Serine-type Protease Research Articles

A SDD1-like subtilase is exuded by tobacco roots.

Hydroponically grown tobacco (Nicotiana tabacum L. cv. Samsun) roots exude proteases under non-stressed conditions. Ten different proteases could be distinguished by 2D-zymography of root exudate. The majority of the gelatinolytic activity was susceptible to serine protease inhibitors. One of the proteases could be assigned to an EST (SGN-P361478) by mass spectrometry of immune-purified root exudate. The sequence was completed by RACE-PCR and shows typical serine protease features of subtilase family S8A. Thermostability and SDS-insensitivity indicate a kinetically stable enzyme. Phylogenetic classification of this highly gelatinolytic subtilase showed SDD1 to be the closest relative in Arabidopsis thaliana (L. Heynh.). Even closer related protein sequences could be found in other distant plant genera indicating a high conservation of the subtilase. A 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase-like protein and suberisation-associated anionic peroxidase-like protein were co-immune-purified and identified by mass spectrometry and may constitute potential interaction partners.

Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of- flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50 °C and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni(2+), Mn(2+), and Cu(2+) to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni.

BackgroundSchistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.MethodsWe have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.ResultsSmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.ConclusionsWe have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1022-z) contains supplementary material, which is available to authorized users.

Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato multicystatin by CLso infected tubers. The disease-induced loss of patatin and protease inhibitors therefore appears to be modulated by ser-type protease(s). The selective catabolism of proteins in ZC-afflicted tubers undoubtedly affects downstream aspects of carbohydrate and amino acid metabolism, which is ultimately reflected by the accumulation of reducing sugars, free amino acids and reduced sprouting capacity.

Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat

Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10–14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5–3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26–30kDa Ca2+-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not leak into or is inactivated in the blood. The role of this proteinase remains to be determined, but its possible interaction with extracellular glycosaminoglycans could focus its proteolytic activity in the tumor microenvironment and affect tumor growth.

Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum.

Cyclotides are head-to-tail cyclized peptides comprising a stabilizing cystine-knot motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 μM. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 μM. The inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson’s disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability.

Characterization of an alkaline protease with high quality bating potential in leather processing from Bacillus licheniformis MZK05M9 mutant

An alkaline protease from Bacillus licheniformis MZK05M9 (BlM9), a mutant strain developed in our laboratory, has been partially purified and characterized for its robustness and eco-friendly application potential in processing of hides and skins for leather manufacturing and detergent industries. The enzyme was purified 2.70 fold with specific activity of 1624U/mg in comparison to crude enzyme extract by using ammonium sulfate precipitation, dialysis and Sephadex G-75 column chromatography. The molecular mass of the enzyme was 27.2 kDa as judged by SDS–PAGE. The purified protease had a pH optimum of 8.5 and temperature optimum of 55°C. According to the inhibition profiles obtained with the various protease inhibitors, it was confirmed that the partially purified protease belongs to the serine protease type. The activity of partially purified enzyme was enhanced by calcium, magnesium, barium, potassium and manganese ions and strongly inhibited by mercury ion. In addition, the protease showed remarkable stability in the presence of 1% SDS; 1, 3 and 5% Triton X-100 and H2O2, which comprise the common bleach-based detergent formulation. The enzyme was found equally efficient to a commercial enzyme Oropon K (one of the commercial enzymes imported into Bangladesh for bating purpose) in bating of animal hide as proved by different comparative qualitative tests such as tensile strength, percent of elongation, stitch tears strength, water vapor permeability, grain crack strength and tongue tear strength tests. In addition, the stability profile (pH, temperature and surfactants) and blood stain removal data also revealed its suitability for application in detergent industry.

Downregulation of chloroplast protease AtDeg5 leads to changes in chronological progression of ontogenetic stages, leaf morphology and chloroplast ultrastructure in Arabidopsis

The chloroplast protein AtDeg5 is a serine-type protease peripherally attached to thylakoid membrane at its lumenal side. Since reliable data regarding the role of AtDeg5 in controlling the course of growth and developmental processes are extremely limited, two independent T-DNA insertional lines with different extent of AtDeg5 reduction were prepared and ontogenesis stage-based analysis performed. Both mutant lines displayed a compensatory overaccumulation of AtDeg8. The repression of AtDeg5 protease altered a range of phenotypic features in at least one of the mutants, with the most prominent being changes in chronological progression of development and growth of individual rosette leaves, flower production and silique ripening as well as in the area of fully expanded leaves and chloroplast ultrastructure. By analyzing the results of parallel-mutant screening we conclude that AtDeg8 overdose may rescue 23% of AtDeg5 deficiency with regard to some AtDeg5-controlled traits; alternatively AtDeg5 may have catalytic sites in excess so that these traits might remain unaltered when AtDeg5 pool is reduced by 23%. For some other AtDeg5-dependent traits the absence of excessive amount of AtDeg5 catalytic sites, lack of AtDeg5 dosage effect and inability of AtDeg8 to compensate deficiency or absence of AtDeg5 occurred.

Insights into the Mechanism of Organ-Specific Cancer Metastasis

Lucas and colleagues nominate transmembrane serine protease type II (TMPRSS2) as an important player in the initiation of epithelial-mesenchymal transition (EMT) in prostate cancer. Cancer cells maintain androgen receptor-regulated cytoplasmic TMPRSS2 expression, which facilitates EMT invasion and metastasis in model systems through hepatocyte growth factor and c-MET signaling. In addition to providing a rationale for potentially targeting this organ-specific enabler of metastatic disease progression, this study also highlights the importance of understanding how organ/tissue-specific genes are co-opted in the context of cancer.

Abstract 3313: 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells

Abstract Prostate cancer (PCa) is the most common and the second leading cause of cancer-related death among males in the Western world. TMPRSS2, a gene that encodes serine-protease type II transmembrane domain, has been shown to be frequently prevalent as a TMPRSS2-ERG gene fusion during early event in the genesis of PCa. However, TMPRESS2 function remains largely unknown, especially in castration-resistant PCa cells. While TMPRESS2 has previously been shown to be an androgen-regulated gene, TMPRSS2-ERG expression is regulated by a novel ERβ-dependent mechanism. The objectives of this study are to determine the mechanisms of expression and the functional significance of TMPRSS2 in growth and migration of castration-resistant AR-null PC-3 cells. 17β-Estradiol (E2; 10nM) induced TMPRSS2 expression (4-fold higher) in an ERβ-dependent manner as evidenced by shRNA silencing, but not by ERα-specific shRNA, in PC-3 cells. Intriguingly, the E2-induced TMPRSS2 expression was blocked by AG1024 (IGF-1R inhibitor) or knockdown of IGF-1R by siRNA, but not by AG1478 (EGFR inhibitor), suggesting that IGF-1R mediates expression of TMPRESS2 by the E2-ERβ axis in PC-3 cells. Moreover, TMPRSS2-specific siRNA markedly attenuated E2-induced cell growth and migration of PC-3 cells, suggesting that TMPRSS2 may be pivotal to E2-mediated progression of castration-resistant disease in vivo. The effects of TMPRSS2 silencing seem to be mediated through inhibition of E2-induced activation of NF-κB-signaling in these cells. Taken together, our results unraveled the underlying mechanisms that govern the expression and function of TMPRSS2 in mediating E2-induced growth and migration of castration-resistant PC-3 cells. Delineation of such critical players may culminate in identifying therapeutic targets to selectively circumvent castration-resistant outgrowth in PCa patients. Citation Format: Hogyoung Kim, Amrita Datta, Zakaria Y. Abd Elmageed, Asim B. Abdel-Mageed. 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2014-3313

Fighting a losing battle: vigorous immune response countered by pathogen suppression of host defenses in the chytridiomycosis-susceptible frog Atelopus zeteki.

The emergence of the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in dramatic global amphibian declines. Although many species have undergone catastrophic declines and/or extinctions, others appear to be unaffected or persist at reduced frequencies after Bd outbreaks. The reasons behind this variance in disease outcomes are poorly understood: differences in host immune responses have been proposed, yet previous studies suggest a lack of robust immune responses to Bd in susceptible species. Here, we sequenced transcriptomes from clutch-mates of a highly susceptible amphibian, Atelopus zeteki, with different infection histories. We found significant changes in expression of numerous genes involved in innate and inflammatory responses in infected frogs despite high susceptibility to chytridiomycosis. We show evidence of acquired immune responses generated against Bd, including increased expression of immunoglobulins and major histocompatibility complex genes. In addition, fungal-killing genes had significantly greater expression in frogs previously exposed to Bd compared with Bd-naïve frogs, including chitinase and serine-type proteases. However, our results appear to confirm recent in vitro evidence of immune suppression by Bd, demonstrated by decreased expression of lymphocyte genes in the spleen of infected compared with control frogs. We propose susceptibility to chytridiomycosis is not due to lack of Bd-specific immune responses but instead is caused by failure of those responses to be effective. Ineffective immune pathway activation and timing of antibody production are discussed as potential mechanisms. However, in light of our findings, suppression of key immune responses by Bd is likely an important factor in the lethality of this fungus.

Autolysis of krill protein from North Pacific krill Euphausia pacifica during protein recovery via isoelectric solubilization/precipitation

Autolysis of North Pacific krill protein in acidic and alkaline solubilization during protein recovery via isoelectric solubilization/precipitation was investigated. Pronounced auto-degradation of myosin heavy chain and actin from krill protein was observed at alkaline pH values (pH 9.0–12.0), with maximum autolytic activity at about pH 12.0. Meanwhile, proteolytic activity in krill protein at pH 12.0 was observed at low temperatures, suggesting a possible cause for the autolysis of krill protein during the protein recovery process. Three major proteinases were detected by zymographic analysis of myofibrillar protein, with approximate molecular weights of 26 kDa (KP-1), 18 kDa (KP-2) and 17 kDa (KP-3). KP-3 was active over a pH range from pH 5.0–12.0, suggesting that it may be responsible for the autolysis of krill protein during alkaline-induced protein recovery. Further study on substrate specificity and inhibitory specificity of KP-3 showed that KP-3 is serine type proteinase.

Smart cleaning-in-place process through crude keratinase: an eco-friendly cleaning techniques towards dairy industries

Abstract Crude keratinolytic protease produced from Bacillus tequilensis hsTKB2 was screened for its ability to clean milk foul generated upon the stainless steel panels as deposit in dairy plants. Enzyme yield was maximized by optimizing the culture conditions of a low-cost culture medium under solid culture condition with a try bed reactor. The highest keratinolytic protease activity of (1770.6 U g−1) was obtained by maintaining fermentation conditions of keratinase production at 45 °C, chicken feathers moisture ratio of 1:1 and an initial pH of 10 of the media. The crude keratinase was active in broad range of pH (8–11) with keratin as a substrate. However, it exhibited highest activities at pH 10.5. The crude keratinase which was strongly inactivated by Phenylmethylsulfonyl fluoride (PMSF) indicated that it was a serine type protease. The crude enzyme showed remarkable stability with Triton X-100 making it suitable for CIP process. The surface immobilized enzyme alginate beads prepared by 3% glutaraldehide fixation with beads improved significant cleaning activity rendering it suitable for commercial exploitation. The proteases produced were assessed on a lab-scale in terms of their potential suitability for cleaning-in-place (CIP) in the dairy industry. Cleaning performance was assessed by determining the ability of the enzymes to remove an industrial-like milk fouling deposit from stainless steel. Satisfactory cleaning, judged by quantification of residual organic matter and protein on the stainless steel surface after cleaning, was achieved using the developed CIP procedure at 70 °C. In addition this CIP procedure based on biodegradable enzymes working at high temperature is more environmentally favorable than conventional CIP methods using caustic based cleaning solutions. Potential environmental benefits of the developed enzymatic CIP procedure include decreased chemical usage of the resultant waste prior to release. This cleaning not only produces alkaline and acidic waste water with detergents but also takes significant time and reduction of odorous compounds.

Identification of predominant genes involved in regulation and execution of senescence-associated nitrogen remobilization in flag leaves of field grown barley

The transcriptomes of senescing flag leaves collected from barley field plots with standard or high nitrogen supply were compared to identify genes specifically associated with nitrogen remobilization during leaf senescence under agronomically relevant conditions. In flag leaves collected in field plots with high nitrogen supply, the decline in chlorophyll content was delayed. By comparing changes in gene expression for the two nitrogen levels, it was possible to discriminate genes related to nitrogen remobilization during senescence and genes involved in other processes associated with the late development of leaves under field conditions. Predominant genes that were more strongly upregulated during senescence of flag leaves from plants with standard nitrogen supply included genes encoding the transcription factor HvNAC026, serine type protease SCPL51, and the autophagy factors APG7 and ATG18F. Elevated expression of these genes in senescing leaves from plants with standard nitrogen supply indicates important roles of the corresponding proteins in nitrogen remobilization. In comparison, the genes upregulated in both flag leaf samples might have roles in general senescence processes associated with late leaf development. Among these genes were the transcription factor genes HvNAC001, HvNAC005, HvNAC013, HvWRKY12 and MYB, genes encoding the papain-like cysteine peptidases HvPAP14 and HvPAP20, as well as a subtilase gene.

Recombinant Drosophila prophenoloxidase 1 is sequentially cleaved by α-chymotrypsin during in vitro activation

Insect prophenoloxidase (PPO) is an essential innate immunity protein to induce pathogen into melanization. In Bombyx mori, pro-phenoloxidase-activating enzyme (PPAE) can directly cleave and activate PPO. However, PPO in Manduca sexta cannot be cleaved into active phenoloxidase (PO) by serine proteases unless cofactors are involved, which indicates that PPO activation is complicated. Here we use recombinant Drosophila melanogaster prophenoloxidase 1 (rPPO1) to study the mechanism of PPO activation induced by a typical serine protease, α-chymotrypsin. Small amounts of α-chymotrypsin cleave rPPO1 at the N- and C-terminus to produce a large fragment rPPO1(N1/C1) that needs further cleavage by α-chymotrypsin to produce a smaller fragment rPO1(60-kD) with PO activity. rPO1(60-kD) oxidizes dopamine without being affected by high temperature, or by having salt and Ethylene diamine tetraacetic acid (EDTA) in the solution. After incubation with dopamine, rPO1(60-kD) cannot be detected using reducing SDS-PAGE due to formation of a large complex. Trypsin, another typical serine protease, cleaves rPPO1 at the N- and C-terminus to produce a small fragment rPPO1(N'/C') without PO activity. Several rPPO1 mutants were created through over-expressing active fragments that have direct PO activity. They are easily cleaved by low amounts of α-chymotrypsin without increasing PO activity. Therefore, rPPO1 can be sequentially cleaved in at least three places by α-chymotrypsin to produce activated rPO1(60-kD).

HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers

Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation.

Functional Analysis of Environmental DNA-Derived Microviridins Provides New Insights into the Diversity of the Tricyclic Peptide Family

Microviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization.

Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

An efficient cloth cleaning properties of a crude keratinase combined with detergent: towards industrial viewpoint

Abstract Production of alkaline keratinolytic protease related to various industrial applications, especially for enzyme detergents combination, was done from a selected bacterial strain Paenibacillus woosongensis TKB2. Enzyme yield was maximized by optimizing the culture conditions of a low-cost culture medium under submerged condition with a lab scale fermentor of 5 L capacity. The highest keratinolytic activity was obtained 76.05 U/mL by maintaining fermentation conditions of keratinase production at 30 °C, 40% dissolved oxygen level at 500 rotations per minute (rpm) and an initial pH of media of 8.5 supplemented with 0.76% chicken feathers. In fact, the crude enzyme showed four proteolytic zones in casein zymography which revealed that the crude keratinase contained four hydrolytic enzymes. The crude keratinase was active in broad range of pH (7–10.5) with keratin as a substrate. However, it exhibited highest activities at pH 9. The crude keratinase which was strongly inactivated by Phenylmethylsulfonyl fluoride (PMSF), aprotinin and leupeptin, indicated that it was a serine type protease. The crude enzyme showed remarkable stability with EDTA and was compatible with commercially available detergents. The enzyme bead prepared by 1.5% CMC improved significant storage stability with commercial detergent powder making it suitable for commercial exploitation. The alkaline keratinase of P. woosongensis TKB2 showed a potential application in the laundry industry by removing the blood stains from the surgical cloths and gave a remarkable result towards removing the composite stain of blood, egg yolk and chocolate stain in a short period without changing texture, strength (27.18 g/tex) of cloths and cloths fibers. The effluents generates after cloth washing with this detergent formulation does not effect on aquatic ecosystem. Thus, this aqua-friendly detergent formulation can be used to avoid pollution problems associated with effluent generates after cloths washing.

Thermostable alkaline protease from Thermomyces lanuginosus: optimization, purification and characterization

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-100 and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K m), maximal reaction velocity (V max) and turnover number (K cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s−1, respectively. The best enzyme activators were K+, Ca2+ and Mn2, respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.