Purification and characterization of α-L-arabinofuranosidases from Geobacillus stearothermophilus strain 12

• Published in: Biológia Vol. 72; no. 8; pp. 831 - 839
• Main Authors: Sevim, Elif, Inan Bektas, Kadriye, Sevim, Ali, Canakci, Sabriye, Sahin, Iclal, Belduz, Ali Osman
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.08.2017; De Gruyter; Springer Nature B.V
• Subjects: Amino acid sequence; Arabinose; Carbon dioxide; Cell Biology; Cobalt; Copper; E coli; Enzymes; Gene expression; Genes; Geobacillus stearothermophilus; Glycoside hydrolase; High temperature; Hydrolase; L-Arabinofuranosidases; L-Arabinose; Life Sciences; Mercury (metal); Microbiology; p-Nitrophenyl-a-L-arabinofuranoside; pH effects; Plant Sciences; Proteins; Substrates; Temperature; Temperature effects; thermophilic bacteria; thermostable enzymes; Zoology; Arabinofuranosidases; -; thermophilic bacteria; thermostable enzymes
• ISSN: 0006-3088; 1336-9563
• DOI: 10.1515/biolog-2017-0099

In order to characterize two α - L -arabinofuranosidases ( α - L -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 -L-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 -L-AFases were produced in E.coli BL21(DE3): pLysS. Characterization of recombinant α -L-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 -L-AFases from G. stearothermophilus . Using p -nitrophenyl α -L-arabinofuranoside as a substrate, the K m and V max 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 L-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.