OBJECTIVETo study the effect of companion fungus on several enzymatic activities of Grifola umbellata.

METHODChitinase, beta-1,3-glucanase, proteinase and extracellular enzymes of G. umbellata were measured during dual culturing with companion fungus.

RESULTCompanion fungus could induce the increase of chitinase and beta-1, 3-glucanase activities of G. umbellata. noevident changeswere found in proteinase activity. When in liquid culture, the activities of extracellular enzymes in dual cultured filtrate were between of these of G. umbellata and companion fungus in monocultures.

CONCLUSIONSclerotia differentiation related materials supplied by mutual nutritional supplement between G. umbellata and companion fungus conduce to sclerotial formation of G. umbellata.

Catechol Oxidase ; analysis ; Chitinases ; analysis ; Coculture Techniques ; Glucan 1,3-beta-Glucosidase ; analysis ; Grifola ; growth & development ; physiology ; Peptide Hydrolases ; analysis ; Polyporaceae ; enzymology ; growth & development

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E. coli. The gene open reading frame was 1311 bp and it codes for 436 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I. The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%). From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group. The DNASTAR program was used to predict the secondary structure. According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns. 14 of the 35 Pro were located at the second sites of beta-turns. Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; Glycoside Hydrolases ; biosynthesis ; classification ; genetics ; Hot Temperature ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Phylogeny ; Protein Structure, Secondary ; physiology ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods ; Sequence Homology ; Thermus ; enzymology ; beta-Glucosidase