Biologia 64/4: 1—, 2009 Section Cellular and Molecular Biology DOI: 10.2478/s11756-009-0140-5 Cloning of a xylanase gene xyn2A from rumen fungus Neocallimastix sp. GMLF2 in Escherichia coli and its partial characterization Ismail Akyol, Ugur Comlekcioglu, Bulent Kar, M. Sait Ekinci & Emin Ozkose* Kahramanmaras Sutcu Imam University, Agriculture Faculty, Animal Science Department, Biotechnology and Gene Engi- neering Laboratory, 46100 Bahcelievler, Kahramanmaras, Turkey; e-mail: eozkose@ksu.edu.tr Abstract: Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50 ◦ C, respectively. The enzyme was stable at 40 ◦ C and 50 ◦ C for 20 min but lost most of its activity when temperature reached 60 ◦ C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. Key words: Neocallimastigaceae; Neocallimastix; xylanase; gene expression; rumen; E. coli. Abbreviations: CTAB, N-cetyl-N,N,N-trimethyl ammonium bromide; GH, glycoside hydrolase; LB, Lysogeny broth; ORF, open reading frame; PCR, polymerase chain reaction. Introduction Plant cell wall is the major carbon source in nature and it is composed mainly from cellulose, hemicellu- lose, pectin and lignin (Thomson 1993). Hemicelluloses are the second abundant polysaccharide after cellulose in the world (Wong et al. 1988) and xylan is the basic polymeric compound of hemicellulose. Xylans possess a backbone consisting of β-1,4-linked xylose units with varying degrees of polymerization (Hespell & White- head 1990). Their structures are relatively more hetero- geneous compared with cellulose. Because of this com- plex structure, complete degradation of xylan requires the synergistic actions of more than one enzyme to gen- erate xylose (Biely 1985). Degradation of xylan by ru- minant herbivores is mainly occurred in the rumen by the synergistic activity of the fibrolytic gut microorgan- isms, such as protozoa, bacteria and fungi which have symbiotic association with the host animals (for a re- view, see Orpin & Joblin 1997). In these habitats rumen microbes, in particular anaerobic fungi, play an important role in the physical and chemical breakdown of ingested plant cell walls by means of their rhizoidal systems and powerful arrays of glycoside hydrolases (GH). This unique group of fungi secretes various plant biomass degrading enzymes, such as cellulase (Barichievich & Calza 1990), hemicellulase (Lowe et al. 1987), amylase (Mountfort & Asher 1989) and protease (Wallace & Joblin 1985). These enzymes are either localized on thalli (Lowe et al. 1987), or are released into the culture medium (Williams & Or- pin 1987). Xylanase is one of the most active enzymes among the endo-acting polysaccharide hydrolases stud- ied from the Neocallimastix frontalis and activity of this enzyme mainly resulted with the releasing of xylobiose as end-product (Mountfort & Asher 1989). Conversion of xylose from the xylooligosaccharides had also been reported for the isolates of the genus Neocallimastix (Pearce & Bauchop 1985). Remarkably high enzymatic activity of rumen fungi was received great impetus by the researchers to express and characterize the genes encoding hemicellu- lolytic enzymes in Escherichia coli. Xylanase genes of xynA (Gilbert et al. 1992), xynB (Black et al. 1994), xyn3 (Durand et al. 1996), xynC (Liu et al. 1999), xyn11A and xyn11B (Huang et al. 2005) from Neocal- limastix sp. were studied extensively. In current study, a fungal isolate Neocallimastix GMLF2, isolated from freshly collected fecal sample of cattle (purified culture has been deposited into the cul- * Corresponding author c 2009 Institute of Molecular Biology, Slovak Academy of Sciences