枯草芽孢杆菌WB600角蛋白酶重组菌发酵条件优化

doi:10.16431/j.cnki.1671-7236.2020.01.006

摘要/Abstract

摘要： 本试验旨在对已构建枯草芽孢杆菌WB600角蛋白酶重组菌WB600-K进行发酵条件优化，获得最佳产酶条件，以期提高重组菌角蛋白酶的产量；同时通过研究不同种类碳源和氮源间的组合优化获得成本低廉的重组菌发酵培养基配方。结果显示，当发酵液D_{600 nm}=1.0时添加0.5%木糖诱导发酵WB600-K分泌表达角蛋白酶酶活最佳（P<0.05）；山梨醇作为碳源可显著提高WB600-K角蛋白酶酶活（P<0.05），但成本较高不宜作为混合碳源来源；不同种类碳源（葡萄糖、玉米粉、糖蜜、甘油）组合添加能显著提高重组菌角蛋白酶酶活（P<0.05）。不同种类氮源（蛋白胨、酵母粉、豆粕粉、尿素）组合添加对于重组菌提高角蛋白酶酶活效果不如单一氮源（蛋白胨）（P<0.05）。在诱导发酵48 h时WB600-K产角蛋白酶酶活最高（P<0.05）。对发酵配方进行正交试验优化，WB600-K在玉米粉5.1 g/L、葡萄糖5.9 g/L、糖蜜5.9 g/L、甘油3.0 g/L、蛋白胨20 g/L、NaCl 10 g/L，发酵液D_{600 nm}=1.0时加0.5%木糖诱导发酵48 h能显著提高角蛋白酶酶活（P<0.05），达到56.9 U/mL，较初始培养条件提高49.74%。综上，在优化发酵条件下进行发酵及不同种类碳源间组合能提高WB600-K角蛋白酶活性，可为角蛋白酶工业化生产提供试验依据。

关键词: 枯草芽孢杆菌WB600; 角蛋白酶; 发酵条件; 优化

Abstract: The aim of this experiment was to optimize the fermentation conditions of the constructed keratinase recombinant B.subtilis WB600 strain WB600-K,and obtain the optimal enzyme production conditions in order to increase the yield of recombinant keratinase;At the same time,the combination optimization between different types of carbon sources and different nitrogen sources was studied in order to obtain a low-cost recombinant fermentative medium formula.The results showed that when the fermentation D_{600 nm}=1.0,and 0.5% xylose was added to induce fermentation,the expression of keratinase was best in the recombinant WB600-K (P<0.05).As a carbon source,sorbitol could significantly increase the activity of the recombinant strain WB600-K keratinase (P<0.05),but the cost was too high to be a source of mixed carbon sources.The combination of different carbon sources (glucose,corn flour,molasses and glycerin) could significantly increase the activity of keratinase (P<0.05).The combination of different kinds of nitrogen sources (peptone,yeast powder,soybean meal and urea) significantly increased the activity of keratinase less than single nitrogen source (peptone) (P<0.05).The WB600-K had the highest keratinase activity at 48 h after induction of fermentation (P<0.05).The overall experimental optimization results showed that,at the condition of corn flour 5.1 g/L,glucose 5.9 g/L,molasses 5.9 g/L,glycerol 3.0 g/L,peptone 20 g/L,NaCl 10 g/L,0.5% xylose was added when fermentation D_{600 nm}=1.0,and 48 h fermentation,the activity of keratinase could be increased significantly (P<0.05),which was 56.9 U/mL and 49.74% higher than that in initial culture conditions.In summary,fermentation under optimized fermentation conditions could increase the activity of recombinant strain WB600-K keratinase,which laid a foundation for the industrial production of keratinase.

Key words: B.subtilis WB600; keratinase; fermentative conditions; optimization

中图分类号:

S616.3

廖朝勇, 张铁鹰, 刘俊丽, 隋景巍. 枯草芽孢杆菌WB600角蛋白酶重组菌发酵条件优化[J]. 中国畜牧兽医, 2020, 47(1): 44-52.

LIAO Chaoyong, ZHANG Tieying, LIU Junli, SUI Jingwei. Optimization of the Fermentation Conditions of Recombinant B.subtilis WB600 Produced Keratinase[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 44-52.

参考文献

[1] CAI C G,CHEN J S,QI J J,et al.Purification and characterization of keratinase from a new Bacillus subtilis strain[J].Journal of Zhejiang University Science B,2008,9(9):713-720.
[2] MATSUI T,YAMADA Y,MITSUYA H,et al.Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328[J].Applied Microbiology and Biotechnology,2009,82(5):941-950.
[3] NICKERSON W J,NOVAL J J,ROBISON R S.Keratinase:Ⅰ.Properties of the enzyme conjugate elaborated by Streptomyces fradiae[J].Biochimica Et Biophysica Acta,1963,77:73-86.
[4] RAI S K,KONWARH R,MUKHERJEE A K.Purification,characterization and biotechnological application of an alkaline β-keratinase produced by Bacillus subtilis RM-01 in solid-state fermentation using chicken-feather as substrate[J].Biochemical Engineering Journal,2009,45(3):218-225.
[5] COMMICHAU F M,ALZINGER A,SANDE R,et al.Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine[J].Metabolic Engineering,2014,25:38-49.
[6] ZAFAR M,AHMED S,KHAN M I M,et al.Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli[J].Molecular Biology Reports,2014,41(5):3295-3302.
[7] ZAGHLOUL T I,ABDELAZIZ A,MOSTAFA M H.High level of expression and stability of the cloned alkaline protease (aprA) gene in Bacillus subtilis[J].Enzyme and Microbial Technology,1994,16(6):534-537.
[8] LIU B,ZHANG J,LI B,et al.Expression and characterization of extreme alkaline,oxidation-resistant keratinase from Bacillus licheniformisin recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing[J].World Journal of Microbiology and Biotechnology,2013,29(5):825-832.
[9] JUNG J,YU K O,RAMZI A B,et al.Improvement of surfactin production in Bacillus subtilis using synthetic wastewater by overexpression of specific extracellular signaling peptides,comX and phrC[J].Biotechnology & Bioengineering,2012,109(9):2349-2356.
[10] HARTZ P,MATTES C,SCHAD M,et al.Expanding the promoter toolbox of Bacillus megaterium[J].Journal of Biotechnology,2019,294:38-48.
[11] 李志敏,潘兴亮,杨雅麟,等.溶解性多糖单加氧酶CBP21的高效分泌表达及与几丁质酶的协同作用研究[J].中国农业科技导报,2017,1:58-65. LI Z M,PAN X L,YANG Y L,et al.Studies on the efficient secretion of lytic polysaccharide monooxygenases CBP21 and its synergism with chitinase[J].Journal of Agricultural Science and Technology,2017,1:58-65.(in Chinese)
[12] 刘柏宏.Bacillus licheniformis角蛋白酶的高效表达、热稳定性及底物特异性改造[D].无锡:江南大学,2015. LIU B H.Over expression of Bacillus licheniformis keratinase,its molecular modification for enhanced thermostability and substrate specificity[D].Wuxi:Jiangnan University,2015.(in Chinese)
[13] 郭自涛,张梁,李由然,等.重组枯草芽孢杆菌分泌表达腺苷酸脱氨酶[J].食品科学,2015,36(21):135-139. GUO Z T,ZHANG L,LI Y R,et al.Expression of adenosine deaminase in recombinant Bacillus subtilis[J].Food Science,2015,36(21):135-139.(in Chinese)
[14] 张云霄.海藻糖合酶在枯草芽孢杆菌中异源表达,调控元件的适配及发酵条件的优化[D].济南:齐鲁工业大学,2017. ZHANG Y X.The heterologous expression of trehalose synthase,regulatory element adaptation and fermentation conditions optimization[D].Jinan:Qilu University of Technology,2017.(in Chinese)
[15] 宋皖.长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的重组表达及条件优化[D].无锡:江南大学,2016. SONG W.Recombinant expression and optimization of Bacillus naganoensis pullulanase from Bacillus subtilis[D].Wuxi:Jiangnan university,2016.(in Chinese)
[16] 张秀江,孙小玉,谷立峰,等.角蛋白酶工程菌株产酶条件的优化研究[J].河南科学,2018,36(12):1935-1940. ZHANG X J,SUN X Y,GU L F,et al.Optimization of enzyme production conditions in keratinase engineering strain[J].Henan Science,2018,36(12):1935-1940.(in Chinese)
[17] 王法富,刘梦涵,刘利利,等.枯草芽孢杆菌出芽培养条件优化[J].当代化工,2018,47(2):286-289. WANG F F,LIU M H,LIU L L,et al.Optimization of Bacillus Subtilis sprouting culture conditions[J].Contemporary Chemical Industry,2018,47(2):286-289.(in Chinese)
[18] 张玲,王男,金吕华,等.双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究[J].中国生物工程杂志,2018,38(12):21-31. ZHANG L,WANG N,JIN L H,et al.To Promote the expression of leucine dehydrogenase in Bacillus subtilis via dual-promoter and fermentation research[J].China Biotechnology,2018,38(12):21-31.(in Chinese)
[19] 陈卫,陈海琴,田丰伟,等.耐热β-半乳糖苷酶重组菌WB600/pMA5-bgaB发酵条件的研究[J].食品与发酵工业,2007,9:1-5. CHEN W,CHEN H Q,TIAN F W,et al.Optimization on fermentation conditions of a recombinant Bacillus subtilis WB600/PMAS-bgaB for thermostableβ-galaetosidase produetion[J].Food and Fermentation Industries,2007,9:1-5.(in Chinese)
[20] 李伟,孙静,林福兴,等.枯草芽孢杆菌M364高产抗菌肽Bacillomycin D工业培养基优化[J].食品工业科技,2018,39(22):192-199. LI W,SUN J,LIN F X,et al.Optimization of Bacillomycin D high-yield industrial fermentation medium of Bacillus subtilis M364[J].Science and Technology of Food Industry,2018,39(22):192-199.(in Chinese)
[21] 代阳,魏利,王继华,等.枯草芽孢杆菌发酵条件优化及其破乳效能[J].微生物学通报,2010,37(4):580-585. DAI Y,WEI L,WANG J H,et al.Optimization of fermentation conditions for Bacillus subtilis and its demulsification efficiency[J].Microbiology China,2010,37(4):580-585.(in Chinese)
[22] 王树香,李术娜,李红亚.产植酸酶枯草芽孢杆菌ZX-29产芽孢条件的优化[J].湖北农业科学,2014,53(1):160-163. WANG S X,LI S N,LI H Y.Optimization of spore production conditions of strain of producing phytase Bacillus subtilis ZX-29[J].Hubei Agricultural Sciences,2014,53(1):160-163.(in Chinese)
[23] 张海玲,刘超,程欲,等.超氧化物歧化酶重组酵母的构建及其发酵条件的优化[J].食品与生物技术学报,2017,36(9):918-921. ZHANG H L,LIU C,CHENG Y,et al.Construction and expression of recombinant superoxide dismutase in Pichia pastoris[J].Journal of Food Science And Biotechnology,2017,36(9):918-921.(in Chinese)