卢伟东,2016.1株产纤维素酶嗜盐放线菌的鉴定、发酵优化及酶学性质的研究.青岛农业大学学报(自然科学版),33(1):27-34
王垚,韩燕峰,梁宗琦,2016.采自武夷山高温木质素降解菌优良菌株筛选及其木质素降解效果测定.菌物学报,35(10):1169-1177
王垚,韩燕峰,梁宗琦,2017a.两株戴氏霉对水稻秸秆的降解及产酶研究.菌物学报,36(5):598-603
王垚,韩燕峰,虞泓,梁宗琦,2017b.爱尔兰帚霉产低温纤维素酶的酶学性质和发酵工艺.菌物学报,36(8):1132-1140
王垚,王玉荣,罗韵,陈万浩,韩燕峰,梁宗琦,2014.一株在低温条件下产纤维素酶的草生帚霉.山地农业生物学报,33(3):50-54
吴曰福,顾爱星,王洪凯,2018.碱蓬根系嗜盐耐盐真菌的分离与鉴定.浙江农业学报,30(4):649-655
张波,刘开辉,陈文强,杨方玉,丁小维,邓百万,2016.陕北花马盐湖1株产纤维素酶真菌的鉴定及产酶条件研究.西北农林科技大学学报(自然科学版),44(4):149-156
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