Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa.

ABSTRACT: An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns. The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions. The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium. The H. lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues. This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates. This enzyme represents a unique example of a thiol proteinase obtained from a fungal source.

SUBMITTER: Shenolikar S

PROVIDER: S-EPMC1158457 | biostudies-other | 1982 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Purification and partial characterization of ?1-proteinase inhibitor in the common marmoset (Callithrix jacchus).

Project description:Fecal alpha1-proteinase inhibitor (?1-PI) concentration has been to diagnose enteric protein loss in dogs and cats. Chronic lymphocytic enteritis is commonly seen in the marmoset (Callithrix jaccus) and is characterized by hypoalbuminemia. As a prelude to immunoassay development for detecting enteric protein loss, marmoset serum ?1-PI was purified using immunoaffinity chromatography and ceramic hydroxyapatite chromatography. Partial characterization was performed by reducing gel electrophoresis and enzyme inhibitory assays. Protein identity was confirmed with peptide mass fingerprinting and N-terminal amino acid sequencing. Molecular mass, relative molecular mass, and isoelectric point for marmoset ?1-PI were 54?kDa, 51,677, and 4.8-5.4, respectively. Trypsin, chymotrypsin, and elastase inhibitory activity were observed. N-terminal amino acid sequence for marmoset ?1-PI was EDPQGDAAQKMDTSHH. In conclusion, marmoset ?1-PI was successfully purified from serum with an overall yield of 12% using a rapid and efficient method. Purified marmoset ?1-PI has characteristics similar to those of ?1-PI reported for other species.

| S-EPMC4378525 | biostudies-literature

Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

Project description:The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

| S-EPMC4897640 | biostudies-literature

A Novel CreA-Mediated Regulation Mechanism of Cellulase Expression in the Thermophilic Fungus Humicola insolens.

Project description:The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (β-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolenscreA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

| S-EPMC6696435 | biostudies-literature

Purification of an inducible DNase from a thermophilic fungus.

Project description:The ability to induce an extracellular DNase from a novel thermophilic fungus was studied and the DNAse purified using both traditional and innovative purification techniques. The isolate produced sterile hyphae under all attempted growing conditions, with an average diameter of 2 µm and was found to have an optimal temperature of 45 °C and a maximum of 65 °C. Sequencing of the internal transcribed region resulted in a 91% match with Chaetomium sp., suggesting a new species, but further clarification on this point is needed. The optimal temperature for DNase production was found to be 55 °C and was induced by the presence of DNA and/or deoxyribose. Static growth of the organism resulted in significantly higher DNase production than agitated growth. The DNase was purified 145-fold using a novel affinity membrane purification system with 25% of the initial enzyme activity remaining. Electrophoresis of the purified enzyme resulted in a single protein band, indicating DNase homogeneity.

| S-EPMC3907870 | biostudies-literature

The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

Project description:Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/10(6) conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.

| S-EPMC4979032 | biostudies-literature

Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

Project description:Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.

| S-EPMC1162616 | biostudies-other

Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

Project description:Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

| S-EPMC91380 | biostudies-literature

CRISPR/Cas9-mediated genome editing directed by a 5S rRNA-tRNA<sup>Gly</sup> hybrid promoter in the thermophilic filamentous fungus Humicola insolens.

Project description:BackgroundHumicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism.ResultsHerein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA-tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation.ConclusionsThe established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

| S-EPMC8542335 | biostudies-literature

Partial purification and characterization of a novel neutral proteinase from human uterine cervix.

Project description:1. Human uterine cervical stroma was found to contain a Ca(2+)-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein-Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4-8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4x10(5) by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10mum) or leupeptin (10mum) was also found to be inhibitory, but chymostatin (40mug/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.

| S-EPMC1161371 | biostudies-other

Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus.

Project description:Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus. The isolated enzymes were all homogeneous on polyacrylamide-disc-gel electrophoresis. Data from chromatography on Bio-Gel P-60 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated mol.wts. of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0, 5.5, 2.6 and 1.8% (w/w) respectively. Although the three purified cellulases were active towards filter paper, only cellulases I and III were active towards CM(carboxymethyl)-cellulose. Cellulase I was also active towards yeast glucan. The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mumol/ml and 6.5 X 10(4) for beta-glucosidase on p-nitrophenyl beta-D-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 35.5 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.

| S-EPMC1162184 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data