title: Rapid purification and characterization of γ-glutamyl-transpeptidase from shiitake mushroom (Lentinus edodes).
abstract: γ-Glutamyl-transpeptidase (GGT) is one of the important enzymes in the pathway of odor formation in shiitake mushroom (Lentinus edodes). Rapid purification and characterization of GGT from shiitake mushroom were studied in this work. The GGT was purified 179-fold after 3 primary steps: precipitation by ammonium sulfate, isolation by Phenyl Sepharose 6 FF, and desalting by Sephadex G-25. The enzyme, consisting of a small and a large subunit with Mr 28 KDa and 60 KDa, respectively, is composed of 17 kinds of amino acids with the ratio of basic and acidic residues 1: 1.84, and its secondary structure was also determined by Fourier transform infrared spectroscopy. The properties of GGT were studied with γ-glutamyl-p-nitroanilide as the substrate. The results showed Km value of 2.601μM, optimal temperature of 40 °C, and isoelectric point of 6.4. In addition, the activity of GGT was promoted by Na⁺, K⁺, and Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Zn²⁺, and Fe³⁺.
  
  
  CONCLUSIONS
  In this work, the γ-glutamyl-transpeptidase (GGT) from shiitake mushroom was purified with a simple scheme. Through the characterization of GGT, the relationship between endogenous formaldehyde and odor formation is to be clarified and assist in finding means of formaldehyde control in shiitake mushroom.
authors.affiliation: School of Food Science and Biotechnology, Zhejiang Gongshang Univ, Hangzhou 310035, China. jincheng527@yahoo.com.cn
authors.forename: Jianrong
authors.initials: J
authors.lastname: Li
authors.forename: Ju
authors.initials: J
authors.lastname: Huang
authors.forename: Jie
authors.initials: J
authors.lastname: Yin
authors.forename: Ning
authors.initials: N
authors.lastname: Wu
authors.forename: Jun
authors.initials: J
authors.lastname: Song
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Tianjia
authors.initials: T
authors.lastname: Jiang
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D011232:Chemical Precipitation; D002847:Chromatography, Agarose; D003911:Dextrans / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048690:Fruiting Bodies, Fungal / Q000201:enzymology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D011188:Potassium / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D020942:Shiitake Mushrooms / Q000201:enzymology*; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D003911:Dextrans; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D021122:Protein Subunits; D005973:Glutamine; C020927:phenyl-sepharose; C001300:gamma-glutamine-4-nitroanilide; D012685:Sepharose; C025614:sephadex; D012964:Sodium; D005723:gamma-Glutamyltransferase; D011188:Potassium; D002118:Calcium