Extracellular Aspartic Proteinase Research Articles

Investigation of optimal parameters for aspartic protease production from Aspergillus terreus using solid-state fermentation

The current study presents extracellular Aspartic protease production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus terreus, and parameters that largely affect enzyme production, namely fermentation time, pH, moistening solution, temperature, inoculum volume, and different solutions, were utilized for enzyme extraction. The isolated fungus was identified as A. terreus (registered as A. terreus SFH5 in the gene bank) by phenotypic and molecular characterization, showed the highest aspartic protease activity of 258.6 units/mL and 272.2 units/mg using a solid-state fermentation technique during screening, and was further used for media component screening and optimization studies. The study employed wheat bran as the preferred production medium and casein as the nitrogen source. The fermentation process was conducted at a temperature of 30°C and a pH of 5. The moistening solution to fermentation medium ratio was set at 3:1, while an inoculum volume of 2 mL (containing 1 × 106 spores/10 g solid) was used. Distilled water was selected as the most effective solution for extracting the enzyme. It has been discovered that the locally isolated novel strain of A. terreus, SFH5, can efficiently produce a substantial amount of aspartic protease by utilizing agro-industrial waste. This makes it a viable candidate for large-scale optimized production of the enzyme to satisfy industrial requirements.

Effects of Oxygen Transference on Protease Production by Rhodotorula mucilaginosa CBMAI 1528 in a Stirred Tank Bioreactor.

Microbial proteases, especially aspartic proteases, are an essential group of enzymes produced from different microorganisms. Microbial proteases have several applications, mainly in the food, beverage, cosmetic, and pharmaceutical industries, due to their efficiency in the processing and in the manufacturing stages. The yeast Rhodotorula mucilaginosa CBMAI 1528 was isolated from the Antarctic environment and was previously reported to have higher extracellular aspartic protease production. In addition, advances in the operational conditions of bioreactors for enzyme production are important to reduce the gap associated with scaling-up processes. This is the first study that evaluates the influence of oxygen transference (kLa) on the protease production of R. mucilaginosa yeast. To that end, batch cultures were created in a stirred tank bioreactor using Sabouraud dextrose broth at 25 °C for 72 h under kLa values from 18 to 135 h-1. The results show that kLa (121 h-1) obtained at 500 rpm and 1.5 vvm plays an important role in protease production (124.9 U/mL) and productivity (6.784 U/L.h) as well as biomass (10.4 g/L), μmax (0.14 h-1) and Yx/s (0.484 g/g). In conclusion, R. mucilaginosa showed high yield production in aerobic culture with the efficiency of protease expression and secretion influenced by kLa. In this sense, our results could be used for further industrial investment.

Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ± 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.

Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from <i>Lactobacillus casei</i> WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

Identification and partial characterization of extracellular aspartic protease genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384.

The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.

An HrpB‐dependent but type III‐independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum

The host specificity of Ralstonia solanacearum, the causal organism of bacterial wilt on many solanaceous crops, is poorly understood. To identify a gene conferring host specificity of the bacterium, SL341 (virulent to hot pepper but avirulent to potato) and SL2029 (virulent to potato but avirulent to hot pepper) were chosen as representative strains. We identified a gene, rsa1, from SL2029 that confers avirulence to SL341 in hot pepper. The rsa1 gene encoding an 11.8-kDa protein possessed the perfect consensus hrp(II) box motif upstream of the gene. Although the expression of rsa1 was activated by HrpB, a transcriptional activator for hrp gene expression, Rsa1 protein was secreted in an Hrp type III secretion-independent manner. Rsa1 exhibited weak homology with an aspartic protease, cathepsin D, and possessed protease activity. Two specific aspartic protease inhibitors, pepstatin A and diazoacetyl-d,l-norleucine methyl ester, inhibited the protease activity of Rsa1. Substitution of two aspartic acid residues with alanine at positions 54 and 59 abolished protease activity. The SL2029 rsa1 mutant was much less virulent than the wild-type strain, but did not induce disease symptoms in hot pepper. These data indicate that Rsa1 is an extracellular aspartic protease and plays an important role for the virulence of SL2029 in potato.

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied, mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.

An extracellular aspartic protease functions in Arabidopsis disease resistance signaling.

We have used activation tagging with T-DNA carrying cauliflower mosaic virus 35S enhancers to investigate the complex signaling networks underlying disease resistance in Arabidopsis. From a screen of approximately 5000 lines, we identified constitutive disease resistance (CDR1) encoding an apoplastic aspartic protease, the overexpression of which causes dwarfing and resistance to virulent Pseudomonas syringae. These phenotypes reflect salicylic-acid-dependent activation of micro-oxidative bursts and various defense-related genes. Antisense CDR1 plants were compromised for resistance to avirulent P. syringae and more susceptible to virulent strains than wild type. CDR1 accumulates in intercellular fluid in response to pathogen attacks. Induction of CDR1 generates a small mobile signal, and CDR1 action is blocked by the protease inhibitor pepstatin and by mutations in the protease active sites. We propose that CDR1 mediates a peptide signal system involved in the activation of inducible resistance mechanisms.

Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection

Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection

Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection.

An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region. Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro. Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.

Investigation of -glucosidase as a potential virulence factor of Candida albicans

In this study, the α-glucosidase of C. albicans as a potential virulence factor was investigated. We compared the intracellular and intact-cellular activities of two C. albicans strains: a clinical isolate (FS) and a laboratory mutant (FR) derived from the former. Although the FR strain was superior in all the virulence factors examined as well as in its in vivo virulence, similar intact-cellular α-glucosidase activities were obtained for the two strains. Moreover, the intracellular α-glucosidase activity of the FR strain was lower than that of the FS strain. Making a survey on 20 clinical isolates of C. albicans, the α-glucosidase activities did not correlate with either the extracellular aspartic proteinase (EAP) or the extracellular phospholipase (EP) activities, although we found a correlation between the EAP and EP activities. In conclusion, our experiments do not support the view that the inducible α-glucosidase is a virulence factor of C. albicans.

Investigation of α ‐glucosidase as a potential virulence factor of Candida albicans

In this study, the α-glucosidase of C. albicans as a potential virulence factor was investigated. We compared the intracellular and intact-cellular activities of two C. albicans strains: a clinical isolate (FS) and a laboratory mutant (FR) derived from the former. Although the FR strain was superior in all the virulence factors examined as well as in its in vivo virulence, similar intact-cellular α-glucosidase activities were obtained for the two strains. Moreover, the intracellular α-glucosidase activity of the FR strain was lower than that of the FS strain. Making a survey on 20 clinical isolates of C. albicans, the α-glucosidase activities did not correlate with either the extracellular aspartic proteinase (EAP) or the extracellular phospholipase (EP) activities, although we found a correlation between the EAP and EP activities. In conclusion, our experiments do not support the view that the inducible α-glucosidase is a virulence factor of C. albicans.

Production, characterization, and epitope mapping of a monoclonal antibody against aspartic proteinase of Candida albicans.

A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp77 to Gly103 sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase fromAspergillus fumigatus

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

Purification and characterization of an extracellular aspartic proteinase from Aspergillus fumigatus.

An aspartic proteinase (PEP) from the culture supernatant of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 24%. The procedure involved affinity chromatography on pepstatin agarose, the interaction requiring a chaotropic salt (sodium trifluoroacetate) for complete elution of the enzyme. Among 11 amino acids of the N-terminal region, nine were identical with the corresponding sequence of the aspartic proteinase aspergillopepsin A from Aspergillus niger var. awamori (previously called Aspergillus awamori). Thus PEP belongs to the aspergillopepsins, a family of closely related aspartic proteinases produced by aspergilli. Specific antibodies against PEP were detected by dot blot assay in sera of two patients with aspergillosis. In addition, PEP antigen was demonstrated by immunofluorescence in mycotic human lung, using specifically elicited antibodies from guinea-pigs. PEP had an estimated molecular mass of 38 kDa and the pI was determined at pH 4.2. PEP is therefore likely to be closely related to an acid proteinase of A. fumigatus which was originally described in 1981.

Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.

A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni,G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichia coli. Recombinant canditropsinogen (Ctg), which was expressed as inclusion bodies in the cytosol of E. coli, was refolded by dialysis from an 8 M urea solution and purified to homogeneity using chromatographies on Sephacryl S-300 and on MonoQ columns. The purified Ctg was converted into canditropsin by either acid activation or trypsin conversion. The specificity of the resulting recombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxidized insulin B chain between Ala-Leu and many other minor cleavage sites. Canditropsin also hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections. Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. Canditropsin is weakly inhibited by leupeptin and antipain, with an apparent Ki of 1.74 x 10(-4)M and 1.5 x 10(-5) M, respectively.