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Humans and other animals consume commercially produced fungus and fungal products. Different secondary metabolites like enzymes, organic acids, and biocontrol agents are produced by fungal groups like decomposers and antibiotic makers. The variety, location, and commercial significance of the fungus population's potential to produce extracellular enzymes in soil samples from Kodaikanal Shola are the subjects of the current study. A total of 34 different fungus species, including Aspergillus, Absidia, Penicillium, Trichoderma, Rhizopus, Drexchelara, and Mucor, were discovered. Aspergillus niger was the most prevalent species among them. Based on their predominance enzymatic features, Aspergillus niger was utilized for future research. Under several circumstances of temperature (15°C, 37°C, and 45°C) and pH, the isolated organism's best growth parameters for producing enzymes were investigated (5,7 and 9). Amylase (0.0068 IU/ml), followed by lipase (0.005 IU/ml), protease (0.0032 IU/ml), and cellulase (0.0026 IU/ml) enzymes, was the enzyme most abundantly generated from A. niger at 45 °C and pH 7 (0.0078 IU/ml). This study raises the possibility of using A. niger as a key source for the industrial production of vital enzymes. [ABSTRACT FROM AUTHOR]

Xylanase especially Endo-1,4-beta-xylanase (EC. 3.2.1.8) is a very important enzyme in various industries. The thermostable xylanase is one of the main enzymes used in the pulp industry, in order to reduce the chemicals substances, such as chlorine dioxide and chlorine. This thermostable enzyme can be isolated from thermophilic bacteria which isolated from hot springs. This research was focus on characterization of thermostable-bacterial xylanase enzymes isolated from Bora hot springs. There were 9 out of 21 isolates of thermophilic bacteria that could produce xylanase and showed xylanolytic index, during screening step on Xylan Agar Plates. Among these 9 isolates, 3 isolates were selected and further characterized in this work, namely BHS 011, BHS 014, and BHS 018. These three bacteria could produce extracellular Xylanase enzyme which can be characterized based on: xylanolytic index were in range of 0.695 - 2.632; xylanase enzyme activity was in range of 8.370 U/ml - 11.359 U/ml; specific activity of xylanase crude enzyme was in range of 463.429 U/mg - 1913.538 U/mg. These produced xylanase enzymes were active in range temperature of 32°C - 80°C which the optimum temperature was in range of 55.9°C - 64.3°C. In addition, its xylanase enzymes were active in pH of 5 – 8.6, which the optimum pH were in the range of 5.2 - 6.8. The properties of the xylanase enzyme produced by these thermophilic isolates, indicating the appropriate potential for its application in the various industrial sector. [ABSTRACT FROM AUTHOR]

Chitinases were extracellular enzymes that hydrolyze chitin compounds into monomers of N-acetyl-glucosamine. It has antagonistic effects on spore germination and hyphal growth of pathogenic fungi and involved in the degradation of fungi cell walls consisting of Chitin. A chitinolytic bacteria used was isolated from the soil in Indonesia and identified by 16S rRNA gene sequence analysis as Bacillus cereus WS4F. The aims of this study were the characterization of WS4F Chitinase and the improvement of its activity by random mutagenesis to be applied as a biocontrol agent on pathogenic fungi. Its activity was improved by random mutagenesis using N-Methyl-N'-Nitro-N-Nitrosoguanidine (NTG) and UV-light irradiation. Extracellular WS4F Chitinase was produced in chitin flakes containing media and optimally operated by the agitation of 200 rpm, pH 7, and temperature of 37°C. Chitinase activity was measured by the Bailey method. The result showed that the highest WS4F Chitinase volumetric activity was 0.167 U/mL from the NTG mutant strain compared to the UV-light Irradiation mutant strain of 0.079 U/mL and wild-type strain of 0.048 U/mL. The purification of WS4F wild-type Chitinase using the ion-exchange method obtained a purity level of 3.397 times compared to crude chitinase, with the highest specific activity value of 4.750 U/mg, protein content of 0.028 mg/mL and volumetric activity 0.133 U/mL. The molecular masses of WS4F Chitinase were at 63.718 kDa and 40.379 kDa. The WS4F Chitinase has the potential to be used as a biocontrol agent due to its antagonistic activity against pathogenic fungi of Ganoderma boninense. [ABSTRACT FROM AUTHOR]

Hermetia illucens L. is known as Black Soldier Fly (BSF), a voracious feeders during larval stages. Its role as bioconversion of organic waste is assumed correlate with the bacteria in BSF larvae. The bacteria are expected to have enzyme activity such as amylase, lipase, and protease. In the present study, the bacteria from BSF larvae were isolated using 6 different media. The isolated bacteria will be screened for their amylolytic, lipolytic, and proteolytic activities. Based on the formation of a clear zone and good growth appearance, total 22 representative bacterial isolates were obtained. Characterization of the isolates was consist of the morphological and biochemical tests. According to the result of the enzymatic test, five isolates have positive result on the amylolytic test, two isolates have a positive result on the lipolytic test, and eleven isolates have a positive result on the proteolytic test. Among those isolates, the B18 isolate has positive result on the three enzymatic tests. Molecular identification based on 16S rRNA showed that B18 was closely related to Bacillus paramycoides with 99.93% sequences similarity. [ABSTRACT FROM AUTHOR]

Litter is a suitable habitat for organisms including fungi, because fungi are considered as the main decomposers of leaf biomass and dead plants. One of the contributing factors is its ability to produce various kinds of extracellular enzymes. Many previous fungi researchers used conventional methods to identify fungi by morphological characterization. However, the application of the conventional method has drawbacks such as requiring hard work and a long time, especially in characterizing these fungi to the species level, and morphological characterization often leads to misperceptions. So, DNA Barcoding is an effective method to identify the fungi based on short gene sequence such as Internal Transcribed Spacer region. The aims of this study was to identify of the prospective isolates, IFDt2 isolates that had been previously isolated in Samanea saman leaf litter. The methods used were refreshment isolates, macroscopic and microscopic morphological characterization. Molecular characterization was carried out by processing isolated DNA samples using the classical DNA barcoding approach by amplification and sequencing using universal primers. According to the results, IFDt2 isolate has similar morphology characteristics with genus Trichoderma. DNA barcoding analysis by using ITS marker showed that the isolate IFDt2 has 100% similarity with T. longibrachiatum. It could be concluded that DNA barcoding effectively could identify the IFDt2 at the species level. [ABSTRACT FROM AUTHOR]

Exploration and isolation of the roots, stems and leaves of wild plants is one way that can be done to obtain endophytic bacteria that has potential as biological control agents. The study aims to obtain endophytic bacterial isolates that have the potential as biological agents to control Colletotrichum sp. based on the in-vitro test. This experiment was conducted at the Plant Protection Laboratory Unit Phytopathology, Faculty of Agriculture, Halu Oleo University, Kendari, from September 2020 until January 2021 used a randomized complete design with three replications. The test includes exploration and isolation, screening for antagonists, testing for inhibition, the activity of cell wall degrading enzymes and volatile compounds. The results showed that ASR-3 (4) and ASR-7 (4) isolates were the best isolates that have several antagonistic mechanisms, including being able to inhibit the growth of Colletotrichum sp. through antibiosis, able to secrete extracellular enzymes that degrade cell walls in the form of proteases and amylases, also can produce volatile compounds and have a very strong inhibition category with inhibition percentages respectively 51.11% and 50.54%, and volatile compound test respectively 52.73% and 54.55% [ABSTRACT FROM AUTHOR]

Endophytic communities provide benefits to their host plants such as protecting plants against herbivores, insects, or pathogens, as well as being able to stimulate plant growth. The association of endophytic bacteria with plants can affect plant productivity directly or indirectly. One of the contributions of endophytic bacteria is as a producer of extracellular enzymes. Mangrove areas are the main ecosystems that support important life in coastal and marine areas. Many benefits are obtained from mangrove plants. One of them is microorganisms associated with mangrove plants. Enzymes from microorganisms are more beneficial and their production is faster than enzymes derived from plants and animals. Endophytic bacteria associated with a mangrove species, Rhizophora apiculata, are a source of hydrolytic enzymes such as proteases and cellulases which are produced for their ecological contribution and also to support the growth of their host plants. The aims of this research were isolate endophytic bacteria from the R. apiculata and screening qualitatively the proteolytic and cellulolytic activities of the endophytic bacterial isolates. The isolates were screened for protease activity on skimmed milk agar (1%) and cellulase activity on Carboxymethyl Cellulose (CMC) agar. In this study, 18 isolates of endophytic bacteria were isolated from the leaves of the mangrove plant R. apiculata and 9 isolates had proteolytic activity and 13 isolates had cellulolytic activity or could produce cellulase. Endophytic bacteria R. apiculata which has the ability to produce proteases and cellulases qualitatively can be explored further to be developed as a source of hydrolytic enzymes from endophytic bacteria. This works contributes to our knowledge of the endophytic bacterial as source of enzymes present in mangrove plants. [ABSTRACT FROM AUTHOR]

Potential sources of thermostable enzymes are thermophilic bacteria. In the research, nucleotide base sequences of 16S rRNA gene from thermophilic bacteria have been identified. The bacteria showed activity of hydrolytic enzyme exo-type on inulin substrate. The bacteria were isolated from 'Batu Bajanjang' hot spring, Solok (West Sumatra) that labeled as BB3 isolate. Gene of 16S rRNA fragment was isolated using PCR technique with BactF1 and UniB1 primers. The nucleotide base sequences of 16S rRNA gene were determined using the dideoxy-Sanger method. Analysis of the gene fragment (1348 bp) from BB3 isolate indicated as Fictibacillus gelatini or Bacillus gelatini species. Activity of extracellular enzyme indicated exo-type action based on analysis of the inulin hydrolysis end products using Thin Layer Chromatography (TLC). So, the enzyme is the potential to produce high fructose syrup from inulin. [ABSTRACT FROM AUTHOR]

Laccase is an enzyme that has the ability to oxidize substances. It is one of those enzymes that has innate qualities of reactive radical generation, and its use in many domains has been overlooked due to its commercial unavailability. The ability of Marasimus palmivorus MG717877.1 to produce extracellular enzymes (Laccase enzyme) utilizing media containing substrate, named Guaiacol agar medium, was tested. In submerged culture, the ideal pH, incubation duration, and temperature for laccase synthesis were examined. The maximum enzyme activity was reported when the pH of the media was 5.5; laccase activity was (1.03U/ml). The maximum enzyme activity for laccase enzyme was 1.040 U/ml when the temperature was 25°, and (0.922U/ml) after the third day of incubation. [ABSTRACT FROM AUTHOR]

Fifty-nine endophytic bacterial isolates from rat taro have been obtained; thirty-four bacterial isolates from rat taro type 3 (TF3) and twenty-five bacterial isolates were from type 4 (TF4). The extracellular enzyme activity was observed qualitatively from rat taro endophytic bacterial isolates. The amylase activity test used starch as substrate, whereas the protease test used gelatin. A cellulose activity test was carried out using Carboxy Methyl Cellulase (CMC) media. The results showed that twenty-seven endophytic bacterial isolates of rat taro TF3 and TF4 showed extracellular enzyme activity. Endophytic bacterial isolate TF3Bt4 had the highest protease activity, indicated by its lytic index of about 11.304 mm in the gelatin-containing medium. The highest amylase activity was produced by isolate TF3U3, where the lytic index was 23.875 mm in the starch containing medium. Bacterial isolate TF3A4 showed a lytic index of about 6.9653 mm in the CMC medium, indicating the highest cellulase activity. The endophytic bacteria with protease activity are potentially utilized in the food industry, whereas endophytic bacteria with cellulase activity can be used in the textile industry. [ABSTRACT FROM AUTHOR]

Laccase (EC 1.10.3.2) is an extracellular enzymes that is widely used in the decolorization process of textile dye wastewater. The aims of this study was to isolate, purify, and characterize the laccase produced by Trametes pavonia EDN 134 and to explore the laccase ability to decolorize azo textile dyes. Laccase purification process was carried out by fractionation using ammonium sulfate and continued with Hi Trap desalting column chromatography method. The results revealed that the highest laccase specific activity was 8268 U/g and detected in the ammonium sulfate fraction 40-60%. The purification using AKTA-Prime showed that the laccase specific activity enhanced to 98976.61 U/g. The purified laccase activity was optimum at pH 4 and temperature 27°C. The addition of heavy metal ions Hg2+ and Zn2+ reduced the activity of laccase, while Cu2+ and Mn2+ enhanced the activity. In addition, the purified laccase decolorized 200 ppm of Methyl Red (MR), Reactive Black 5 (RB5), and Reactive Red 120 (RR 120) of azo dyes as much as 50.7%, 21.9%, and 35.5%, respectively. This study proposes the potency of the laccase from T. pavonia EDN 134 for decolorization of textile dye wastewater. [ABSTRACT FROM AUTHOR]

Three potential Bacillus sp. strain, KRG, KRT, and LDR from cocopeat was known to have antagonistic activity against fungal pathogen. The antagonistic activity represented the potency of three isolates as biocontrol agent. In this study, the growth and extracellular enzyme of three isolates will be characterized and also the selected bacterium was identified based on 16S rRNA. The growth of Bacillus sp. strains were characterized in Potato Dextrose Broth (PDB) with various pH medium and temperature incubation. The three isolates can growth at 30–45 °C, meanwhile KRG can grew up to 50 °C. The optimum temperature growth of LDR was 30–45 °C. All of isolates can grow in medium with pH 5–8 with optimal growth for LDR in pH 7–8. Extracellular enzyme assay showed all isolates can produce amylase, lipase, protease, and gelatinase. Molecular identification showed that selected strain LDR is closed to Bacillus siamensis and has 99.52 % similarity with Bacillus siamensis KCTC 13613 strain PD-A10. [ABSTRACT FROM AUTHOR]

High salt-tolerant extracellular protease is an enzyme that plays a crucial role in industries and requires high salt conditions. Some protease enzymes are not resistant to high salt conditions because they may denature under high salt concentrations. Halophilic microbes produce extracellular enzymes that are stable at high salts. This research has succeeded isolating several halophilic microbes capable of producing extracellular proteases from Bledug Kuwu, Grobogan, Central Java, Indonesia. Extracellular proteases producing bacteria screened with skim milk selective medium and purified with the quadrant streak method. Then, the pure isolate and the resulting extracellular protease were characterized BK1B isolate was a Gram-positive bacterium, while BK1D was a Gram-negative bacterium. BK1B isolate is a halotolerant bacterium and BK1D is a moderate halophilic bacterium. BK1B and BK1D had the largest proteolytic index values, capable of producing extracellular proteases with an optimal production time of three days and pH 6. Extracellular proteases from BK1B and BK1D worked optimally at pH 6 and temperatures of 45 and 42 °C with 2.541 and 3.505 U/mL activity and resistance to NaCl concentrations ranging from 3 - 13% NaCl. This suggests that extracellular proteases from BK1B and BK1D strains can be stable at high salt concentrations. [ABSTRACT FROM AUTHOR]

Tributyrin in solid media can be used to screen lipolytic activity. Lipolytic activity of many strains from Rhizopus has been described and some enzymes have been isolated. Members of Rhizopus play an important role in the decomposition of organic materials due to their effective extracellular enzyme production. Extracellular esterases have been produced by a member of Rhizopus. Esterases are used for degradation of domestic and agricultural biomass. There is always a demand for esterase enzymes with increased stability and selectivity for a variety of applications in industrial biotechnology. This study was carried out to detect the ability of Rhizopus azygosporus UICC 539 to use 1 % (v/v) and 2 % (v/v) tributyrin at various temperatures. Agar block (diameter 6 mm) containing a concentration of R. azygosporus cells at approximately 106 cell/mL was prepared from a 5-day old fungus in Potato Sucrose Agar (PSA) at 30 °C. The agar block was inoculated into 1 % (v/v) or 2 % (v/v) commercial tributyrin in Tributyrin agar base plates and incubated for 3 and 5 days at various temperatures (30 °C, 35 °C, 40 °C, 45 °C and 50 °C). Tributyrin agar base plates without the fungus served as control. Clear zones were indicative of tributyrin hydrolysis. The following formula was employed to calculate the Enzymatic Index (EI): R/r, where R was the diameter of the entire clear zone, and r was the diameter of the fungal colony. The results showed that R. azygosporus UICC 539 was able to use both tributyrin concentrations at all tested temperatures. High EI was detected at 50 °C at both tributyrin concentrations, with the highest EI at 1 % tributyrin after day-5 of incubation. In conclusion, R. azygosporus UICC 539 secreted esterolytic enzyme (esterase) into the medium when grown in 1 % and 2 % tributyrin. The strain utilized tributyrin in the temperature range of 30–50 °C. This study indicated that the enzymatic ability of R. azygosporus UICC 539 can be exploited in converting lipid-containing agricultural biomass in higher temperatures. [ABSTRACT FROM AUTHOR]

Reutealis trisperma plant is widely used for nature conservation and the oil seeds have been studied for biodiesel production. The press cake is a solid waste which contains 34.03% protein, 18.56% total sugar, 15.58% lipid, 6.32% moisture and 25.51% others. Since its high content of protein, the cake is used as additional nitrogen source for xylanase production using Trichoderma reesei. Xylanase has a wide range of applications such as in pre-bleaching of pulp, improving the digestibility of animal feed stocks, bioconversion of lignocellulosic material to fermentable products and clarification of fruit juices. In pulp and paper industries, pre-bleaching of pulp by xylanase can save 35-40% of chlorine used which would reduce the potential hazard to environment. T. reesei is a mesophyllic, filamentous fungus which has been well studied for its ability to produce extracellular enzymes capable of degrading cell wall polysaccharides. The aims of the study are: 1) to formulate media at various C/N ratio in defined medium and various initial pH, and to formulate media containing ground press cake at both optimum C/N ratio and initial pH from defined medium. 2) to calculate xylanase activity in both media. In this study, T.reesei was cultivated in shake flask at 28°C in defined medium at various C/N ratio and initial pH and in the culture medium contained ground press cake at optimum C/N ratio and initial pH. The defined medium composition contained cellulose as carbon source at various concentration; organic and inorganic nitrogen sources and mineral salts. Ground cake was used to replace some inorganic nitrogen used in defined medium for media containing ground cake. Sample was withdrawn periodically to measure the pH and xyalanse activity. The best C/N ratio and initial pH in defined medium were 7.8/1 and 6.0 respectively which resulted in 66.93 U/mL of xylanase activity. The xylanase activity was 30.75 U/mL in media containing ground press cake. [ABSTRACT FROM AUTHOR]

Our previous study reveal that single culture of Lactobacillus plantarum has ability to ferment cassava tuber in relation to produce modified cassava flour (mocaf). It was used to accelerate a fermentation process. L. plantarum grow well and produce some extracellular enzymes i.e. cellulase to change the structure and breakdown the cell wall of cassava tuber. Then, the starchy materials will be hydrolyzed by i.e. amylase into simple sugar and convert to organic acid. All of these process will give new characteristic of cassava i.e. lower fiber content, good flavor, taste, aroma and texture and the amount of cyanide acid is lower. Therefore this present study was to analyze Michaelis kinetics of extracellular carboxymethyl cellulase and amylase production by L. plantarum during cassava fermentation. The maximum carboxymethyl cellulase and amylase activity of 8.60 U/ml and 14.07 U/ml, respectively, were obtained from filtrate which has been incubated at 37°C for 18 h under stationary conditions. The Vmax and Km of CMCase were 0.8506 x 10-3 U/ml and 0.9594 x 10-3 g/mL, respectively. For amylase were 9.291 x 10-3 U/ml and 0.9163 x 10-3 g/ml, respectively. [ABSTRACT FROM AUTHOR]

Halophilic microorganisms, either bacteria or archaea, flourish in media with salinity levels varying from negligible until to saturation in NaCl and thus are considered extremophiles. Such kinds of media as salt lakes, salted soils, salt deposits or salt mines are widely distributed over the entire Romanian landscape. Several strains of halophilic bacteria and archaea have been isolated from such environments and characterized either by polyphasic taxonomy approach or by their ability to produce extracellular enzymes with putative potential for use in several domains like industry, agriculture and biotechnology. Even if sodium chloride is widely used in Romanian agriculture fields, little is known about salt microbiota, and its effect on the agricultural processes. This paper approaches the wide spectrum of extracellular hydrolytic enzymes with putative use in agriculture for treatment of polluted soils or recovery of salted soils. The strains isolated from inside of salt crystal are divided in two groups, one consisting of six strains belonging to Halorubrum, Haloarcula and Halobacterium genera and characterized by the absence of detectable extracellular enzymatic activity for the tested substrates and the other group consisting of four strains, all belonging to Halorubrum genus and characterized by the presence of amylolytic activity and the absence of other activities. The starch degrading enzymes appear to have a wide distribution both among the strains associated with salt lakes and among those isolated from rock salt. [ABSTRACT FROM AUTHOR]

It has been suggested that extracellular ATP signalling is implicated in protecting the auditory epithelium of the inner ear from noise-induced damage (Housley et al., 2009). Although ATP receptors have been found on several types of cells in the organ of Corti, these studies have focussed on either dissociated or a limited class of cells (Lagostena et al., 2001). Supporting cells show parallels with glia (Rio et al., 2002), however it is unclear if and how such cells, positioned nearthe first auditory synapse, might act in a protective manner. Multiple types of supporting cells are present and it is unknown whether ATP affects them similarly or activates different processes in them (Gale et al., 2004). To address these issues we have studied features of ATP-dependent signalling in the adult organ of Corti in an in situ system. The temporal bone of adult mice (ages P30-P90) was removed after killing the animals by cervical dislocation and the organ of Corti was exposed through a small opening at the apical end of the cochlea. For immunochemistry, antibodies against P2X2 (Alomone Labs) were used on 4% PFA fixed tissue. For calcium imaging, opened cochlear bones were incubated for 40 min in buffer containing 20 μM OGB1-AM, washed and then fluorescent signals were collected by widefield epifluorescence microscopy. Whole cell tight seal patch clamp recordings were made from inner sulcus and phalangeal cells identified visually. In some cases cells were decoupled with the gap junction blocker flufenamic acid (100 μM) to improve voltage clamp. There was extensive P2X2 immunoreactivity in the supporting cells of the organ of Corti. Comparatively, outer hair cells showed a limited degree of P2X2 on their apical surface. Consistent with these observations, 100 μM ATP puff applied to the organ of Corti produced a rise in intracellular (Cai) in supporting cells measured by OGB1. There was no detectable increase in Cai levels in the inner hair cells. ATP (100μM) applied to the inner sulcus and phalangeal cells surrounding the inner hair cells produced inward currents in both types of cells. The ATP gated currents showed inward rectification in both types of cells consistent with the properties of recombinant P2X channels. The ATP dependent currents in inner sulcus cells desensitized (τ1/2 = 1.6s) whereas little or no desensitization to prolonged application (15s) was observed in recorded phalangeal cells. We suggest that these results indicate differential expression of P2X receptor subunits and could reflect different roles for extracellular ATP signalling in these two cell types. [ABSTRACT FROM AUTHOR]

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC). Cleavage at specific sites in the extracellular domains of α-, δ-, and γENaC is essential for channel activation [1; 2]. However, the (patho-)physiologically relevant proteases and molecular mechanisms involved in proteolytic channel activation remain to be determined. Inappropriate ENaC activation by proteases may be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. Recently, we and others reported that the serine protease plasmin can proteolytically activate ENaC which may contribute to renal sodium retention in nephrotic syndrome [3; 4]. In mouse γENaC a putative plasmin cleavage site (K194) has been reported [4]. For human ENaC, the relevant cleavage site(s) for plasmin remain to be determined. In addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. Our aims were (a) to identify functionally relevant plasmin cleavage sites in human γENaC [5] and (b) to investigate the effect of Cat-S on human ENaC [6]. Mutant human γENaC constructs were generated by site-directed mutagenesis. Human wild-type or mutant αβγENaC was expressed in Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents (ΔIaml) were determined by two-electrode voltage-clamp before and after 30 min incubation of the oocytes in human plasmin (10 μg/ml), chymotrypsin (2 μg/ml) or Cat-S (1 μM). Biotinylated cell surface γENaC cleavage products were detected by western blot analysis using a γENaC antibody. (a) Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type αβγENaC and αβγK189AENaC in oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole-cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189 the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wild-type or double mutant ENaC. This indicates a causal link between proteolytic cleavage and channel activation. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near silent channels was confirmed in single-channel recordings from outside-out patches. Interestingly, mutating two phenylalanines (γFF174) adjacent to the prostasin cleavage site pre vented proteolytic activation by chymotrypsin. This indicates that the two phenylalanines constitute a preferential cleavage site for ENaC activation by chymotrypsin. (b) We demonstrated that Cat-S activates ΔIaml in ENaC-expressing oocytes. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutat ing two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. The stimulatory effect of Cat-S on ENaC activity and the con comitant appearance of a γENaC cleavage product at the cell surface were prevented by the Cat-S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl). In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. To our knowledge, this is the first report that the cysteine protease Cat-S can activate ENaC which may be relevant under patho physiological conditions. In this study, we identified cleavage sites in a region critical for proteolytic channel activation in the γ-subunit of human ENaC with functional importance for its proteolytic activation by plasmin, chymotrypsin and Cat-S. The fact that γENaC contains several different cleavage sites in this region is likely to have physiological implications. Indeed, it may provide a mechanism for differential ENaC regulation by tissue-specific proteases. [ABSTRACT FROM AUTHOR]