Extraction, Sequence Alignment and Cloning of Recombinant Papain from Carica papaya

Musa Sale Makeri *

Department of Biological Sciences, Federal University of Kashere, Gombe State, Nigeria.

John Otumala Egbere

Department of Microbiology, University of Jos, Plateau State, Nigeria.

Victoria Kaneng Pam

Department of Microbiology, University of Jos, Plateau State, Nigeria.

Margaret Musa Atsen Danladi

Department of Microbiology, Plateau State University, Bokkos, Nigeria.

Ayika Philomena Dan

Department of Microbiology, Plateau State University, Bokkos, Nigeria.

Munir Umar

Technical Unit, National Biotechnology Development Agency (NABDA), Nigeria.

Usman Usman Musa

Department of pharmaceutical Microbiology/Biotechnology University of Jos, Nigeria.

Hussein Ridwan Abdulsalam

Department of Basic-Science Federal College of Forestry Mechanization Afaka, Kaduna State, Nigeria.

*Author to whom correspondence should be addressed.


Abstract

Papain is a proteolytic enzyme obtained from the fruits of carica papaya with abundant therapeutic, food, industrial and analytical applications. The challenges related to the development of papain technology on industrial scale include cost of production and downstream processing of the enzyme. In the present research, the responsible for gene encoding papain enzyme was obtained from the carica papaya plant, sequenced and amplified using polymerase chain reaction. This papain gene was initially converted to cDNA and then the papain gene was sequence to identify the region of similarity that may be a classify base on functional, structure, and  phylogenic relationships between the known papain DNA sequence and the sample Papain gene. The papain gene was then clone into the pBR322 cloning vector to create recombinant protein molecules. This showed that the DNA sequencing of the extracted genes exhibited an acceptable level of similarity to the corresponding gene (MEROO647) with identity of 98% and E-value 0.246 X10-9 from the public database NCBI (National Centre for Biotechnology information) In contrast, the similar papain genes sequence (P00784, MEROO647, and AT3G5470) in NCBI extracted from other sources in this study, the papaya gene sequences was obtained from carica papaya (fruit) and in the sequence similarity analysis because the product was amplified from mRNA that was extracted from carica papaya fruit.

Keywords: Recombinant papain, extraction, sequencing, cloning, Carica papaya


How to Cite

Makeri, M. S., Egbere, J. O., Pam, V. K., Danladi , M. M. A., Dan, A. P., Umar, M., Musa, U. U., & Abdulsalam, H. R. (2024). Extraction, Sequence Alignment and Cloning of Recombinant Papain from Carica papaya. Asian Journal of Biotechnology and Genetic Engineering, 7(1), 79–84. Retrieved from https://journalajbge.com/index.php/AJBGE/article/view/126

Downloads

Download data is not yet available.

References

Abernethy JL, Lovett Jr, CM, Hoddad A, Felberg JD (20100.: Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides. Bioorg. Chem. ll 251-261.

Arnon R, Shapira E. Antibodies to papain. A selective fractionation according to inhibitory capacity. Biochemistry. 2011; 6:3942-3950.

Behrens OK, Bergmann M. Cosubstrates in proteolysis. J. Bioi. Chem. 1999;129:587-602.

Amon R. The reaction of papain with antipapain. Immunochemistry. 2000;2:107-114.

Nallamsetty S, Waugh D. Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 2006;45: 175–182.

Palma JM, Sandalio LM, Corpas FJ, Romero-Puertas MC, McCarthy I, & Del- Rio, L.A. (2002). Plant proteases, protein degradation, and oxidative stress: role of peroxisomes. Plant Physiol. Biochem., 40, 521–30.

Aehle W. Enzymes in industry: Production and applications (2nd edn). Weinheim: Wiley -VCH/Verlag GmbH & Co; 2013.

Harrach T, Eckert K, Schulze-Forster K, Nuck R, Grunow D, Maurer HR. Isolation and partial characterization of basic proteinases from stem bromelain. J Protein Chem. 1995;14:41–52.

Roep BO, Van den Engel NK, Van Halteren AGS, Duinkerken G, Martin S. Modulation of autoimmunity to beta-cell antigens by proteases. Diabetologia. 2002;45: 686-692.

Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada K. L-trans- Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L, Biochem. J., 1982;201:189–198.

Benucci I, Liburdi K, Garzillo AMV, Esti M. Bromelain from pineapple stem in alcoholic–acidic buffers for wine application. Food Chem. 2011;124:1349–1353.

Beuth J, Braun JM. Modulation of murine tumor growth and colonization by bromelaine, an extract of the pineapple plant (Ananas comosus L.). In Vivo. 2015;19:483–485.

Hoffmann A, Roeder RG. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res 1991;19:6337–8.

Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci USA. 1991;88:8972–6.

Ahmad B, Khan RH. Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: A comparison with catalytically inactive form. J. Biochem. 2016;140 (4): 501-508.

Alloue WAM, Destain J, Amighi K, Thonart P. Storage of Yarrowia lipolytica lipase after spray-drying in the presence of additives. Process Biochemistry. 2017;42:1357–1361.

Baker EN, Drenth J. Active sites of enzymes, In: Jurnak, F.A. & McPherson, A. (eds); 1987.

Abdulmajid FA, Abdulgani M, Talib SZ, Hasyim KK. Stability of bromelain-polyphenol complex in pineapple juice. Jurnal Teknologi. 2018; 49:27-38.