title: Heterologous expression of a GH3 β-glucosidase from Neurospora crassa in Pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides.
abstract: Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 β-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60 °C, and had its maximum activity against p-nitrophenyl-β-d-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-β-d-cellobioside (pNPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 μM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries.
authors.affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Pei
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Pengli
authors.initials: P
authors.lastname: Cai
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Wenliang
authors.initials: W
authors.lastname: Sun
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Ren
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wu
authors.affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors.forename: Shihong
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: tian_cg@tib.cas.cn.
authors.forename: Chaoguang
authors.initials: C
authors.lastname: Tian
mesh_terms: D000595:Amino Acid Sequence; D002475:Cellobiose / Q000737:chemistry; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007529:Isoflavones / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013025:Glycine max / Q000737:chemistry; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D006027:Glycosides; D007529:Isoflavones; D011994:Recombinant Proteins; D002475:Cellobiose; D001617:beta-Glucosidase; D005947:Glucose
keywords: Isoflavone glycosides; Neurospora crassa; Pichia pastoris; β-glucosidase