Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α

BAYLAN, MAKBULE; ÖZCAN, Numan; Guzel, Ali İrfan; ÖZCAN, BAHRİ DEVRİM

Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α

Bahri Devrim ÖZCAN, (Korkut Ata Üniversitesi Fen Edebiyat Fakültesi, Biyoloji, Bölümü, Osmaniye, Türkiye)

Numan ÖZCAN, (Çukurova Üniversitesi, Ziraat Fakültesi, Zootekni Bölümü, Adana, Türkiye)

Makbule BAYLAN, (Çukurova Üniversitesi, Su Ürünleri Fakültesi, Temel Bilimler Bölümü, Adana, Türkiye)

Ali İrfan GÜZEL (Recep Tayyip Erdoğan Üniversitesi, Temel Tıp Bilimleri Bölümü Rize, Türkiye)

Kafkas Üniversitesi Veteriner Fakültesi Dergisi

6 0

Yıl: 2013 Cilt: 19 Sayı: 3 Sayfa Aralığı: 523 - 528 Metin Dili: Türkçe İndeks Tarihi: 29-07-2022

Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α

Öz:

Bu çalışmada Cellulosimicrobium cellulans bakterisine ait β-1,3-glukanaz geni PCR ile amplifiye edilerek pUC18 klonlama vektörüne klonlanmış, böylece pTEG5 ve pTEG11 rekombinant plazmit DNAlar elde edilmiştir. Rekombinant plazmit DNAlar pTEG5 ve pTEG11 kompetent Escherichia coli hücrelerine transfer edilmişlerdir. SacI enzimi ile kesilmiş rekombinant plazmitlerin agaroz jelde elektroforezi sonucu 1.9 kbç büyüklüğündeki β-1,3-glukanaz gen bandının görünmesi, gen entegrasyonunun tamam olduğunu göstermiştir. β-1,3-glukanaz geninin rekombinant vektörlerden amplifikasyonu da 1.9 kbç büyüklüğündeki geni göstermiştir. Rekombinant enzim E. coli tarafından hücre içi olarak üretilmiştir. LB-laminarin-agar plağına damlatılan rekombinant E. coli suşlarının intraselüler içerikleri Congo-red boyaması ile pozitif zonlar üretmiş, böylece rekombinant bakterilerce üretilen proteinin aktif olduğu anlaşılmıştır. Zimogram analizinde, hücre içi üretilen rekombinant β-1,3-glukanaz enzimleri C. cellulans enzimi ile aynı moleküler ağırlığa ait aktivite bantları sergilemişlerdir.

Anahtar Kelime: DNA klonlama enzimler plazmitler enzim aktivitesi Escherichia coli gen ifadesi genler

Konular: Veterinerlik

Cellulosimicrobium cellulans dan β-1,3-glukanaz geninin Escherichia coli DH5α da klonlanması ve ekspresyonu

Öz:

In this study, β-1,3-glucanase gene of Cellulosimicrobium cellulans was amplified by PCR and cloned in pUC18 cloning vector to construct the recombinant plasmids pTEG5 and pTEG11. The recombinant plasmids pTEG5 and pTEG11 were transformed into competent Escherichia coli cells. Digestion of recombinant plasmids with SacI produced 1.9 kbp β-1,3-glucanase gene band on agarose gel which indicated the gene integration. β-1,3-Glucanase gene amplification on the recombinant vectors also indicated 1.9 kbp gene insert. Recombinant enzyme was produced by E. coli intracellularly. Intracellular components of recombinant E. coli strains with pTEG5 or pTEG11 dropped on LB-laminarin-agar plate, showed clear positive zones by Congo-red staining revealing the activity of secreted protein. Based on the zymogram analysis, the intracellular produced recombinant β-1,3-glucanase enzymes exhibited the same activity bands with C. cellulans enzyme with respect to molecular weight.

Anahtar Kelime: enzyme activity Escherichia coli gene expression genes DNA cloning enzymes plasmids

Konular: Veterinerlik

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

1. Manners DJ, Masson AS, Patterson JC: The structure of a β-(1 leads to 3)-D-glucan from yeast cell walls. Biochem J, 135 (1): 19-30, 1973.
2. Shen S-H, Chretien P, Bastien L, Slilaty SN: Primary sequence of the glucanase gene from Oerskovia xanthineolytica. J Biol Chem, 266 (2): 1058-1063, 1991.
3. Salazar O, Molitor J, Lienqueo ME, Asenjo JA: Overproduction, purification, and characterization of β-1,3-glucanase type II in Escherichia coli. Protein Express Purif, 23 (2): 219-225, 2001.
4. Tanabe Y, Pang Z, Oda M: Cloning and sequencing of endo-1,3-β-glucanase from Cellulosimicrobium cellulans. J Biol Macromol, 8 (3): 60-63, 2008.
5. Ferrer P, Halkier T, Hedegaard L, Savva D, Diers I, Asenjo JA: Nucleotide sequence of a β-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis. J Bacteriol, 178 (15): 4751-4757, 1996.
6. Ferrer P: Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review. Microb Cell Fact, 5 (10): 1-8, 2006.
7. Salazar O, Asenjo JA: Enzymatic lysis of microbial cells. Biotechnol Lett, 29 (7): 985-994, 2007.
8. Kitamura K, Yamamoto Y: Purification and properties of an enzyme, zymolyase, which lyses viable yeast cells. Arch Biochem Biophys, 153 (1): 403-406, 1972.
9. Teather RM, Wood PJ: Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol, 43 (4): 777-780, 1982.
10. Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 7 (6): 1513-1523, 1979.
11. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 1989.
12. Hanahan D: Techniques for transformation of Escherichia coli. In, Glover DM (Ed): DNA Cloning: A Practical Approach. (1): 109-135, IRL. Press Inc., Oxford, United Kingdom, 1985.
13. Laemmli UK: Cleauage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227 (5259): 680-685, 1970.
14. Saul DJ, Williams LC, Grayling RA, Chamley LW, Love DR, Bergquist PL: celB, a gene coding for a bifunctional cellulase from the extreme thermophile Caldocellum saccharolyticum. Appl Environ Microbiol, 56 (10): 3117-3124, 1990.
15. Ozcan N, Cunningham C, Haris WJ: Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product. Lett Appl Microbiol, 22 (1): 85-89, 1996.
16. Lee SP, Morikawa M, Takagi M, Imanaka T: Cloning of the aapT gene and characterization of its product, α-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601. Appl Environ Microbiol, 60 (10): 3764-3773, 1994.
17. Ikeda T, Yamazaki H, Yamashita K, Shinke R: The tetracycline inducible expression of alpha-amylase in Bacillus subtilis. J Ferment Bioengineer, 74, 58-60, 1992.
18. Coral G, Arikan B, Unaldi MN, Guvenmez H: Some properties of crude carboxymethyl cellulase of Aspergillus niger Z10 wild-type strain. Turk J Biol, 26, 209-213, 2002.
19. Scott JH, Schekman R: Lyticase: Endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol, 142 (2): 414-423, 1980.
20. Cheong YH, Kim CY, Chun HJ, Moon BC, Park HC, Kim JK, Lee S, Han C, Lee SY, Cho MJ: Molecular cloning of a soybean class III beta-1,3-glucanase gene that is regulated both developmentally and in response to pathogen infection. Plant Sci, 154 (1): 71-81, 2000.
21. Tian SP, Yao EJ, Deng X, Xu XB, Qin GZ, Chan ZL: Characterization and expression of β-1,3-glucanase genes in jujube fruit induced by the microbial biocontrol agent Cryptococcus laurentii. Phytopathology, 97 (3): 260-268, 2007.
22. Yamaguchi T, Nakayama K, Hayashi T, Tanaka Y, Koike S: Molecular cloning and characterization of a novel β-1,3-glucanase gene from rice. Biosci Biotechnol Biochem, 66 (6): 1403-1406, 2002.
23. Ferrer P, Hedegaard L, Halkier T, Diers I, Savva D, Asenjo JA: Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall. Ann NY Acad Sci, 782, 555-565, 1996.
24. Sandkvist M, Bagdasarian M: Secretion of recombinant proteins by Gram-negative bacteria. Curr Opin Biotechnol, 7 (5): 505-511, 1996.
25. Waegeman H, De Mey M: Increasing recombinant protein production in E. coli by an alternative method to reduce acetate. Advances in Applied Biotechnology, (6):127-144, 2012.
26. Pugsley AP: The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev, 57 (1): 50-108, 1993.
27. Economou A: Following the leader: Bacterial protein export through the Sec pathway. Trends Microbiol, 7 (8): 315-320, 1999.
28. Mergulhão FJM, Summers DK, Monteiro GA: Recombinant protein secretion in Escherichia coli. Biotech Adv, 23, 177-202, 2005.
29. Baneyx F: Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol, 10 (5): 411-421, 1999.
30. Mergulhão F, Monteiro G: Secretion capacity limitations of the Sec pathway in Escherichia coli. J Microb Biotechnol, 14, 128-133, 2004.
31. Rosenberg HF: Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques, 24 (2): 188-190, 1998.
32. Huang HC, Sherman MY, Kandror O, Goldberg AL: The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli. J Biol Chem, 276 (6): 3920-3928, 2001.
33. Koster M, Bitter W, Tommassen J: Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol, 290 (4-5): 325-331, 2000.
34. Nakai K, Kanehisa M: Expert systems for predicting protein localization sites in gram-negative bacteria. Proteins, 11 (2): 95-110, 1991.
35. Aftab S, Aftab MN, Haq IU, Javed MM, Zafar A, Iqbal I: Cloning and expression of endo-1,4-β-glucanase gene from Bacillus licheniformis ATCC 14580 into Escherichia coli BL21 (DE 3). Afr J Biotechnol, 11 (12): 2846-2854, 2012.
36. Doi K, Doi A: Cloning and expression in Escherichia coli of the gene for an Arthrobacter β-(1→3)-glucanase. J Bacteriol, 168 (3): 1272-1276, 1986.
37. Nagata S, Maru I, Ishihara F, Misono H, Nagasaki S: Cloning and expression of endo-beta-1,3-glucanase gene from Flavobacterium dormitator in Escherichia coli and characterization of the gene product. Agric Biol Chem, 54 (10): 2675-2680, 1990.
38. Choi JH, Lee ST: Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol, 64 (5): 625-635, 2004.
39. Jeang CL, Chen LS, Chen MY, Shiau RJ: Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli. Appl Environ Microbiol, 68 (7): 3651-3654, 2002.
40. Robson LM, Chambliss GH: Cloning of the Bacillus subtilis DLG β-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis. J Bacteriol, 165 (2): 612-619, 1986.

APA	ÖZCAN B, ÖZCAN N, BAYLAN M, Guzel A (2013). Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. , 523 - 528.
Chicago	ÖZCAN BAHRİ DEVRİM,ÖZCAN Numan,BAYLAN MAKBULE,Guzel Ali İrfan Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. (2013): 523 - 528.
MLA	ÖZCAN BAHRİ DEVRİM,ÖZCAN Numan,BAYLAN MAKBULE,Guzel Ali İrfan Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. , 2013, ss.523 - 528.
AMA	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. . 2013; 523 - 528.
Vancouver	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. . 2013; 523 - 528.
IEEE	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A "Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α." , ss.523 - 528, 2013.
ISNAD	ÖZCAN, BAHRİ DEVRİM vd. "Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α". (2013), 523-528.

APA	ÖZCAN B, ÖZCAN N, BAYLAN M, Guzel A (2013). Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 19(3), 523 - 528.
Chicago	ÖZCAN BAHRİ DEVRİM,ÖZCAN Numan,BAYLAN MAKBULE,Guzel Ali İrfan Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 19, no.3 (2013): 523 - 528.
MLA	ÖZCAN BAHRİ DEVRİM,ÖZCAN Numan,BAYLAN MAKBULE,Guzel Ali İrfan Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, vol.19, no.3, 2013, ss.523 - 528.
AMA	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2013; 19(3): 523 - 528.
Vancouver	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2013; 19(3): 523 - 528.
IEEE	ÖZCAN B,ÖZCAN N,BAYLAN M,Guzel A "Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α." Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 19, ss.523 - 528, 2013.
ISNAD	ÖZCAN, BAHRİ DEVRİM vd. "Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α". Kafkas Üniversitesi Veteriner Fakültesi Dergisi 19/3 (2013), 523-528.