First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity.

Carola Schrã¶Der,Saskia Blank,Garabed Antranikian

doi:10.3389/fbioe.2015.00076

Abstract

Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2.

Highlights

Glycoside hydrolases (GHs) (EC 3.2.1.X) hydrolyze glycosidic bonds between carbohydrates or between carbohydrate and non-carbohydrate components, for instance alcohols or phenols
The majority of identified β-glucosidases is grouped in GH family 1 (Cairns and Esen, 2010). β-Galactosidase activity can be found in different GH families (1, 2, 3, 16, 20, 35, 42, 43, 50, and 59)
Identification of Novel GH-Encoding Genes Gene libraries were constructed from pure cultures of T. antranikianii and T. brockianus, and screened for genes coding for enzymes active toward esculin

Summary

Introduction

Glycoside hydrolases (GHs) (EC 3.2.1.X) hydrolyze glycosidic bonds between carbohydrates or between carbohydrate and non-carbohydrate components, for instance alcohols or phenols. Enzyme classification is based on amino acid similarities rather than substrate specificity (Henrissat, 1991). GHs are assigned to different groups based on conserved domains, and the families are numbered from 1 to 131 that are listed in the Carbohydrate-Active Enzymes Database (Lombard et al, 2014). The majority of identified β-glucosidases is grouped in GH family 1 (Cairns and Esen, 2010). Β-Galactosidase activity can be found in different GH families (1, 2, 3, 16, 20, 35, 42, 43, 50, and 59). Glycoside hydrolases classified as GH2 were described to function as β-galactosidase

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Conclusion