枯草芽胞杆菌FJ-3-16产角蛋白酶条件优化及在生猪脱毛中的应用
|
摘要
枯草芽胞杆菌(Bacillus subtilis)FJ-3-16是一株角蛋白酶高产菌株,在畜禽废弃物降解和生物脱毛方面有潜在应用价值。为提高菌株B.subtilis FJ-3-16胞外角蛋白酶产量,本研究综合利用单因子实验、Plackett-Burman(PB)设计和中心组合设计(Central composite design,CCD)优化了B.subtilis FJ-3-16的摇瓶产酶发酵条件,优化后的培养基配方为玉米粉1.41%、豆粉3.62%、酪素2%、CaC_l20.16%、K_2HPO_4 0.3%、KH_2PO_4 0.1%、pH 8.0,温度37℃,转速250 r/min,培养时间48 h,胞外角蛋白酶酶活由原有培养基的73.82U/mL提高至166.08 U/mL。且建立了利用50 L发酵罐生产角蛋白酶的发酵工艺,酶活在摇瓶发酵的基础上进一步提高,达207.6 U/mL,同时产酶时间缩短至18 h。同时利用菌株发酵液对巴马香猪(Sus scrofa)的猪蹄、猪头皮张及猪蹄整蹄进行脱毛,效果显著,表明B.subtilis FJ-3-16在巴马香猪为代表的难脱毛生猪的脱毛应用中具有极好的应用前景。本研究为实现角蛋白酶的工业化应用提供了基础资料。
Bacillus subtilis FJ-3-16 is an efficient keratinase-producing bacterium strain and its extracellular keratinase has many prospective applications in livestock wastes degradation, animal furs dehairing, detergent additive and so on. In order to increase the industrial yield of the extracellular keratinase produced by the strain, its fermentative condition was optimized using one-variable-at-a-time method, Plackett-Burman(PB)design and central composite design(CCD). The optimized medium contained corn meal 1.41%, soybean flour 3.62%, casein 2%, CaCl_2 0.16%, K_2HPO_4 0.3%, K_2HPO_4 0.1%(pH 8.0) and optimized condition was 48 h cultivation at 37 ℃ and a rotate shaking of 250 r/min. The total activity of the extracellular keratinase was increased from 73.82 to 166.08 U/mL. Then, the optimized cultivation condition was further amplified in 50 L fermenter. The total activity of the extracellular keratinase was further increased to 207.6 U/mL in theamplified 50 L fermenter, and meanwhile the fermentative time was reduced from 48 to 18 h. The dehairing capacity of the ferment toward the skins of the Bama pig's(Sus scrofa) hoof and head was also evaluated using enzymatic dehairing experiments. Pieces of skins obtained from the hoof and head of the Bama pig, and the whole hoof of the Bama pig were treated by the crude extracellular keratinase at 50 ℃ for 6 h, 10 h,respectively, and dehairing effects were obvious. This study would pave the way for the application of strain FJ-3-16 in pig dehairing.
Bacillus subtilis FJ-3-16 is an efficient keratinase-producing bacterium strain and its extracellular keratinase has many prospective applications in livestock wastes degradation, animal furs dehairing, detergent additive and so on. In order to increase the industrial yield of the extracellular keratinase produced by the strain, its fermentative condition was optimized using one-variable-at-a-time method, Plackett-Burman(PB)design and central composite design(CCD). The optimized medium contained corn meal 1.41%, soybean flour 3.62%, casein 2%, CaCl_2 0.16%, K_2HPO_4 0.3%, K_2HPO_4 0.1%(pH 8.0) and optimized condition was 48 h cultivation at 37 ℃ and a rotate shaking of 250 r/min. The total activity of the extracellular keratinase was increased from 73.82 to 166.08 U/mL. Then, the optimized cultivation condition was further amplified in 50 L fermenter. The total activity of the extracellular keratinase was further increased to 207.6 U/mL in theamplified 50 L fermenter, and meanwhile the fermentative time was reduced from 48 to 18 h. The dehairing capacity of the ferment toward the skins of the Bama pig's(Sus scrofa) hoof and head was also evaluated using enzymatic dehairing experiments. Pieces of skins obtained from the hoof and head of the Bama pig, and the whole hoof of the Bama pig were treated by the crude extracellular keratinase at 50 ℃ for 6 h, 10 h,respectively, and dehairing effects were obvious. This study would pave the way for the application of strain FJ-3-16 in pig dehairing.
引文
韩瑞枝,刘孟华,吴重德,等.2010.角蛋白酶在牛皮脱毛工序中的应用研究[J].中国皮革,39(5):5-8.(Han R Z,Liu M H,Wu C D,et al.2010.Application of keratinase in bovine hides depilcation[J].China Leather,39(5):5-8.)
刘冬敏,王建辉,刘永乐,等.2015.食用猪皮脱毛工艺优化研究[J].湖南农业科学,7:56-59.(Liu D M,Wang J H,Liu Y L,et al.2015.Optimization of edible pigskin depilating process[J].Hunan Agricultural Sciences,7:56-59.)
刘彦,陈倩,刘建泽.2013.具有脱毛性能的角蛋白酶产生菌的筛选[J].中国皮革,42(23):15-18.(Liu Y,Chen Q,Liu J Z.2013.Isolation and identification of dehairing keratinase’s producing strain[J].China Leather,42(23):15-18.)
王学川,李飞虎,任龙芳,等.2012.酶法脱毛技术的研究进展与展望[J].中国皮革,41(13):49-52.(Wang X C,Li F H,Ren L F,et al.2012.Present situations and prospect of enzymatic[J].Unhairing Technology,41(13):49-52.)
谢菲,李从虎,郑佳,等.2010.角蛋白酶生产菌株的分离筛选与鉴定[J].微生物学报,50(4):537-541.(Xie F,Li CH,Zheng J,et al.2010.Screening and identification of a new bacillus strain producing keratinase[J].Acta Microbiologica Sinica,50(4):537-541.)
张红见,赵静,张虎.2010.短小芽胞杆菌(Bacillus pumilus UN-31-C-42)对青海藏系羊蹄脱毛效果研究[J],食品研究与开发,31(8):167-169.(Zhang H J,Zhao J,Zhang H.2010.Effects of Bacillus pumilus UN-31-C-42used in unhairing of tibeteau sheep hooves in qinghai province[J].Food Researchand Development,31(8):167-169.)
张玉红,但年华,但卫华,等.2015.固定化复合脱毛酶脱毛废液的循环利用[J].皮革科学与工程,25(4):42-49.(Zhang Y H,Dan N H,Dan W H,et al.2015.Unhairing effluent recycling technology with immobilized multienzymes[J].Leather Science and Engineering,25(4):42-49.)
Awad G E A,Esawy M A,Salam W A,et al.2011.Keratinase production by Bacillus pumilus GHD in solid-state fermentation using sugar cane bagasse:Optimisation of culture conditions using a box-behnken experimental design[J].Annals of Microbiology,61(3):663-672.
Casarin F,Cladera-Olivera F,Brandelli A.2008.Use of poultry byproduct for production of keratinolyticenzymes[J].Food and Bioprocess Technology,1(3):301-305.
Daroit D J,Brandelli A.2014.A current assessment on the production of bacterial keratinases[J].Critical Reviews in Biotechnology,34(4):372-84.
El-Gendy M M.2010.Keratinase production by endophytic Penicillium spp.Morsy1 under solid-state fermentation using rice straw[J].Applied Biochemistry and Biotechnology,162(3):780-794.
Gradisar H,Friedrich J,Krizaj I,et al.2005.Similarities and specificities of fungal keratinolytic proteases:Comparison of keratinases of Paecilomycesmarquandii and Doratomycesmicrosporus to some known proteases[J].Applied and Environmental Microbiology,71(7):3420-3426.
Gupta R,Rajput R,Sharma R,et al.2013.Biotechnological applications and prospective market of microbial keratinases[J].Appl Microbiol Biotechnol,97(23):9931-9940.
Gupta R,Ramnani P.2006.Microbial keratinases and their prospective applications:An overview[J].Applied Microbiology and Biotechnology,70(1):21-33.
Jaouadi N Z,Rekik H,Badis A,et al.2013.Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities[J].Plo S One,8(10):e76722.
Lo W H,Too J R,Wu J Y.2012.Production of keratinolytic enzyme by an indigenous feather-degrading strain Bacillus cereus Wu2[J].Journal of Bioscience and Bioengineering,114(6):640-647.
Rai S K,Mukherjee A K.2011.Optimization of production of an oxidant and detergent-stable alkalineβ-keratinase from Brevibacillus,sp.strain AS-S10-II:Application of enzyme in laundry detergent formulations and in leather industry[J].Biochemical Engineering Journal,54(1):47-56.
Shrinivas D,Naik G R.2011.Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairingactivity[J].International Biodeterioration&Biodegradation,65(1):29-35.
Suntornsuk W,Suntornsuk L.2003.Feather degradation by Bacillus sp.FK 46 in submerged cultivation[J].Bioresource Technology,86(3):239-43.
Tiwary E,Gupta R.2010.Medium optimization for a novel58 k D dimeric keratinase from Bacillus licheniformis ER-15:Biochemical characterization and application in feather degradation and dehairing of hides[J].Bioresource Technology,101(15):6103-6110.
Wang S L,Hsu W T,Liang T W,et al.2008.Purification andcharacterization of three novel keratinolytic metalloproteases producedby Chryseobacteriumindologenes TKU014 in a shrimp shellpowder medium[J].Bioresource Technology,99(13):5679-86.
Yusuf I,Ahmad S A,Lai Y P,et al.2016.Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-alcaligenes,sp.AQ05-001[J].Journal of Environmental Management,183:182-195.
刘冬敏,王建辉,刘永乐,等.2015.食用猪皮脱毛工艺优化研究[J].湖南农业科学,7:56-59.(Liu D M,Wang J H,Liu Y L,et al.2015.Optimization of edible pigskin depilating process[J].Hunan Agricultural Sciences,7:56-59.)
刘彦,陈倩,刘建泽.2013.具有脱毛性能的角蛋白酶产生菌的筛选[J].中国皮革,42(23):15-18.(Liu Y,Chen Q,Liu J Z.2013.Isolation and identification of dehairing keratinase’s producing strain[J].China Leather,42(23):15-18.)
王学川,李飞虎,任龙芳,等.2012.酶法脱毛技术的研究进展与展望[J].中国皮革,41(13):49-52.(Wang X C,Li F H,Ren L F,et al.2012.Present situations and prospect of enzymatic[J].Unhairing Technology,41(13):49-52.)
谢菲,李从虎,郑佳,等.2010.角蛋白酶生产菌株的分离筛选与鉴定[J].微生物学报,50(4):537-541.(Xie F,Li CH,Zheng J,et al.2010.Screening and identification of a new bacillus strain producing keratinase[J].Acta Microbiologica Sinica,50(4):537-541.)
张红见,赵静,张虎.2010.短小芽胞杆菌(Bacillus pumilus UN-31-C-42)对青海藏系羊蹄脱毛效果研究[J],食品研究与开发,31(8):167-169.(Zhang H J,Zhao J,Zhang H.2010.Effects of Bacillus pumilus UN-31-C-42used in unhairing of tibeteau sheep hooves in qinghai province[J].Food Researchand Development,31(8):167-169.)
张玉红,但年华,但卫华,等.2015.固定化复合脱毛酶脱毛废液的循环利用[J].皮革科学与工程,25(4):42-49.(Zhang Y H,Dan N H,Dan W H,et al.2015.Unhairing effluent recycling technology with immobilized multienzymes[J].Leather Science and Engineering,25(4):42-49.)
Awad G E A,Esawy M A,Salam W A,et al.2011.Keratinase production by Bacillus pumilus GHD in solid-state fermentation using sugar cane bagasse:Optimisation of culture conditions using a box-behnken experimental design[J].Annals of Microbiology,61(3):663-672.
Casarin F,Cladera-Olivera F,Brandelli A.2008.Use of poultry byproduct for production of keratinolyticenzymes[J].Food and Bioprocess Technology,1(3):301-305.
Daroit D J,Brandelli A.2014.A current assessment on the production of bacterial keratinases[J].Critical Reviews in Biotechnology,34(4):372-84.
El-Gendy M M.2010.Keratinase production by endophytic Penicillium spp.Morsy1 under solid-state fermentation using rice straw[J].Applied Biochemistry and Biotechnology,162(3):780-794.
Gradisar H,Friedrich J,Krizaj I,et al.2005.Similarities and specificities of fungal keratinolytic proteases:Comparison of keratinases of Paecilomycesmarquandii and Doratomycesmicrosporus to some known proteases[J].Applied and Environmental Microbiology,71(7):3420-3426.
Gupta R,Rajput R,Sharma R,et al.2013.Biotechnological applications and prospective market of microbial keratinases[J].Appl Microbiol Biotechnol,97(23):9931-9940.
Gupta R,Ramnani P.2006.Microbial keratinases and their prospective applications:An overview[J].Applied Microbiology and Biotechnology,70(1):21-33.
Jaouadi N Z,Rekik H,Badis A,et al.2013.Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities[J].Plo S One,8(10):e76722.
Lo W H,Too J R,Wu J Y.2012.Production of keratinolytic enzyme by an indigenous feather-degrading strain Bacillus cereus Wu2[J].Journal of Bioscience and Bioengineering,114(6):640-647.
Rai S K,Mukherjee A K.2011.Optimization of production of an oxidant and detergent-stable alkalineβ-keratinase from Brevibacillus,sp.strain AS-S10-II:Application of enzyme in laundry detergent formulations and in leather industry[J].Biochemical Engineering Journal,54(1):47-56.
Shrinivas D,Naik G R.2011.Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairingactivity[J].International Biodeterioration&Biodegradation,65(1):29-35.
Suntornsuk W,Suntornsuk L.2003.Feather degradation by Bacillus sp.FK 46 in submerged cultivation[J].Bioresource Technology,86(3):239-43.
Tiwary E,Gupta R.2010.Medium optimization for a novel58 k D dimeric keratinase from Bacillus licheniformis ER-15:Biochemical characterization and application in feather degradation and dehairing of hides[J].Bioresource Technology,101(15):6103-6110.
Wang S L,Hsu W T,Liang T W,et al.2008.Purification andcharacterization of three novel keratinolytic metalloproteases producedby Chryseobacteriumindologenes TKU014 in a shrimp shellpowder medium[J].Bioresource Technology,99(13):5679-86.
Yusuf I,Ahmad S A,Lai Y P,et al.2016.Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-alcaligenes,sp.AQ05-001[J].Journal of Environmental Management,183:182-195.