[1]王 军,康利琴,刘中华,等.一种灰盖鬼伞外切β-1,3葡聚糖酶基因的克隆与表达[J].南京师范大学学报(自然科学版),2016,39(03):96.[doi:10.3969/j.issn.1001-4616.2016.03.016]
Wang Jun,Kang Liqin,Liu Zhonghua,et al.Cloning and Expression of an Exo-β-1,3 glucanaseGene from Coprinopsis cinerea[J].Journal of Nanjing Normal University(Natural Science Edition),2016,39(03):96.[doi:10.3969/j.issn.1001-4616.2016.03.016]
点击复制
一种灰盖鬼伞外切β-1,3葡聚糖酶基因的克隆与表达(
)
《南京师范大学学报》(自然科学版)[ISSN:1001-4616/CN:32-1239/N]
- 卷:
-
第39卷
- 期数:
-
2016年03期
- 页码:
-
96
- 栏目:
-
·生命科学·
- 出版日期:
-
2016-09-30
文章信息/Info
- Title:
-
Cloning and Expression of an Exo-β-1,3 glucanaseGene from Coprinopsis cinerea
- 文章编号:
-
1001-4616(2016)03-0096-06
- 作者:
-
王 军; 康利琴; 刘中华; 袁 生
-
南京师范大学生命科学学院,江苏省微生物与微生物功能基因组学重点实验室,江苏省微生物资源产业化工程技术研究中心,江苏 南京 210023
- Author(s):
-
Wang Jun; Kang Liqin; Liu Zhonghua; Yuan Sheng
-
School of Life Sciences,Nanjing Normal University,Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics,Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources,Nanjing 210023,China
-
- 关键词:
-
外切β-1; 3葡聚糖酶; 基因克隆; 异源表达; 生化特性; 灰盖鬼伞
- Keywords:
-
exo-β-1; 3 glucanase; gene cloning; heterologous expression; biochemical characterization; Coprinopsis cinerea
- 分类号:
-
Q786
- DOI:
-
10.3969/j.issn.1001-4616.2016.03.016
- 文献标志码:
-
A
- 摘要:
-
为了简单快速地获取大量的β-1,3葡聚糖酶以研究其在真菌形态发生过程中所发挥的作用,本研究以灰盖鬼伞的cDNA为模板克隆了一种编码外切β-1,3葡聚糖酶的基因,并将该基因插入表达质粒pET28A(+)中,获得了重组表达质粒pET28A(+)-exg,转化到E. coli Rosetta菌株中并进行异源重组表达. 结果显示,克隆基因的cDNA全长2 415 bp,共编码786个氨基酸;SDS-PAGE电泳表明该基因在E.coli Rosetta菌株中得到了高效表达,重组表达的酶蛋白表观分子量为85 kDa;纯化后获得的表达酶经DNS法测得比活力为45 U/mg. 酶学性质测定结果表明,该酶具有β-1,3葡聚糖外切酶活性,以昆布多糖为底物时,最适反应条件为pH 6.0、温度60 ℃,且有一定的耐热能力和较好的pH稳定性,具有较好的应用前景.
- Abstract:
-
To acquire enough β-1,3 glucanase for investigating its role in morphological changes of fungus,an exo-β-1,3 glucanase gene(exg)was cloned from cDNA of Coprinopsis cinerea okayama 7#130 and inserted into plasmid pET28A(+)to generate the recombinant expression plasmid pET28A(+)-exg. pET28A(+)-exg was transformed into Escherichia coli Rosetta strain for heterologous expression. The result showed that the length of exg gene is 2 415 bp,which encodes 786 amino acids. SDS-PAGE analysis showed that the exo-β-1,3 glucanase was effectively expressed in E. coli Rosetta with an apparent molecular masses of 85 kDa. The specific enzyme activity was 45 U/mg after purification. Characterization of the recombinant enzyme showed that it had an exo-hydrolases activity and the optimum pH value was 6.0,and the optimum reaction temperature was at 60 ℃ when laminarin as substrate. Besides,the thermostability and pH stability give it some prospect in the future.
参考文献/References:
[1] MARTIN K,MCDOUGALL B M,MCILROY S,et al. Biochemistry and molecular biology of exocellular fungal β-(1,3)- and β-(1,6)-glucanases[J]. FEMS microbiology reviews,2007,31(2):168-192.
[2] REESE E. Degradation of polymeric carbohydrates by microbial enzymes[G]//The structure,biosynthesis,and degradation of wood. New York:Plenum Press,1977,11:311-367.
[3] YAMAMOTO R,NEVINS D J. Degradation of a glucan containing β-(1→3)and β-(1→6)linkages by exo-(1→3)-β-d-glucanase[J]. Carbohydrate research,1983,122(2):217-226.
[4] PITSON S,SEVIOUR R,MCDOUGALL B. Noncellulolytic fungal β-glucanases:their physiology and regulation[J]. Enzyme and microbial technology,1993,15(3):178-192.
[5] REESE E T,MANDELS M. β-d-1,3 glucanases in fungi[J]. Canadian journal of microbiology,1959,5(2):173-185.
[6] LATGé J P. The cell wall:a carbohydrate armour for the fungal cell[J]. Molecular microbiology,2007,66(2):279-290.
[7] ADAMS D J. Fungal cell wall chitinases and glucanases[J]. Microbiology,2004,150(7):2 029-2 035.
[8] BOWMAN S M,FREE S J. The structure and synthesis of the fungal cell wall[J]. Bioessays,2006,28(8):799-808.
[9] CHET I,ORDENTLICH A,SHAPIRA R,et al. Mechanisms of biocontrol of soil-borne plant pathogens by rhizobacteria[J]. Plant and soil,1990,129(1):85-92.
[10] PEBERDY J. Fungal cell walls—a review[G]//Biochemistry of cell walls and membranes in fungi. Berlin:Springer,1990:5-30.
[11] SAKURAI N. Cell wall functions in growth and development—a physical and chemical point of view[J]. The botanical magazine=Shokubutsu-gaku-zasshi,1991,104(3):235-251.
[12] FONTAINE T,HARTLAND R P,DIAQUIN M,et al. Differential patterns of activity displayed by two exo-beta-1,3-glucanases associated with the Aspergillus fumigatus cell wall[J]. Journal of bacteriology,1997,179(10):3 154-3 163.
[13] MOUYNA I,SARFATI J,RECCO P,et al. Molecular characterization of a cell wall-associated β(1-3)endoglucanase of Aspergillus fumigatus[J]. Medical mycology,2002,40(5):455-464.
[14] DELGADO N,XUE J,YU J J,et al. A recombinant β-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis[J]. Infection and immunity,2003,71(6):3 010-3 019.
[15] COLE G T,HUNG C Y. The parasitic cell wall of Coccidioides immitis[J]. Medical mycology,2001,39(1):31-40.
[16] ZHOU Y,ZHANG W,LIU Z,et al. Purification,characterization and synergism in autolysis of a group of β-1,3-glucan hydrolases from the pilei of Coprinopsis cinerea fruiting bodies[J]. Microbiology,2015,161(10):1 978-1 989.
[17] AMANN E,BROSIUS J,PTASHNE M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli[J]. Gene,1983,25(2):167-178.
[18] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature,1970,227(5259):680-685.
[19] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical biochemistry,1976,72(1):248-254.
[20] MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical chemistry,1959,31(3):426-428.
[21] O’CONNELL E,PIGGOTT C,TUOHY M. Purification of exo-1,3-beta-glucanase,a new extracellular glucanolytic enzyme from Talaromyces emersonii[J]. Applied microbiology and biotechnology,2011,89(3):685-696.
[22] KUMAGAI Y,SATOH T,INOUE A,et al. A laminaribiose-hydrolyzing enzyme,AkLab,from the common sea hare Aplysia kurodai and its transglycosylation activity[J]. Comparative biochemistry and physiology part B:biochemistry and molecular biology,2014,167:1-7.
[23] SAHA B C,BOTHAST R J. Production,purification,and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata[J]. Applied and environmental microbiology,1996,62(9):3 165-3 170.
备注/Memo
- 备注/Memo:
-
收稿日期:2015-09-08.
基金项目:国家自然科学基金面上项目(31170028)、江苏省自然科学基金青年基金(BK20140918)、江苏省高校自然科学研究项目(14KJB180013).
通讯联系人:袁生,教授,博士生导师,研究方向:微生物生长代谢调控和酶工程研究. E-mail:yuansheng@njnu.edu.cn
更新日期/Last Update:
2016-09-30