一株高产内切纤维素酶贝莱斯芽孢杆菌的产酶条件优化及酶学性质分析
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摘要
试验旨在对一株杜仲树皮内生菌贝莱斯芽孢杆菌(Bacillus velezensis)的内切纤维素酶(CMCase)进行产酶条件优化及酶学性质分析,为探究贝莱斯芽孢杆菌CMCase的相关特性提供参考依据。采用单因子试验设计,分别优化pH、接种量、温度、时间、碳源和氮源等发酵条件,并探讨温度、pH、底物专一性、金属离子及表面活性剂对CMCase的影响。结果表明,以玉米秸秆为碳源(30 g/L)、豆粕为氮源(30 g/L)、3%接菌量、pH 7.0的条件下37℃培养48 h,贝莱斯芽孢杆菌157的CMCase最高可达(5.14±0.18) U/mL。经CMCase酶学性质分析发现,贝莱斯芽孢杆菌157的最适酶反应温度为60℃,最适pH为5.0;温度稳定性试验发现,在40和50℃时,相对剩余酶活力均高于80%,随着温度的升高,酶活力逐渐降低,当温度高于55℃,相对剩余酶活力约为50%;pH稳定性试验发现,在pH 5.0～10.0之间耐受性良好,相对剩余酶活力均高于90%,随作用时间的延长,相对剩余酶活力变化不大。贝莱斯芽孢杆菌157 CMCase属于典型内切型纤维素酶。Na~+、Mg~(2+)、Ca~(2+)可促进贝莱斯芽孢杆菌157 CMCase酶活力,而Co~(2+)、Hg~(2+)、Fe~(2+)、Cu~(2+)和SDS抑制CMCase酶活力;表面活性剂Tween-80、Tween-20、Triton X-100对贝莱斯芽孢杆菌157 CMCase无影响。本试验通过对贝莱斯芽孢杆菌157进行产酶条件优化及CMCase酶学性质分析发现,贝莱斯芽孢杆菌157 CMCase具有产酶量高,耐受pH范围广的特点,在饲料添加剂、洗涤剂和造纸领域中具有一定的应用价值。
This study was aimed to test the optimization and characterization of high-yield cellulase produced by an endophytic Bacillus velezensis(B.velezensis) 157 from Eucommia bark,which provided a reference for exploring the relevant characteristics of B.velezensis 157 CMCase.Single-factor experiment was designed to optimize fermentation conditions such as pH,inoculation amount,temperature,fermentation time,carbon source and nitrogen source,and to explore the effects of temperature,pH,substrate specificity,metal ions and surfactants on the enzymatic properties of CMCase.The optimized conditions of CMCase production by B.velezensis 157 were as follows:Maize straw as carbon source(30 g/L),soybean meal as nitrogen source(30 g/L),3% inoculation,and pH 7.0 under the condition of 37 ℃ for 48 h.After the optimization,the CMCase activity of B.velezensis 157 was up to(5.14±0.18) U/mL.The CMCase properties analysis of B.velezensis 157 found that the optimal temperature of enzyme reaction was 60 ℃,it could still maintain above 80% cellulase activity when temperature was from 40 to 50 ℃;With the increase of temperature,the relative residual enzyme activity decreased,and it decreased to 50% when the temperature was higher than 55 ℃.The optimal pH of cellulase was 5.0,and it could still maintain over 90% cellulase activity in a pH range of 5.0 to 10.0;The pH had little effect on the relative residual enzyme activity with the extension of the action time.The cellulase from B.velezensis 157 belonged to typical endoglueanases.Na~+,Mg~(2+),Ca~(2+)could increase the activity of B.velezensis 157 CMCase,Co~(2+),Hg~(2+),Fe~(2+),Cu~(2+) and SDS inhibited the activity of CMCase;The surfactant Tween-80,Tween-20 and Triton X-100 had no effect on CMCase activity.In this study,the enzyme production conditions and enzymatic properties of B.velezensis 157 CMCase were optimized,and it was concluded that B.velezensis 157 had a high yield of CMCase and tolerated a wide range of pH,which had certain application value in feed additive,detergent and paper industry.
This study was aimed to test the optimization and characterization of high-yield cellulase produced by an endophytic Bacillus velezensis(B.velezensis) 157 from Eucommia bark,which provided a reference for exploring the relevant characteristics of B.velezensis 157 CMCase.Single-factor experiment was designed to optimize fermentation conditions such as pH,inoculation amount,temperature,fermentation time,carbon source and nitrogen source,and to explore the effects of temperature,pH,substrate specificity,metal ions and surfactants on the enzymatic properties of CMCase.The optimized conditions of CMCase production by B.velezensis 157 were as follows:Maize straw as carbon source(30 g/L),soybean meal as nitrogen source(30 g/L),3% inoculation,and pH 7.0 under the condition of 37 ℃ for 48 h.After the optimization,the CMCase activity of B.velezensis 157 was up to(5.14±0.18) U/mL.The CMCase properties analysis of B.velezensis 157 found that the optimal temperature of enzyme reaction was 60 ℃,it could still maintain above 80% cellulase activity when temperature was from 40 to 50 ℃;With the increase of temperature,the relative residual enzyme activity decreased,and it decreased to 50% when the temperature was higher than 55 ℃.The optimal pH of cellulase was 5.0,and it could still maintain over 90% cellulase activity in a pH range of 5.0 to 10.0;The pH had little effect on the relative residual enzyme activity with the extension of the action time.The cellulase from B.velezensis 157 belonged to typical endoglueanases.Na~+,Mg~(2+),Ca~(2+)could increase the activity of B.velezensis 157 CMCase,Co~(2+),Hg~(2+),Fe~(2+),Cu~(2+) and SDS inhibited the activity of CMCase;The surfactant Tween-80,Tween-20 and Triton X-100 had no effect on CMCase activity.In this study,the enzyme production conditions and enzymatic properties of B.velezensis 157 CMCase were optimized,and it was concluded that B.velezensis 157 had a high yield of CMCase and tolerated a wide range of pH,which had certain application value in feed additive,detergent and paper industry.
引文
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[21] RAWAT R,TEWARI L.Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3[J].Extremophiles,2012,16(4):637-644.
[22] MANFREDI A P,PISA J H,VALDEON D H,et al.Synergistic effect of simple sugars and carboxymethyl cellulose on the production of a cellulolytic cocktail from Bacillus sp.AR03 and enzyme activity characterization[J].Applied Biochemistry and Biotechnology,2016,179(1):16-32.
[23] IRFAN M,TAYYAB A,HASAN F,et al.Production andcharacterization of organic solvent-tolerant cellulase from Bacillus amyloliquefaciens AK9 isolated from hot spring[J].Applied Biochemistry and Biotechnology,2017,182(4):1390-1402.
[24] ZHANG G,LI S,XUE Y,et al.Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp.BG-CS10[J].Extremophiles,2012,16(1):35-43.
[25] LI X,YU H Y.Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate,Bacillus sp.L1[J].Journal of Industral Microbiology and Biotechnology,2012,39(8):1117-1124.
[26] KIM B K,LEE B H,LEE Y J,et al.Purification and characterization of carboxymethylcellulase isolated from a marine bacterium,Bacillus subtilis subsp.subtilis A-53[J].Enzyme and Microbial Technology,2009,44(7):411-416.
[27] GAUR R,TIWARI S.Isolation,production,purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07[J].BMC Biotechnol,2015,15(1):19.
[28] LAMED R,TORMO J,CHIRINO A J,et al.Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum[J].Journal of Moloecular Biology,1994,244(2):236-237.
[2] SANCHEZ C.Lignocellulosic residues:Biodegradation and bioconversion by fungi[J].Biotechnology Advances,2009,27(2):185-194.
[3] 栾素军,邢焕,孙永波,等.解淀粉芽孢杆菌在畜禽养殖中的应用研究进展[J].中国畜牧兽医,2016,43(10):2615-2620.LUAN S J,XING H,SUN Y B,et al.Research progress on Bacillus amloliquefacines in animal breeding industry[J].China Animal Husbandry & Veterinary Medicine,2016,43(10):2615-2620.(in Chinese)
[4] SALIM A A,GRBAVCIC S,SEKULJICA N,et al.Production of enzymes by a newly isolated Bacillus sp.TMF-1 in solid state fermentation on agricultural by-products:The evaluation of substrate pretreatment methods[J].Bioresource Technology,2017,228:193-200.
[5] CHEN L,GU W,XU H Y,et al.Complete genome sequence of Bacillus velezensis 157 isolated from Eucommia ulmoides with pathogenic bacteria inhibiting and lignocellulolytic enzymes production by SSF[J].3 Biotech,2018,8(2):114.
[6] MENG F,MA L,JI S,et al.Isolation and characterization of Bacillus subtilis strain BY-3,a thermophilic and efficient cellulase-producing bacterium on untreated plant biomass[J].Letters in Applied Microbiology,2014,59(3):306-312.
[7] MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Chemistry,1959,31(3):426-428.
[8] LIANG Y L,ZHANG Z,WU M,et al.Isolation,screening,and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1[J].Biomed Research International,2014,2014:512497.
[9] YE M,TANG X,YANG R,et al.Characteristics and application of a novel species of Bacillus:Bacillus velezensis[J].ACS Chemical Biology,2018,13(3):500-505.
[10] GAUTAM S P,BUNDELA P S,PABDEY A K,et al.Optimization for the production of cellulase enzyme from municipal solid waste residue by two novel cellulolytic fungi[J].Wiley Interdisciplinary Reviews- Nanomedicine and Nanobiotechnology,2011,2011:810425.
[11] SHANKAR T,ISAIARASU L.Cellulase production by Bacillus pumilus EWBCM1 under varying cultural conditions[J].Middle East Journal of Scientific Research,2011,8(1):40-45.
[12] AMRITKAR N,KAMAT M,LALI A.Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue-cellulose matrices[J].Process Biochemistry,2004,39(5):565-570.
[13] GUEDON E,DESVAUX M,PETITDEMANGE H.Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering[J].Applied and Environmental Microbiology,2002,68(1):53-58.
[14] 陈光,吴卓夫,张兆业.秸秆综合利用研究动态及展望[J].吉林农业大学学报,2016,38(5):505-510.CHEN G,WU Z F,ZHANG Z Y.Research trend and prospect of comprehensive utilization of straw[J].Journal of Jilin Agricultural University,2016,38(5):505-510.(in Chinese)
[15] YANG W,MENG F,PENG J,et al.Isolation and identification of a cellulolytic bacterium from the Tibetan pig’s intestine and investigation of its cellulase production[J].Electronic Journal of Biotechnology,2014,17(6):262-267.
[16] RASTOGI G,BHALLA A,ADHIKARI A,et al.Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains[J].Bioresource Technology,2010,101(22):8798-8806.
[17] LI W,ZHANG W W,YANG M M,et al.Cloning of the thermostable cellulase gene from newly isolated Bacillus subtilis and its expression in Escherichia coli[J].Molecular Biotechnology,2008,40(2):195-201.
[18] PAUDEL Y P,QIN W.Characterization of novel cellulase-producing bacteria isolated from rotting wood samples[J].Applied Biochemistry and Biotechnology,2015,177(5):1186-1198.
[19] MAWADZA C,HATTI-KAUL R,ZVAUYA R,et al.Purification and characterization of cellulases produced by two Bacillus strains[J].Journal of Biotechnology,2000,83(3):177-187.
[20] ANNAMALAI N,RAJESWARI M V,ELAYARAJA S,et al.Thermostable,haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes[J].Carbohydrate Polymers,2013,94(1):409-415.
[21] RAWAT R,TEWARI L.Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3[J].Extremophiles,2012,16(4):637-644.
[22] MANFREDI A P,PISA J H,VALDEON D H,et al.Synergistic effect of simple sugars and carboxymethyl cellulose on the production of a cellulolytic cocktail from Bacillus sp.AR03 and enzyme activity characterization[J].Applied Biochemistry and Biotechnology,2016,179(1):16-32.
[23] IRFAN M,TAYYAB A,HASAN F,et al.Production andcharacterization of organic solvent-tolerant cellulase from Bacillus amyloliquefaciens AK9 isolated from hot spring[J].Applied Biochemistry and Biotechnology,2017,182(4):1390-1402.
[24] ZHANG G,LI S,XUE Y,et al.Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp.BG-CS10[J].Extremophiles,2012,16(1):35-43.
[25] LI X,YU H Y.Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate,Bacillus sp.L1[J].Journal of Industral Microbiology and Biotechnology,2012,39(8):1117-1124.
[26] KIM B K,LEE B H,LEE Y J,et al.Purification and characterization of carboxymethylcellulase isolated from a marine bacterium,Bacillus subtilis subsp.subtilis A-53[J].Enzyme and Microbial Technology,2009,44(7):411-416.
[27] GAUR R,TIWARI S.Isolation,production,purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07[J].BMC Biotechnol,2015,15(1):19.
[28] LAMED R,TORMO J,CHIRINO A J,et al.Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum[J].Journal of Moloecular Biology,1994,244(2):236-237.