A keratinolytic proteinase secreted by Microsporum canis in a broth containing human hair was purified 134-fold from the culture filtrate by ion-exchange chromatography using DEAE-Sephacel, CM-Sephadex C-50, and by Sephadex G-75 gel filtration. The purified enzyme was electrophoretically homogeneous with a molecular weight of 33,000. The enzyme had an optimum pH of 8.0, and the activity was stable in the alkaline pH range. Enzyme activity increased with temperature up to 35 degrees C and was stable up to 45 degrees C. The keratinolytic activity was not affected by the addition of nonionic detergents, was activated by Mg2+, but inhibited by Zn2+. The purified enzyme was used to obtain guinea pig antiserum. The antiserum tested by double diffusion against the purified enzyme showed a single line of precipitation and completely neutralized the proteinase activity. This study reaffirms that the proteinase from M. canis may be a biochemical mechanism for the invasion of keratinized tissue, and could possibly play a role in the hypersensitivity reactions arising from superficial infections of this fungus.
Microsporum/enzymology* ; Peptide Hydrolases/isolation & purification*

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.
Bacillus subtilis ; classification ; enzymology ; Enzyme Activation ; Enzyme Stability ; Peptide Hydrolases ; chemistry ; isolation & purification ; Species Specificity ; Substrate Specificity

AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Mutation ; Peptide Hydrolases ; chemistry ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo investigate the effect of andrographolide on virulence factors production in Pseudomonas aeruginosa.

METHODGrowth rate, pyocyanin, proteolytic activity and elastase activity were measured with or without the presence of andrographolide. The effect of andrographolide on pyocyanin production, proteolytic activity and elastase activity in PAO-JP2 was investigated simultaneously.

RESULTThe andrographolide did not affect the growth of PAO1 in planktonic culture. The production of pyocyanin, proteolytic activity and elastase activity were significanthy suppressed in P. aeruginosa cultures grown in the presence of andrographolide. However, these effects were not observed in PAO-JP2.

CONCLUSIONThe inhibiting effect of andrographolide on virulence factors production in P. aeruginosa may play a role in its anti-infection activity.

Andrographis ; chemistry ; Anti-Bacterial Agents ; pharmacology ; Diterpenes ; isolation & purification ; pharmacology ; Pancreatic Elastase ; metabolism ; Peptide Hydrolases ; metabolism ; Plants, Medicinal ; chemistry ; Pseudomonas aeruginosa ; growth & development ; metabolism ; pathogenicity ; Pyocyanine ; metabolism ; Virulence Factors ; metabolism

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.
Antiviral Agents ; pharmacology ; Enterovirus A, Human ; enzymology ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; drug therapy ; virology ; Humans ; Molecular Targeted Therapy ; Peptide Hydrolases ; chemistry ; metabolism ; physiology ; Protein Kinase Inhibitors ; pharmacology ; RNA, Viral ; genetics ; Viral Nonstructural Proteins ; chemistry ; metabolism ; physiology ; Virus Replication ; drug effects