ABSTRACT
A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60 degrees C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu(2+) ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.
Subject(s)
Bacillus cereus/enzymology , Metalloproteases/chemistry , Bacillus cereus/classification , Bacillus cereus/isolation & purification , Detergents/pharmacology , Enzyme Stability/drug effects , Hydrogen-Ion Concentration , Ions/pharmacology , Metalloproteases/drug effects , Metalloproteases/isolation & purification , Phylogeny , Protease Inhibitors/pharmacology , Solvents/pharmacology , TemperatureABSTRACT
The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett-Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.
Subject(s)
Culture Media/chemistry , Pichia/genetics , Pichia/metabolism , Streptokinase/biosynthesis , Carbon/chemistry , Cloning, Molecular , Gene Expression , Genetic Vectors/genetics , Nitrogen/chemistry , Streptokinase/geneticsABSTRACT
BACKGROUND: Two cases of paternity dispute, examined with 17 autosomal short tandem repeats signified a possible single and double maternal mismatch at vWA and D8S1179/D21S11 loci in the children under investigation. METHODS: Seventeen autosomal STR loci were analyzed using AmpFlSTR Identifiler, PowerPlex 16 kits. Six STR markers on X chromosome were amplified and analyzed. Mutated alleles were amplified, cloned in pCR(R)II-Topo vector, sequenced and investigated. RESULTS: In case S1 the vWA locus indicated an allele mismatch with the mother. All the vWA alleles on amplification, cloning and sequencing depicted an increase of 2 repeats in the child. In case D1 maternal child inconsistency at D8S1179 and D21S11 loci was observed. The alleles were amplified, cloned and sequenced to analyze the repeat structure. Increase of 1 repeat in D8S1179 locus and an insertion mutation in D21S11 locus between the mother and questioned child were confirmed. A complete match with the 17 autosomal loci of the father and 6 X chromosome STR loci of the mother was observed in both the cases. CONCLUSION: This is the first report of a maternally transmitted single mismatch at vWA locus and double mismatch at D8S1179 and D21S11 loci due to increase/mutation of the repeat in the paternity DNA testing. The results of nucleotide sequencing and STR analyses convincingly established that the suspected father and the mother are undeniably the biological parents of the questioned child.
Subject(s)
DNA Fingerprinting , Paternity , Tandem Repeat Sequences/genetics , Alleles , Chromosomes, Human, X/genetics , Female , Gene Frequency , Humans , Infant , Male , Mothers , Polymerase Chain Reaction/methods , Predictive Value of Tests , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, DNA/methods , von Willebrand Factor/geneticsABSTRACT
A protease producing bacterial culture ('S7') was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Streptomyces sp. on the basis of biochemical properties and 16S rRNA gene sequencing. Purification of the protease present in the culture medium supernatant on sephacryl S-100 indicated that it contains a keratinase with 67% recovery, 2.5-fold purification and an estimated molecular mass of approximately 44,000 Da. Keratinase showed an optimal activity at 45 degrees C and pH 11. Keratinase activity increased substantially in presence of Ca(2+) and was inhibited in presence of PMSF and EDTA identifying it as a serine metalloprotease. Stability in the presence of detergents, surfactants and solvents make this keratinase extremely useful for biotechnological process involving keratin hydrolysis or in the leather industry.
Subject(s)
Biotechnology/methods , Peptide Hydrolases/chemistry , Peptide Hydrolases/isolation & purification , Streptomyces/enzymology , Acrylic Resins/chemistry , Animals , Calcium/chemistry , Chickens , Edetic Acid/chemistry , Feathers , Hydrogen-Ion Concentration , Hydrolysis , Keratins/chemistry , RNA, Ribosomal, 16S/chemistry , TemperatureABSTRACT
An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus 'S' gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited approximately 20-22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Hepatitis B Surface Antigens/genetics , Pichia/genetics , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/genetics , Blotting, Southern , Blotting, Western , Gene Expression Regulation, Fungal , Hepatitis B Surface Antigens/metabolism , Hepatitis B Surface Antigens/ultrastructure , Microscopy, Electron, Transmission , Models, Genetic , Pichia/enzymology , Saccharomyces cerevisiae/metabolismABSTRACT
This article has been retracted consistent with Elsevier Policy on Article Withdrawal. Please see . The Publisher apologizes for any inconvenience this may cause.
ABSTRACT
This article has been retracted consistent with Elsevier Policy on Article Withdrawal. Please see . The Publisher apologizes for any inconvenience this may cause.
ABSTRACT
The paper entitled "Constitutive expression and characterization of Hepatitis B surface antigen purified by metal affinity precipitation", which was published online on 19 May 2006, was withdrawn at the author's request.
