High Protease Activity Research Articles

Production of bioactive hydrolysate using protease, β-glucosidase and α-amylase of Bacillus spp. isolated from kinema

The aim of this study was the production of soybean bioactive hydrolysate using Bacillus spp. isolated from kinema. Totally 251 bacteria isolated from kinema samples, collected at different time period were screened for protease, β-glucosidase and α-amylase activities and further identified by ARDRA based grouping followed by analysis of 16S rRNA gene sequence similarity. The results showed that Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis were the major Bacillus species. Twelve fermentative strains belonging to these groups and having high protease, α-amylase and β-glucosidase activity were used for solid state fermentation. The best strains for soybean fermentation that result in production of protein hydrolysates rich in polyphenols that have higher bioactivity were B. subtilis KN12C, B. amyloliquefaciens KN2G and B. licheniformis KN13C. Potential isolates can be applied for the production of soybean hydrolysates and can also find application in production of value added products from by-products of soybean processing industries.

Highly active and stable protease production by an extreme halophilic archaeon Haloarcula sp. TG1 isolated from Lake Tuz, Turkey

Abstract Objective Isolation of halophilic microorganisms from Çankırı salt mine and Lake Tuz in Turkey to explore versatile protease producers for industry and characterization of protease enzyme from the best protease producer among the isolated strains. Methods Extreme halophiles were isolated from salt samples of Çankırı salt mine and Lake Tuz. Their protease activities were determined. The isolate with the highest protease activity was characterized. Its protease activity was evaluated in different NaCl concentrations, temperature and pH ranges, and in the presence of different inhibitors and metals. Thermostability and pH stability were also determined. Results The highest protease producer strain was identified as Haloarcula sp. on the basis of 16S rRNA analysis. The isolate namely, Haloarcula sp. TG1, was found to be 99% identical to Haloarcula salaria strain HST01-2R. The TG1 protease was found to possess very high activity and stability over a broad pH and temperature ranges. Its maximum activity was recorded at pH: 4.0, 50°C and 4 M NaCl. Among inhibitors tested, dimethyl sulfoxide (DMSO) and ethanol caused the highest decrease (ca. 25%) in its activity. Conclusion Due to the high activity and stability over a wide range of extreme conditions, Haloarcula sp. TG1 protease reported here is a promising candidate in biotechnology.

Characterization of Aspergillus sojae Isolated from Meju, Korean Traditional Fermented Soybean Brick.

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and α-amylase with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.

Extracellular peptidases as possible markers of fungal ecology

Fungi produce extracellular peptidases to break down proteins and polypeptides into smaller soluble molecules. These molecules are subsequently transported into the fungal cell, supplying osmotrophic nutrition. Although a variety of extracellular peptidases have been isolated from numerous fungal species, it remains largely unknown how the spectrum of secreted peptidases correlates with fungal ecology and taxonomy. In this study, we cultivated 17 fungal species on protein-enriched medium to compare the spectra of secreted peptidases produced by the fungi belonging to different ecological groups (pathogens, saprotrophs, symbionts) and taxonomic phyla (Ascomycota and Basidiomycota). Basidiomycetes predominantly produced metallopeptidases with neutral pH optima, while the species of ascomycetes secreted serine peptidases with alkaline pH optimum, mostly of the subtilisin-type. Fungal species with dominant activity of serine peptidases had, in general, much higher peptidase activity and were better adapted to growth on protein medium compared to the species with dominant metallopeptidase activity. Pathogenic fungi effectively cleaved the synthetic substrate specific for trypsin-like peptidases, while saprotrophic species were characterized by a relatively high aminopeptidase activity. Nevertheless, fungi have numerous genes encoding various types of peptidases, the spectra of fungal enzymes secreted on cultivation medium were to a major extent represented by the production of one class of peptidases that correlated with fungal taxonomic phylum. We suppose fungal strategies for cleaving polypeptides in experimental conditions are largely predetermined both by trophic status and phylogeny.

Biological H2 potential harvested from complex gelatinaceous wastewater via attached versus suspended growth culture anaerobes

The effect of cultural growth treating gelatinaceous wastewater on hydrogen fermentative was assessed using up-flow multi-stage anaerobic sponge reactor (UMASR) and anaerobic sequencing batch reactor (AnSBR). Both reactors were operated at five hydraulic retention times (HRTs). UMASR achieved the maximum COD removal efficiency of 60.2±4.4% at HRT of 48h. Moreover, UMASR exhibited superiority in the course of carbohydrates and proteins removal efficiencies’ of 100 and 52.5±2.4% due to high amylase and protease activities’ of 4.1±0.3 and 0.032±0.002U, respectively. Contrariwise, AnSBR assigned for the peak hydrogen production rate of 1.17±0.14L/L/day at HRT of 24-h. Lipase activity was quite high (0.307±0.023U) in AnSBR resulting in removal efficiency of 35.2±2.1% for lipids. Stover–Kincannon model emphasized that UMASR required lesser volume than AnSBR to sustain the same substrate degradation efficacy. Nevertheless, the net gain energy harvested from AnSBR surpassed UMASR by 4.0-folds at HRT of 24-h.

Effect of soybean flour (Glycine max) and its inhibitors enzyme on endogenous protease activity in surimi-like gel elaborated with jumbo squid (Dosidicus gigas) during the storage at 4°C and 10ºC

Jumbo squid (Dosidicus gigas) is a marine product abundant in the Pacific shores of Mexico. It has high endogenous protease activity that affects gellation process and may to increase its deterioration during storage of muscle mantle fresh or processed. Enzyme inhibitors from soybean may to contribute to the biological regulation of this endogenous alkaline protease. Changes in proteolytic activity during the storage at 4ºC and 10ºC were studied in a model system of alkaline enzyme extracts of mantle muscle and enzyme inhibitors extracts. Surimi-like gel (SLG) elaborated with myofibrillar proteins from muscle mantle and soybean flour was also analyzed. Significant reduction (P<0.05) of proteolytic activity was observed in model system and SLG for both temperatures. During storage time lowest values were 14.3 AU/mg (4ºC, 192 h) and 6.90 AU/mg (10ºC, 120 h) with the treatments of 30 mg/mL protein of enzyme inhibitors extracts/70 mg/mL protein of enzyme extracts and 77.80 AU/mg (4ºC, 144 h) and 80 AU/mg (10ºC, 72 h) for SLG elaborated using 50 g soybean flour (dry base) and 50 g myofibrillar protein (wet base). These results showed feasible to use soybean flour as a source of proteolytic inhibitors in surimi-like gels from jumbo squid during the storage at refrigeration conditions and temperature abuse conditions.

Oral candidal speciation, virulence and antifungal susceptibility in type 2 diabetes mellitus.

To determine the oral candidal carriage (OCC), activity of virulent factors and fluconazole susceptibility in subjects with type 2 diabetes mellitus (T2DM) and investigate their association with HbA1c measurements. A cross sectional study was conducted on 100 diabetics and 100 healthy volunteers. The virulence was assessed by measuring the phospholipase activity and proteolysis index. Fluconazole susceptibility was performed using the gradient diffusion method. The OCC, virulence factors and antifungal susceptibility were correlated with patients' HbA1c measurements. The OCC and candidal density carriage was significantly higher in diabetics. Candida albicans (C. albicans) was the most frequently isolated species followed by Candida tropicalis (C. tropicalis). Relatively uncommon species, Candida lusitaniae (C. lusitaniae) and Candida lipolytica (C. lipolytica) were isolated from the diabetics. Prevalence of virulence factor, proteinase, was greater in diabetic group (p<0.05). Reduced fluconazole susceptibility was noted among the isolates from diabetics; however it was not statistically significant (p=0.593). Except one, all the susceptible-dose dependent and resistant isolates were Candida no-albicans (C. non-albicans). C. albicans remains the predominant pathogen in diabetics, although other species are on the rise. Compared to control group, the isolated species from T2DM group had higher proteinase activity. Resistance to fluconazole was considerably greater among the C. non-albicans isolates from T2DM group. These findings warrant effective treatment modalities to reduce the occurrence of oropharyngeal candidiasis.

Antibacterial serine protease from Wrightia tinctoria: Purification and characterization

A serine protease was purified from the leaves of Wrightia tinctoria by sequential flow through method comprising screening, optimization, ammonium sulfate precipitation, gel filtration and ion exchange column chromatography. The yield and purification fold obtained were 11.58% and 9.56 respectively. A single band of serine protease was visualized on SDS-PAGE and 2-D gel electrophoretic analyses were revealed with the molecular mass of 38.5 kDa. Serine protease had an optimum pH of 8.0 and was stable at 45°C with high relative protease activity. The addition of metal ions such as Mg2+ and Mn2+ exhibits a high relative activity. Serine protease had a potent antibacterial activity against both Gram-positive and Gram-negative bacteria. A 10 μg/ml of serine protease was tested against S. aureus, M. luteus, P. aeruginosa and K. pneumoniae which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis.

Geographic Origin and Host Cultivar Influence on Digestive Physiology ofSpodoptera exigua(Lepidoptera: Noctuidae) Larvae

Digestive enzymatic activity in three geographic strains (Miandiab, Kalposh and Moghan regions) of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) reared on different sugar beet cultivars (Dorothea, Rozier, Persia and Perimer) was studied under laboratory conditions (25 ± 1 °C, 65 ± 5% RH, and a photo period of 16:8 (L:D) h photoperiod). The results of this study demonstrated that digestive protease and amylase activity of S. exigua larvae was affected by both geographic origin of the pest and host plant cultivar. Three strains reared on the same sugar beet cultivars demonstrated different levels of proteolytic and amylolytic activities in fourth and fifth instars. The highest proteolytic and amylolytic activity, in most cases, was observed in larvae collected from Kalposh region. Among different sugar beet cultivars, the highest protease activity in three strains was observed on cultivars Rozier and Perimer. Nevertheless, the highest amylase activity was seen on cultivar Dorothea, and the lowest activity was seen on cultivar Rozier. This study suggested that variations in digestive enzymatic activity of three geographic strains of S. exigua might be attributed to local adaptation with their local host plant and environmental conditions inherent by larvae.

형질전환 벼 현탁세포 배양에서 hCTLA4Ig의 in situ 회수

In this research, recombinant human cytotoxic Tlymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

Evaluation of Imbrasia belina meal as a fishmeal substitute in Oreochromis mossambicus diets: Growth performance, histological analysis and enzyme activity

The main objective of this study was to investigate mopane worm (Imbrasia belina) as a protein source in the diet of Oreochromis mossambicus. One thousand five hundred O. mossambicus fingerlings (40±2.5g) were fed five isonitrogenous, isolipidic and isoenergetic diets formulated to contain 30% crude protein and 20MJ/kggross energy (dry matter basis) for 51days. Fifteen indoor rectangular concrete tanks (1.5m3) connected to a recirculating system were used. Water temperature ranged between 27 and 29°C. The diets were prepared by replacing fishmeal with mopane worm meal at 10%, 20%, 40% and 60%. The diets were coded D2, D3, D4 and D5 respectively. A control diet with no mopane worm meal was coded D1. The diets were fed to triplicate groups of O. mossambicus twice a day. Specific growth rate (SGR), Thermal-unit growth coefficient (TGC), protein efficiency ratio (PER) and apparent digestibility coefficient (ADC) increased with higher inclusion levels of mopane worm meal. Feed conversion ratio (FCR) also improved with higher inclusion levels. However, the highest growth performance (SGR: 3.49%; FCR: 1.29) was recorded in fish fed the fishmeal based control diet. Protease, amylase and lipase activities were determined in the intestines. Both protease and amylase activity were significantly higher (P<0.05) at high mopane worm inclusion levels. It is suggested that the high protein levels of the mopane worm diet elicited high protease activity. The health status of the fish was evaluated by examining the liver and intestine histology. There were no evident histological alterations of either liver or intestine as mopane worm meal inclusion levels increased. This showed that mopane worm meal may be a good candidate for the replacement of fishmeal in O. mossambicus diets. The highest profit index (1.67) was recorded in the 60% mopane worm inclusion level. The lowest profit index was in the control. More studies on mopane worm meal as a substitute of fishmeal are recommended in other fish species.

Characterization of a metalloprotease involved in Vibrio splendidus infection in the sea cucumber, Apostichopus japonicus

Vibrio splendidus is an important aquatic pathogen that infects a broad range of hosts, leading to typical symptoms of “skin ulceration syndrome” in the sea cucumber Apostichopus japonicus. However, there are few reports on the virulence factors and pathogenic mechanism of V. splendidus. In the present study, V. splendidus could survive in the coelomic fluid of A. japonicus but poorly internalized into the coelomocyte under the tested conditions. It was thus postulated that V. splendidus was pathogenic to A. japonicus, mainly due to its extracellular products. The main extracellular proteins of V. splendidus were detected using MALDI-TOF-TOF/MS, and a metalloprotease was identified. The gene encoding a metalloprotease belonging to the thermolysin family, named vsm, was cloned and characterized. Furthermore, the expression of vsm was growth dependent and the supernatant of V. splendidus also showed high protease activity. The vsm gene was conditionally expressed in Escherichia coli BL21(DE3). Enzyme activity analysis showed that the optimal temperature and pH for purified recombinant Vsm were approximately 40 °C and 7.0, respectively. Furthermore, Vsm was determined to be associated with the pathogenesis of V. splendidus due to the following aspects: (1) real-time reverse transcriptase PCR showed that expression of vsm was significantly up-regulated when V. splendidus was co-incubated with the coelomic fluid of A. japonicus; and (2) purified recombinant Vsm showed obvious cytotoxicity to the coelomocyte of A. japonicus. Our results indicated that Vsm is involved in the interaction between V. splendidus and A. japonicus and also contributed to the cytotoxic effects on the coelomocyte of A. japonicus.

Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function

A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

Phospholipase and proteinase activities of Candida spp. isolates from vulvovaginitis in Iran

This study aims to characterize phospholipase and proteinase activities of Candida isolates from 82vulvovaginal candidiasis (VVC) and to study the relationship of these activities with vulvovaginitis. Totally 82Candida isolates from vagina samples of VVC patients were randomly collected over the period between September and December 2014 from hospitalized patients at the general hospitals of Lorestan province, Iran. Isolates were previously identified by conventional mycological methods. The phospholipase and proteinase activities were evaluated by Egg yolk agar, Tween 80 opacity medium and agar plate methods. The most common Candida species was identified Candida albicans (n=34, 41.5%), followed by Candida famata (n=13, 15.8%), Candida tropicalis (n=11, 13.4%), and Candida parapsilosis (n=9, 11%). The most phospholipase activity was observed in Candida colliculosa (40%), followed by C.famata (38.5%), and Candida krusei (33.3%). The findings revealed that the correlation between phospholipase production by Candida spp. and the presence of VVC was not found to be statistically significant (P=0.91). All Candida spp. exhibited considerable proteinase activity; so that 100% of C.colliculosa, C.parapsilosis, Candida kefyr, and Candida intermedia isolates produced high proteinase activity with Pz 4+ scores. There was a significant correlation between proteinase production by Candida spp. and the presence of VVC (P=0.009). The obtained findings revealed that Candida spp. isolates may produce both virulence factors, phospholipase and proteinase. Although the phospholipase production was only observed in <40% of the isolates; however there was a significant association between proteinase production by Candida spp. and VVC.

Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese

We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable availability. Future studies will aim to purify these enzymes to test their suitability for use in similar artisanal cheeses or in large scale commercial cheeses.

Extracellular Proteome Profiling of Bacillus pumilus SCU11 Producing Alkaline Protease for Dehairing

Bacillus pumilus is one of the most characterized microorganisms that are used for high-level production of select industrial enzymes. A novel B. pumilus SCU11 strain possessing high alkaline protease activity was obtained in our previous work. The culture supernatant of this strain showed efficient dehairing capability with minimal collagen damage, indicating promising potential applications in the leather industry. In this study, the strain's extracellular proteome was identified by LC-MS/MS-based shotgun proteomic analysis, and their related secretory pathways were characterized by BLAST searches. A total of 513 proteins, including 100 actual secreted and 413 intracellular proteins, were detected in the extracellular proteome. The functions of these secreted proteins were elucidated and four complete secretory systems (Sec, Tat, Com, and ABC transporter) were proposed for B. pumilus. These data provide B. pumilus a comprehensive extracellular proteome profile, which is a valuable theoretical and applicative basis for future genetic modifications and development of industrial enzymes.

Comparative analysis on the distribution of protease activities among fruits and vegetable resources

In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

Antifungal sourdough lactic acid bacteria as biopreservation tool in quinoa and rice bread

The use of sourdough fermented with specific strains of antifungal lactic acid bacteria can reduce chemical preservatives in bakery products. The main objective of this study was to investigate the production of antifungal carboxylic acids after sourdough fermentation of quinoa and rice flour using the antifungal strains Lactobacillus reuteri R29 and Lactobacillus brevis R2Δ as bioprotective cultures and the non-antifungal L. brevis L1105 as a negative control strain. The impact of the fermentation substrate was evaluated in terms of metabolic activity, acidification pattern and quantity of antifungal carboxylic acids. These in situ produced compounds (n=20) were extracted from the sourdough using a QuEChERS method and detected by a new UHPLC-MS/MS chromatography. Furthermore, the sourdough was applied in situ using durability tests against environmental moulds to investigate the biopreservative potential to prolong the shelf life of bread. Organic acid production and TTA values were lowest in rice sourdough. The sourdough fermentation of the different flour substrates generated a complex and significantly different profile of carboxylic acids. Extracted quinoa sourdough detected the greatest number of carboxylic acids (n=11) at a much higher concentration than what was detected from rice sourdough (n=9). Comparing the lactic acid bacteria strains, L. reuteri R29 fermented sourdoughs contained generally higher concentrations of acetic and lactic acid but also the carboxylic acids. Among them, 3-phenyllactic acid and 2-hydroxyisocaproic acid were present at a significant concentration. This was correlated with the superior protein content of quinoa flour and its high protease activity. With the addition of L. reuteri R29 inoculated sourdough, the shelf life was extended by 2 days for quinoa (+100%) and rice bread (+67%) when compared to the non-acidified controls. The L. brevis R2Δ fermented sourdough bread reached a shelf life of 4 days for quinoa (+100%) and rice (+33%). However, the shelf life was similar to the chemically acidified control indicating that the preservation effect of the carboxylic acids seems to have a minor contribution effect on the antifungal activity in gluten-free breads.

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

Characterisation of yeast and filamentous fungi from Brøggerbreen glaciers, Svalbard

ABSTRACTCryoconite holes have ecological and biotechnological importance. This article presents results on culturable cryophilic yeasts and filamentous fungi isolated from cryoconite holes at Austre and Vestre Brøggerbreen glaciers, Svalbard. Based on DNA sequence data, these were identified asRhodotorulasp.,Thelebolussp., andArticulospora tetracladia. Amongst these,Articulospora tetracladia(88.7–89.4% gene similarity with 5.8S rDNA) is a novel species, yet to be described. Filamentous fungusArticulosporasp. Cry-FB1 and Cry-FB2, expressed high amylase, cellulase, lipase and protease activities while yeastRhodotorulasp. Cry-FB3 showed high amylase and cellulase activity.Thelebolussp. Cry-YB 240 and Cry-YB 241 showed protease and urease activities. The effects of temperature, and salt on the growth of the cultures were studied. Optimum temperature of growth was on 10ºC at pH 7.0. Filamentous fungi and yeast in the cryoconite holes possibly drive the process of organic macromolecule degradation through cold-adapted enzyme secretion, thereby assisting in nutrient cycling in these supraglacial environments. Further, these cryophilic fungi, due to their enzyme producing ability, may provide an opportunity for biotechnological research in the Arctic.