Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Establishment and application of activity determining methods for different subtypes of xanthine oxidoreductase / 药学学报

Xanthine oxidoreductase (XOR), the key enzyme catalyzing purine to produce uric acid, including two subtypes, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), respectively, in vivo. Usually, XDH and XO can transform to each other. In this study, based on the principle that the subtype XO or XDH uses different electron acceptors, the methods for the measuring the activities of bovine milk XOR (pure enzyme) and its subtypes were established. The optimal concentrations of substrate xanthine (50 μmol·L-1) and electron acceptor NAD+ (50 μmol·L-1), pH value (7.80) were investigated. The ranges of the XOR, XO, XDH activity which could be determined were 0.97-17.5 U·L-1, 1-9 U·L-1, and 66-1 191 mU·L-1, respectively. Furthermore, the methods for determining the activities of XOR and its subtypes in mouse liver were established. The preparation of liver samples, the optimal concentrations of xanthine (100 μmol·L-1) and NAD+ (100 μmol·L-1) were researched. And the activity ranges of XOR, XO and XDH in mouse liver which could be determined were 0.67-3.98, 0.19-1.08, and 0.52-3.55 U·gprot-1, respectively. With the methods above, the effects of classic XOR inhibitor allopurinal (Allo) on XOR, XO and XDH from both milk and mouse liver were determined. All animal experiments have been approved by the Animal Experimental Center, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College (00003346). This study established new methods for the determination of XOR and its subtypes activity in pure enzyme system and in mouse liver, respectively, which were accurate and convenient. It laid the experimental foundation for exploring the different pathophysiological effects of XOR in the body and developing new XOR inhibitors.

Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides.

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophilamelanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

Expression of / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by