动物营养学报 >

2017 , Vol. 29 >Issue 11: 4048 - 4057

DOI: https://doi.org/10.3969/j.issn.1006-267x.2017.11.026

饲料资源开发 Feed Resources Development

地衣芽孢杆菌CP-16脂类水解酶的研究

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  • 1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
    2. 甘肃农业大学动物科学技术学院, 兰州 730070
李爽(1992-),女,河南安阳人,硕士研究生,动物营养与饲料科学专业。E-mail:15236286917@163.com

收稿日期: 2017-05-02

  网络出版日期: 2017-10-31

基金资助

国家自然科学基金面上项目（31470122）

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Research on Lipolytic Enzymes from Bacillus licheniformis CP-16

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  • 1. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing 100193, China;
    2. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received date: 2017-05-02

  Online published: 2017-10-31

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摘要

本试验旨在通过异源表达获得地衣芽孢杆菌（Bacillus licheniformis） CP-16的脂类水解酶，并探究其在羽毛降解过程中的作用。试验以地衣芽孢杆菌CP-16基因组DNA为模板，扩增脂类水解酶基因，转化入大肠杆菌中表达，获得重组酶L-4。研究重组酶L-4的适宜pH、pH稳定性、适宜温度、温度稳定性以及有机溶剂和金属离子对其相对活性的影响，同时探究其对角蛋白酶K水解天然羽毛角蛋白的作用。结果显示，获得的脂类水解酶基因大小为747 bp，编码248个氨基酸，在大肠杆菌中成功表达出重组酶L-4，其分子质量约为28.3 ku，酯酶活性为0.42 U/mL，适宜pH为6.5，适宜温度为50℃；在pH 6.5~9.5条件下处理30 min相对活性保持80%以上，在低于50℃温度条件下处理30 min相对活性保持70%以上。二价铁离子（Fe2+）、钠离子（Na+）、锰离子（Mn2+）、钙离子（Ca2+）对重组酶L-4相对活性具有激发作用，钡离子（Ba2+）、锌离子（Zn2+）、铜离子（Cu2+）、镍离子（Ni2+）对重组酶L-4相对活性具有抑制作用。当有机溶剂浓度为30%时，重组酶L-4在二甲基亚砜（DMSO）和甲醇中保存97%和85%的相对活性，在丙酮、乙醇中保存45%以上的相对活性，在异丙醇中保存不到20%的相对活性，而在乙腈中相对活性基本完全丧失。用重组酶L-4预处理天然羽毛底物，可提高角蛋白酶K对底物的水解效率，促进率为4.32%。由此可见，脂类水解酶可降解羽毛表层脂质，可在促进角蛋白酶水解羽毛角蛋白中发挥作用。

关键词： 地衣芽孢杆菌; 脂类水解酶; 角蛋白; 羽毛脂质; 克隆表达

本文引用格式

李爽, 张铁鹰, 廖朝勇, 梁争文, 马霞飞, 刘俊丽 . 地衣芽孢杆菌CP-16脂类水解酶的研究[J]. 动物营养学报, 2017 , 29(11) : 4048 -4057 . DOI: 10.3969/j.issn.1006-267x.2017.11.026

Abstract

In order to explore the influence of lipolytic enzyme on feather degradation, the lipolytic enzyme gene of Bacillus licheniformis CP-16 was cloned and heterologous expressed in this research. The target gene of recombinant enzyme L-4, which was used Bacillus licheniformis CP-16 genome DNA as a template, amplified lipid hydrolase gene, and then transferred into Escherichia coli for expression of the targeted gene. The optimum pH, pH stability, temperature, temperature stability and effects of organic solvents and metal ions on relative activity of the recombinant enzyme L-4 were determined, and its application on keratin K hydrolyzed natural feather keratin was also investigated. The results showed that the length of lipid hydrolase gene was 747 bp, encoded 248 amino acids, the recombinant enzyme L-4 was successfully expressed in Escherichia coli, and the molecular weight was about 28.3 ku, the esterase activity was 0.41 U/mL, the optimum pH was 6.5, the optimum temperature was 50℃. The relative activity of recombinant enzyme L-4 kept above 80% treated with 30 min under pH 6.5 to 9.5 condition, and the relative activity kept above 70% treated with 30 min under below 50℃ condition. The ferrous iron (Fe2+), sodion (Na+),manganese ion (Mn2+) and calcium ion (Ca2+) had an stimulative effect on the relative activity of recombinant enzyme L-4, while the barium ion (Ba2+), zinc ion (Zn2+), copper ion (Cu2+) and nickel ion (Ni2+) had an disincentive effect on the relative activity of recombinant enzyme L-4. When the concentration of organic solvent was 30%, the relative activity of recombinant enzyme L-4 kept 85% and 97% in methanol and dimethyl sulfoxide solutions, the relative activity kept above 45% in acetone and ethanol solutions, the relative activity kept less than 20% in isopropanol solution, while the relative activity was complete loss in acetonitrile solution. The pretreatment of natural feather substrates with recombinant enzyme L-4 promoted the hydrolysis of keratinase K to the substrate, and the promotion rate is 4.32%. In conclusion, lipolytic enzymes can degrade feather surface lipids and it may play a role in promoting keratin hydrolysis of feather keratin.

Key words： Bacillus licheniformis; lipolytic enzymes; keratin; feather lipid; cloning and expression

参考文献

[1] 计成.动物营养学[M].北京:高等教育出版社,2008:75.

[2] 赵全芳.鸟类羽毛疏水、亲水性原因探析[J].管理学刊,2005,18(2):121-122.

[3] SARAVANAN K,DHURAI B.Exploration on amino acid content and morphological structure in chicken feather fiber[J].Journal of Textile and Apparel:Technology and Management,2012,7(3):1-6.

[4] MALL J K,SIMS P,CARR C M.Surface chemical analysis of lipase enzyme treatments on wool and mohair[J].The Journal of the Textile Institute,2002,93(1):43-51.  

[5] 冯宇隆,谢明,闻治国,等.禽类尾脂腺及其生物学功能研究进展[J].中国饲料,2013(9):9-12.

[6] 任露泉,尚广瑞,杨晓东.禽羽结构及羽表脂质对其润湿性能的影响[J].吉林大学学报:工学报,2006,36(2):213-218.

[7] 王德山,张铁鹰,梁争文,等.羽毛中脂质对羽毛角蛋白降解的影响[J].饲料工业,2014,35(19):26-29.

[8] VASILEVA-TONKOVA E,GOUSTEROVA A,NESHEV G.Ecologically safe method for improved feather wastes biodegradation[J].International Biodeterioration & Biodegradation,2009,63(8):1008-1012.  

[9] ZHANG Y H,MENG K,WANG Y R,et al.A novel proteolysis-resistant lipase from keratinolytic Streptomyces fradiae,var. k11[J].Enzyme and Microbial Technology,2008,42(4):346-352.  

[10] 刘春红,冯志彪.以α-乙酸萘酯为底物植物酯酶活力测定条件的优化[J].食品工业科技,2008(6):145-147,151.

[11] 梁争文,张铁鹰,李爽,等.植物角质降解菌的种属鉴定、发酵条件优化及酶学性质研究[J].动物营养学报,2016,28(11):3567-3575.

[12] 王德山.地衣芽孢杆菌CP-16降解羽毛角蛋白相关蛋白酶的分离、表达和相互关系[D].硕士学位论文.北京:中国农业科学院,2014.

[13] NUC P,NUC K.Recombinant protein production in Escherichia coli[J].Postepy Biochemii,2006,52(4):448-456.

[14] JHAMB K,SAHOO D K.Production of soluble recombinant proteins in Escherichia coli:effects of process conditions and chaperone co-expression on cell growth and production of xylanase[J].Bioresource Technology,2012,123:135-143.

[15] LIU Z Q,CHI Z M,WANG C L,et al.Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils[J].Biochemical Engineering Journal,2008,40(3):445-451.  

[16] ÜLKER S,KARAO?LU S A.Purification and characterization of an extracellular lipase from Mucor hiemalis F. corticola isolated from soil[J].Journal of Bioscience and Bioengineering,2012,114(4):385-390.  

[17] DAOUD L,KAMOUN J,ALI M B,et al.Purification and biochemical characterization of a halotolerant Staphylococcus sp. extracellular lipase.[J].International Journal of Biological Macromolecules,2013,57:232-237.

[18] 齐志国.高二硫键还原能力角蛋白降解菌的研究[D].硕士学位论文.北京:中国农业科学院,2012.

[19] GOLDBECK R,FILHO F M.Screening,characterization,and biocatalytic capacity of lipases producing wild yeasts from Brazil biomes[J].Food Science and Biotechnology,2013,22(S1):79-87.  

[20] MHETRAS N C,BASTAWDE K B,GOKHALE D V.Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207[J].Bioresource Technology,2009,100(3):1486-1490.  

[21] MUÑOZ P A,CORREA-LLANTÉN D N,BLAMEY J M.Production,purification and partial characterization of four lipases from a thermophile isolated from Deception Island.[J].Lipids,2013,48(5):527-533.  

[22] LEVISSON M,VAN DER OOST J,KENGEN S W M.Carboxylic ester hydrolases from hyperthermophiles[J].Extremophiles,2009,13:567.

[23] ESTEBAN L,JIMÉNEZ M J,HITA E,et al.Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis[J].Biochemical Engineering Journal,2011,54(1):62-69.  

[24] 阎金勇.白地霉Y162脂肪酶基因克隆与表达[D].硕士学位论文.武汉:华中科技大学,2007.

[25] JOSEPH B,RAMTEKE P W.Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526:partial purification and characterization[J].Annals of Microbiology,2013,63(1):363-370.  

[26] UGUR A,SARAC N,BORAN R,et al.New lipase for biodiesel production:partial purification and characterization of LipSB 25-4[J].ISRN Biochemistry,2014,2014:289749.

[27] SALIBIAN A,MONTALTI D.Physiological and biochemical aspects of the avian uropygial gland[J].Brazilian Journal of Biology,2009,69(2):437-446.  

[28] 陈振林.山羊皮脱脂方法的选择[J].中国皮革,1989(9):12-13.

[29] FRANKA G,MEYER H H,DEUTZ H,et al.Enzyme-catalyzed hydrolysis of 18-methyl eicosanoic acid-cysteine thioester[J].European Journal of Lipid Science and Technology,2003,105(10):627-632.  

[30] DEGANI O,GEPSTEIN S,DOSORETZ C G.Potential use of cutinase in enzymatic scouring of cotton fiber cuticle[J].Applied Biochemistry and Biotechnology,2002,102(1/2/3/4/5/6):277-289.

[31] 王平.羊毛表面生物酶改性及机制研究[D].博士学位论文.无锡:江南大学,2010.
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