In order to prepare antioxidant peptide through hydrolyzing low-value protein resources with bacterial extracellular proteases and to discover novel proteases, crude extracellular protease from Pseudoalteromonas sp. SHK1-2 was obtained through fermentation which was used to hydrolyze collagen extracted from Cirrhinus molitorella skin. Small peptide fraction was isolated from hydrolysate by ultrafiltration and Sephadex LH-20 size exclusion chromatography and showed 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity (35.6%±7%), oxygen radical absorbance capacity and inhibition of DNA oxidation damage. The molecule weight was 776.2 Da, and amino acid sequence was Thr-Ala-Gly-His-Pro- Gly-Thr-His through liquid chromatography mass spectrum. Our findings suggest that peptide obtained from low-value protein of fish waste by hydrolysis with bacterial protease has antioxidant activity.
Amino Acid Sequence ; Animals ; Antioxidants ; chemistry ; Chromatography, Gel ; Collagen ; chemistry ; Cyprinidae ; Dextrans ; Hydrolysis ; Oxidation-Reduction ; Peptide Hydrolases ; Peptides ; chemistry ; Skin ; chemistry

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

OBJECTIVECurrent study was conducted to investigate and compare the impact of temperature and pH on the activities of amylase, protease and lipase in alimentary tract of Whitmania pigra.

METHODThe responses of amylase, protease, and lipase activities were determined over a wide range of temperatures (7-52 degrees C) and pH gradient (2.2-11.2).

RESULTThe highest lipase activity was found under 37 degrees C, pH 8.2, and the highest amylase activity was detected under 37 degrees C, pH 5.2, while protease activity peaked at 42 degrees C, pH 3.2 or pH 9.2.

CONCLUSIONThe optimal temperature in alimentary tract of Wh. pigra for lipase and amylase was 37 degrees C, and the responding temperature for protease was 42 degrees C. The optimal pH value in alimentary tract of Wh. pigra for lipase and amylase was pH 8.2 and pH 5.2, respectively. While pH 3.2 or 9.2 seems to be both favorable for high protease activity.

Amylases ; chemistry ; metabolism ; Animals ; Digestive System ; chemistry ; enzymology ; Enzyme Stability ; Hydrogen-Ion Concentration ; Leeches ; chemistry ; enzymology ; Lipase ; chemistry ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Temperature

The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.
Carcinoma, Hepatocellular ; blood ; diagnosis ; Chromatography, High Pressure Liquid ; Chromatography, Reverse-Phase ; Humans ; Liver Neoplasms ; blood ; diagnosis ; Mass Spectrometry ; Nanotechnology ; Peptide Hydrolases ; metabolism ; Peptides ; blood ; Protein Structure, Tertiary ; Proteome ; analysis ; Proteomics ; Silicon Dioxide ; chemistry ; Time Factors

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

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
Aeropyrum ; chemistry ; enzymology ; Archaeal Proteins ; genetics ; metabolism ; Binding Sites ; Biocatalysis ; Caprylates ; metabolism ; Cloning, Molecular ; Dimethyl Sulfoxide ; chemistry ; Escherichia coli ; Hot Temperature ; Industrial Microbiology ; methods ; Kinetics ; Laurates ; metabolism ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; methods ; Peptide Hydrolases ; genetics ; metabolism ; Protein Binding ; Protein Conformation ; Recombinant Proteins ; genetics ; metabolism ; Solvents ; chemistry ; Substrate Specificity

The aim of the study is to construct two vectors for efficient expression of soluble recombinant proteins. The first vector was constructed by cloning the HisSUMO fragment into an expression vector pET30a(+) to fuse with the gene of interest (designated as HisSUMO Express). The second vector was constructed in the same way, but with a hydroxylamine cleavage site between HisSUMO and the gene of interest for an economic purpose (designated as HisSUMO Economic). The mouse fibroblast growth factor-21(mFGF-21), which was difficult to express in routine-used expression vectors, was taken as an example to test the vectors. The results showed that the mFGF-21 was expressed at high level in both vectors. The Sumo/mFGF-21 fusion protein accounted for more than 40% of the total bacterial protein. The fusion protein was purified with Ni-TNA column, and the HisSUMO was removed by cleavage of the fusion protein with either hydroxylamine solution or SUMO protease I. The concentration of the purified mFGF-21 mature protein was 54 mg/L and the recovery rate was 6%. The purified proteins derived from either hydroxylamine or SUMO protease I cleavage could stimulate glucose up-take by adipocytes. These results indicated that both HisSUMO Express and HisSUMO Economic were useful expression vectors for efficient expression of soluble recombinant proteins.
Animals ; Fibroblast Growth Factors ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Hydroxylamine ; chemistry ; Mice ; Peptide Hydrolases ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility

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

COP9 Signalosome Complex ; Carcinoma, Hepatocellular ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; chemistry ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Peptide Hydrolases ; chemistry ; genetics ; metabolism ; Phosphorylation ; Phosphoserine ; metabolism

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