Biologia 72/8: 831—839, 2017 Section Cellular and Molecular Biology DOI: 10.1515/biolog-2017-0099 Purification and characterization of α-L-arabinofuranosidases from Geobacillus stearothermophilus strain 12 Elif Sevim 1 , Kadriye Inan Bektas 2 , Ali Sevim 1 , Sabriye Canakci 2 *, Iclal Sahin 2 & Ali Osman Belduz 2 1 Genetic and Bioengineering, Faculty of Engineering and Architecture, Ahi Evran University, 40100 Kır¸ sehir, Turkey 2 Department of Biology, Faculty of Science, Karadeniz Technical University, 61000 Trabzon, Turkey; e-mail: sabriye@ktu.edu.tr Abstract: In order to characterize two α- -arabinofuranosidases (α- -AFases), Abf1Geo12 and Abf2Geo12, produced by Geobacillus stearothermophilus strain 12, the genes (abf 1 and abf 2) coding for these enzymes were cloned and sequenced. Based on the protein sequence similarities, approximately 57 kDa two α- -AFases were assigned to the glycoside hydrolase family 51. To obtain pure enzymes, the abf 1 and abf 2 genes were cloned into pET28a+ expression vector and recombinant α- -AFases were produced in E.coli BL21(DE3): pLysS. Characterization of recombinant α- -AFases revealed that Abf1Geo12 and Abf2Geo12 were active in a broad temperature range from 50 to 85 ◦ C and from 40 to 80 ◦ C, respectively. Also, the Abf1Geo12 was active in a broad pH range from 5.0 to 9.0. The optimum pH and temperature for Abf1Geo12 were determined as pH 6.0 and 65 ◦ C, respectively, whereas the optimum pH and temperature for Abf2Geo12 were determined as pH 5.5 and 60 ◦ C, respectively. Based on characterization studies, it was determined that the Abf1Geo12 was more stable than Abf2Geo12 and previously identified α- -AFases from G. stearothermophilus. Using p-nitrophenyl α- -arabinofuranoside as a substrate, the Km and Vmax values for Abf1Geo12 and Abf2Geo12 were determined as 0.31 mM and 290 U/mg for the former enzyme and 0.19 mM and 213.2 U/mg for the latter enzyme, respectively. The activities of Abf1Geo12 and Abf2Geo12 were strongly inhibited by 1 mM Hg 2+ . Interestingly, Cu 2+ and Co 2+ stimulated the activity of Abf1Geo12, but they reduced the activity of Abf2Geo12. The recombinant enzymes released -arabinose from sugar beet arabinan, arabinobiose, arabinotriose, arabinotetraose and arabinopentaose. Consequently, these characterized two enzymes may be used in industrial fields since they are stable at high temperatures. Key words: α- -Arabinofuranosidases; Geobacillus stearothermophilus; thermostable enzymes; thermophilic bacteria. Abbreviations: α- -AFase, α- -arabinofuranosidase; CAZy, Carbohydrate-Active Enzymes database; DNS, dinitrosali- cylic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GH, glycoside hydrolase; IPTG, isopropyl-β-- thiogalactopyranoside; LB, Luria Bertani; PAGE, polyacrylamide gel electrophoresis; pNP, p-nitrophenyl; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TLC, thin-layer chromatography. Introduction Hemicellulose is the second most abundant polymer in the world (Isikgor & Becer 2015). All plant cell walls are composed of lignocelluloses, such as hemicellulose, cellulose, pectin and lignin (Numan & Bhosle 2006). From cereal crops and crop fibre biomass, the break- down of hemicelluloses is achieved by enzymes that are increasingly becoming important due to their pivotal role in the utilization of these renewable energy sources (Wagschal et al. 2008). Many enzymes can perform mi- crobial degradation of hemicellulose materials, such as endoxylanases, β-xylosidase, acetylxylan esterases, glu- curonidases and α-l-arabinofuranosidase (α-l-AFase). While some of these enzymes, e.g. endoxylanases and β-xylosidase, are responsible for the back-bone degra- dation, other group of enzymes that are named as ac- cessory enzymes, e.g. α-l-arabinofuranosidase, are re- sponsible for the cleavage of the side chains (Degrassi et al. 2003). Arabinofuranosidases are hemicellulose degrading enzymes, which cleave l-arabinofuranosyl residues. Within this group of enzymes, the α-l-AFases specif- ically catalyse the hydrolysis of terminal non-reducing α-l-1,2-, α-l-1,3- and α-l-1,5-arabinofuranosyl residues belonging to different hemicellulosic homopolysaccha- rides (branched and debranched arabinans) and het- eropolysaccharides (arabinoxylans, arabinogalactans, etc.) (Saha 2000; Margolles & de los Reyes-Gavilan 2003; Inacio et al. 2008). Endo-arabinase and α-l- AFase are major hydrolytic enzymes essential to gen- erate l-arabinose from arabinan. To produce arabino- oligosaccharides or l-arabinoses, the arabinan backbone of α-1,5-linked l-arabinofuranosyl should be hydrol- * Corresponding author c 2017 Institute of Molecular Biology, Slovak Academy of Sciences Brought to you by | University of Sussex Library Authenticated Download Date | 9/18/17 9:51 AM