BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS One-step purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Stereum hirsutum Ngoc-Phuong-Thao Nguyen & Kyoung-Mi Lee & Kyoung-Min Lee & In-Won Kim & Yeong-Suk Kim & Marimuthu Jeya & Jung-Kul Lee Received: 17 March 2010 / Revised: 4 May 2010 / Accepted: 5 May 2010 / Published online: 8 June 2010 # Springer-Verlag 2010 Abstract A highly efficient β-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and se- quence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-β-D-gluco- pyranoside (V max =3,028 Umg-protein -1 , k cat =4,945 s -1 ) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 Umg-protein -1 using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydro- lase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process. Keywords β-1 . 4-Glucosidase . Glycoside hydrolase family 1 . Glucoside . Stereum hirsutum . Wine fermentation Introduction β-Glucoside glucohydrolases, commonly called β- glucosidases (BGLs), catalyze the hydrolysis of alkyl- and aryl-β-glucosides, as well as diglucosides and oligosac- charides. These enzymes are widely used in a number of biotechnological processes, including the production of fuel ethanol from cellulosic agricultural residues and the synthesis of useful glucosides (Christine et al. 1998). The food industry, and particularly the enological sector, is also now showing an increasing interest in glycohydrolase enzymes, especially pectinases and glycosidases (Barbagallo et al. 2004). This is because many natural compounds that are important compo- nents of flavors and aromas (e.g., monoterpenols, C13- norisoprenoids, and shikimate derivatives) accumulate in many fruits as flavorless mono- or diglycoside-linked precursors. These require chemical or enzymatic hydrolysis for the liberation of their fragrances and flavors (Christine et al. 1998). The enzymatic hydrolysis of glycosides of wine grapes involves sequential operation of rhamnosidase (EC 3.2.1.40), arabinosidase (EC 3.2.1.55), and then BGL (EC 3.2.1.21) reactions; the latter catalyses the release of flavor compounds from the monoglucosides and directly increases wine aroma (Barbagallo et al. 2004). Unlike acidic hydrolysis, enzymatic hydrolysis is highly efficient and does not result in modifications of the N.-P. Nguyen : K.-M. Lee : K.-M. Lee : I.-W. Kim : M. Jeya (*) : J.-K. Lee (*) Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea e-mail: jeya@konkuk.ac.kr e-mail: jkrhee@konkuk.ac.kr Y.-S. Kim Department of Forest Products, Kookmin University, Seoul 136-702, Republic of Korea J.-K. Lee Institute of SK-KU Biomaterials, Konkuk University, Seoul 143-701, Republic of Korea Appl Microbiol Biotechnol (2010) 87:2107–2116 DOI 10.1007/s00253-010-2668-2