ABSTRACT
Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.
Subject(s)
Actinomycetales/enzymology , Cellulases/chemistry , Cellulases/isolation & purification , Cellulases/genetics , Hydrogen-Ion Concentration , Mutagenicity Tests , TemperatureABSTRACT
A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.
Subject(s)
Adult , Female , Humans , Male , Base Sequence , Middle Aged , Models, Molecular , Molecular Sequence Data , Pedigree , Point Mutation , Polymerase Chain Reaction/methods , Protein C/genetics , Protein C/chemistry , Protein C Deficiency/genetics , Protein Conformation , Serine Endopeptidases/genetics , Serine Endopeptidases/chemistry , Venous Thrombosis/geneticsABSTRACT
Objective: To investigate the structural characteristics and the primary functions of antigen gene cC1. Meth-ods: The primary and the secondary structures were analyzed using bioinformatics programs provided by Internet servers.The predicted 3D structure of cC1 was established by homology protein modeling method. Anticoagulant activity of GST-anx32 was assayed by modified kaolin partial thromboplastin time (KPTT). Results: cC1 had high homology to annexinsgenes both at nucleic acid and at amino acid level. It contained 4 homologous regions, and each region included the typical an-nexin motif "G-X-G-T (38 residues)-D/E". The results of KPTT assay showed that the recombinant protein GST-anx32 hadhigh anticoagulant activity. Conclusion: cC1 has the common structures of annexins but the homology to the extant annexinsis no more than 48%, cC1 is a member of a novel annexin subfamily and designed as annexin32.