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 ,  and Ali Osman Belduz 2
  • 1 Genetic and Bioengineering, Faculty of Engineering and Architecture, Ahi Evran University, 40100, Kırşehir, Turkey
  • 2 Department of Biology, Faculty of Science, Karadeniz Technical University, 61000, Trabzon, Turkey
Elif Sevim
  • Genetic and Bioengineering, Faculty of Engineering and Architecture, Ahi Evran University, 40100, Kırşehir, Turkey
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, Kadriye Inan Bektas
  • Department of Biology, Faculty of Science, Karadeniz Technical University, 61000, Trabzon, Turkey
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, Ali Sevim
  • Genetic and Bioengineering, Faculty of Engineering and Architecture, Ahi Evran University, 40100, Kırşehir, Turkey
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, Sabriye Canakci
  • Corresponding author
  • Department of Biology, Faculty of Science, Karadeniz Technical University, 61000, Trabzon, Turkey
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, Iclal Sahin
  • Department of Biology, Faculty of Science, Karadeniz Technical University, 61000, Trabzon, Turkey
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and Ali Osman Belduz
  • Department of Biology, Faculty of Science, Karadeniz Technical University, 61000, Trabzon, Turkey
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Abstract

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 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 Hg2+. Interestingly, Cu2+ and Co2+ 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.

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