Screening, Cloning and Characteristics of the Common Xylanase Gene in Anaerobic Fungi
محورهای موضوعی : Camel
U. Comlekcioglu
1
(Department of Biology, Faculty of Science and Arts, Osmaniye Korkut Ata University, Osmaniye, Turkiye)
کلید واژه: Ruminant, cloning, anaerobic fungi, in silico analysis, xylanase,
چکیده مقاله :
The aim of this study was to screen, clone and characterize the xylanase genes and to determine the common xylanase gene in anaerobic fungi. For this purpose, genomic DNA of 45 anaerobic fungi were used to amplify xylanase genes using 9 different primer pairs. The PCR yield rates of the primers in fungal isolates ranged from 6.6% to 100%. The xynA gene encoding xylanase was amplified from all anaerobic fungal DNAs with OrpXA primers (100%). The xynA was cloned into E. coli and 17 recombinant E. coli strains were obtained. The nucleotide sequences of the cloned genes were determined and characterized. The molecular weight of open reading frames (ORF) regions of the cloned genes varied between 24.7-30.2 kDa and the catalytic domains are members of glycoside hydrolase family 11. The specific activity of xylanase enzymes varied between 4.99-37.6 U/mg. Xylanase enzymes showed remaining activities ranging between 71.52-100% after incubation at 50 ˚C for 1 hour. High correlation was found between specific activity and thermal stability. This study showed that the xynA gene is common in anaerobic fungi, but this finding needs to be validated with further studies including species from the genera not included in this study.
Black G.W., Hazlewood G.P., Xue G.P., Orpin C.G. and Gilbert H.J. (1994). Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide. Biochem. J. 299(2), 381-387.
Borneman W.S., Akin D.E. and Ljungdahl L.G. (1989). Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi. Appl. Environ. Microbiol. 55, 1066-1073.
Chang K.Y. and Yang J.R. (2013). Analysis and prediction of highly effective antiviral peptides based on random forests. PLoS One. 8, e70166.
Collins T., Gerday C. and Feller G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29, 3-23.
Comlekcioglu U., Aygan A., Yazdic F.C. and Ozkose E. (2011). Effects of various agro-wastes on xylanase and b-xylosidase production of anaerobic ruminal fungi. J. Sci. Ind. Res. 70, 293-299.
Comlekcioglu U., Ozkose E., Tutus A., Akyol I. and Ekinci M. (2010). Cloning and characterization of cellulase and xylanase coding genes from anaerobic fungus Neocallimastix sp. GMLF1. Int. J. Agric. Biol. 12, 691-696.
Comlekcioglu U., Yazdic F.C., Keser S., Kelleci B.M. and Battaloglu G. (2012). Effects of carbon sources on enzyme production of Neocallimastix sp. ve Orpinomyces sp. Kafkas Univ. Vet. Fak. Derg. 18, 799-806.
Dagar S.S., Kumar S., Mudgil P. and Puniya A.K. (2018). Comparative evaluation of lignocellulolytic activities of filamentous cultures of monocentric and polycentric anaerobic fungi. Anaerobe. 50, 76-79.
Daniel R.M. and Danson M.J. (2010). A new understanding of how temperature affects the catalytic activity of enzymes. Trends Biochem. Sci. 35, 584-591.
Fanutti C., Ponyi T., Black G.W., Hazlewood G.P. and Gilbert H.J. (1995). The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J. Biol. Chem. 270, 29314-29322.
Gamage D.G., Gunaratne A., Periyannan G.R. and Russell T.G. (2019). Applicability of instability index for in vitro protein stability prediction. Protein Pept. Lett. 26, 339-347.
Garcia-Vallvé S., Romeu A. and Palau J. (2000). Horizontal gene transfer of glycosyl hydrolases of the rumen fungi. Mol. Biol. Evol. 17, 352-361.
Gasteiger E., Hoogland C., Gattiker A., Wilkins M.R., Appel R.D. and Bairoch A. (2005). Protein identification and analysis tools on the ExPASy server. Pp. 571-607 in The Proteomics Protocols Handbook. J.M. Walker, Ed., Humana Press, New York.
Gilbert H.J., Hazlewood G.P., Laurie J.I., Orpin C.G. and Xue G.P. (1992). Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin. Mol. Microbiol. 6, 2065-2072.
Gruninger R.J., Nguyen T.T.M., Reid I.D., Yanke J.L., Wang P., Abbott D.W., Tsang A. and McAllister T. (2018). Application of transcriptomics to compare the carbohydrate active enzymes that are expressed by diverse genera of anaerobic fungi to degrade plant cell wall carbohydrates. Front. Microbiol. 9, 1-15.
Haki G.D. and Rakshit S.K. (2003). Developments in industrially important thermostable enzymes: A review. Bioresour. Technol. 89, 17-34.
Hanafy R.A., Elshahed M.S., Liggenstoffer A.S., Griffith G.W. and Youssef N.H. (2017). Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep. Mycologia. 109, 231-243.
Ikai A. (1980). Thermostability and aliphatic index of globular proteins. J. Biochem. 88, 1895-1898.
Joshi A., Lanjekar V.B., Dhakephalkar P.K., Callaghan T.M., Griffith G.W. and Dagar S.S. (2018). Liebetanzomyces polymorphus gen. et sp. nov., a new anaerobic fungus (Neocallimastigomycota) isolated from the rumen of a goat. MycoKeys. 40, 89-110.
Kulkarni N., Shendye A. and Rao M. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23, 411-456.
Li X.L., Chen H. and Ljungdahl L.G. (1997). Monocentric and polycentric anaerobic fungi produce structurally related cellulases and xylanases. Appl. Environ. Microbiol. 63, 628-635.
Liu J.R., Duan C.H., Zhao X., Tzen J.T.C., Cheng K.J. and Pai C.K. (2008). Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies. Appl. Microbiol. Biotechnol. 79, 225-233.
Ljungdahl L.G. (2008). The cellulase/hemicellulase system of the anaerobic fungus Orpinomyces PC-2 and aspects of its applied use. Ann NY Acad. Sci. 1125, 308-321.
Lombard V., Golaconda Ramulu H., Drula E., Coutinho P.M. and Henrissat B. (2013). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, 490-495.
Lowe S.E., Theodorou M. and Trinci A. (1987). Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan. Appl. Environ. Microbiol. 53, 1216-1223.
Mandel M. and Higa A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159-162.
Matsui H. and Ban-Tokuda T. (2008). Studies on carboxymethyl cellulase and xylanase activities of anaerobic fungal isolate CR4 from the bovine rumen. Curr. Microbiol. 57, 615-619.
Miller G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428.
Mountfort D.O. and Asher R.A. (1989). Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 55, 1016-1022.
Nicholson M.J., Theodorou M.K. and Brookman J.L. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces – insights into an AT-rich genome. Microbiology. 151, 121-133.
Novotná Z., Procházka J., Šimůnek J. and Fliegerová K. (2010). Xylanases of anaerobic fungus Anaeromyces mucronatus. Folia Microbiol. 55, 363-367.
Orpin C.G. (1976). Studies on the rumen flagellate Sphaeromonas communis. J. Gen. Microbiol. 94, 270-280.
Pai C.K., Wu Z.Y., Chen M.J., Zeng Y.F., Chen J.W., Duan C.H., Li M.L. and Liu J.R. (2010). Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum. Appl. Microbiol. Biotechnol. 85, 1451-1462.
Paul S.S., Deb S.M., Punia B.S., Singh D. and Kumar R. (2010). Fibrolytic potential of anaerobic fungi (Piromyces sp.) isolated from wild cattle and blue bulls in pure culture and effect of their addition on in vitro fermentation of wheat straw and methane emission by rumen fluid of buffaloes. J. Sci. Food Agric. 90, 1218-1226.
Sarkar S., Banerjee A., Chakraborty N., Soren K., Chakraborty P. and Bandopadhyay R. (2020). Structural-functional analyses of textile dye degrading azoreductase, laccase and peroxidase: A comparative in silico study. Electron J. Biotechnol. 43, 48-54.
Stabel M., Schweitzer T., Haack K., Gorenflo P., Aliyu H. and Ochsenreither K. (2021). Isolation and biochemical characterization of six anaerobic fungal strains from zoo animal feces. Microorganisms. 9, 1655-1662.
Teather R.M. and Wood P.J. (1982). Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777-780.
Thomson J.A. (1993). Molecular biology of xylan degradation. FEMS Microbiol. Lett. 104, 65-82.
Verma D., Kawarabayasi Y., Miyazaki K. and Satyanarayana T. (2013). Cloning, expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene (Mxyl) retrieved from compost-soil metagenome. PLoS One. 8, e52459.
Xue G.P., Gobius K.S. and Orpin C.G. (1992). A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities. Microbiology. 138, 2397-2403.
Yousefi N. and Abbasi S. (2022). Food proteins: Solubility and thermal stability improvement techniques. Food Chem. Adv. 1, 100090.
