Protease

Endophytic bacteria isolated from internal tissue of surface sterilized jatropha curcas L. leaves, stem, frouts and roots. Collected from field plots produced secondary metabolites in nutrition broth media (NB) that were highly toxic to Meloidogyne spp. especially strains SJS54 followed by RJS175 were highly active among 23 strains producing secondary metabolite toxic to Root-knot nematodes. The mechanism of action of the toxic metabolites produced by the non-pathogenic strains SJS54 and RJS175 with proven biological control of Meloidogyne spp in Syracuse glass experiments was investigated. These metabolites reduced Meloidogyne spp mobility within 6-12 h of exposure. After 6 h, approximately ±50% of juveniles was inactivated. But with exposure of 12 h 80-90% of the juveniles were dead. The SJS54 and RJS175 toxicity were highly effective towards migratory parasites. Most of nematodes have been shown body wall degradation caused by hydrolytic enzymes produced by endophytic bacteria, a...

An in vitro procedure that simulated digestion in growing broilers was tested to predict phosphorus availability and arabinoxylan hydrolysis in samples of nine wheat varieties and in a wheatbased diet. Amounts of dialysable phosphorus freed from wheat samples correlated with activities of endogenous phytase (R = 0.913; p`0.0001), whereas amounts of pentoses released were correlated with viscosities of the digested samples (R = 0.899; p`0.0001). Differences in phosphorus release resulting from graded levels of microbial phytase added to feeds that were either autoclaved or not autoclaved revealed a decreasing role of endogenous phytase in dephosphorylation as levels of microbial phytase supplementation grew. Amounts of pentoses released from feeds containing two different xylanase preparations re¯ected literature data on different in vivo ef®cacies of those preparations. Simultaneous addition of phytase and xylanase affected phosphorus release in a manner that depended upon the form of xylanase preparation used (liquid or powder). There was a positive in¯uence of acid protease on both phytate and arabinoxylan hydrolysis in feeds supplemented with phytase. Effects observed by the in vitro procedures corresponded to in vivo phenomena described in the literature.

The seeds of thirteen varieties of Pearl millet (Pennisetum glaucum (L.)) were evaluated for enzyme activity in response to sulphate base salinity. Sterilized seeds were safely maintain in filter paper lined Petri dishes and irrigated with water or salt solution (00, 40, 80, 120 m.eq/L). Practice was maintained for the filter paper kept moist by periodic additions of salt solution or water for the required treatments. The research carried out on sulphate dominant salinity. Enzymatic study protease and peroxidase activity increased with increase in salinity, while in case alfa amylase activity, it decreased with increasing salinity level. There was significant interacting effect between salinity treatments and varieties of pearl millet with respect to enzyme activities.

Protease production under solid state fermentation (SSF) was investigated using isolated Aspergillus flavus. Different agroindustrial waste products were evaluated to check the possibility of potential utilization of substrates in SSF for protease production by Aspergillus flavus using wheat bran as a substrate. The results showed that the optimum conditions for maximum protease production were found to be 7 th day of incubation at pH 5.0, temperature 30 o C; inoculum size 3%; substrate concentration 3% and 3% KNO 3 as nitrogen source. The purified enzyme produced 5.8 fold with recovery of 3.2% by DEAE-column chromatography and the molecular weight was estimated to be 46kDa by SDS-PAGE. It has a V max value of 60.0 U/mg and K m value of 0.6 mg/ml at pH of 7. The enzyme activity was found to be stable at 50 0 C and it was stimulated by metal ions like Cu 2+ and Zn 2+ and inhibited by Ca 2+ and Mg 2+ .

Psoriasis is an autoimmune skin disorder, which affects approximately 2-3% of the population worldwide. The current conventional therapy cannot offer satisfactory clinical results for most of the patients, largely due to the fact that many antipsoriatic drugs have serious side effects and psoriasis is prone to developing drug resistance after long term exposure. Natural products are used extensively throughout the world, at least in 70% of drugs available in the global market. However, there is a great threat to biodiversity due to overharvesting raw materials for herbal medicines and health care products. Since many medicinal plants are also in the extinct herb list, it is better to adapt mineral-based medicine practiced traditionally in India. An ancient system of traditional Indian medicine 'Siddha' uses herbs, metals, and minerals. Siddha system of medicine is one of the ancient traditional healing therapies known to the mankind since several centuries. It has numerous novel formulations which benefit the society for various ailments. SwarnaPushpa Rasa Chendhuram (SPRC) is kind of novel herbomineral preparation consist of mercury, sulfur, Stannum, Ammonium chloride and Benincasahispida. HaCaT cells are the immortalized human keratinocytes and have been extensively used to study the epidermal homeostasis and its pathophysiology. The main aim of the present investigation is to evaluate the anti-psoriatic and cytotoxic potential of the formulation SPRC using standard optimized HaCaT cell lines. The result obtained from the study reveals that the percentage of HaCaT cell viability decrease with increase in concentration of the siddha formulation SPRC. Least viability of cell was observed at the concentration of 100µg/ml shows 14.92%. Further the highest percentage inhibition of about 85.07% observed at 100µg/ml of SPRC. It was concluded from the present investigation that the siddha formulation SPRC possess significant anti-psoriatic and cytotoxic activity further actual active compounds responsible for potential cytotoxic has to be studied in detail with different models which exactly simulate the cell biology and actual pathophysiology of the psoriasis in humans.

Proses perkecambahan pada pertumbuhan kacang hijau terjadi dalam beberapa tahapan antara lain:
1. Tahap 1 dimana air masuk ke dalam biji atau dikenal dengan istilah imbibisi.
2. Tahap 2 dimana embrio diaktifkan untuk melepaskan hormon bernama giberelin.
3. Tahap 3 dimana terjadi pendorongan terhadap aleueron atau lapisan super tipis yang ada pada endosperm.
4. Tahap 4 ditandai dengan sintesis enzim amilase, maltase dan protease.
5. Tahap 5 yakni fase dimana enzim mencegah cadangan makanan pada endosperm dan menjadikannya energi untuk pertumbuhan dan energi untuk membentuk sel. Setelah tahapan ini, biasanya batang dan daun mulai terlihat.

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT 50 of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60Á75 g/kg rice), faster sedimentation time (ST 50 ) (2.3Á2.65 h in 0.1% (w/v) Tween 80) and lower LC 50 (1.4Á 5.7 )10 3 conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only ‘highly pure’ immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ~100-kDa F(ab′)2 intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab′)2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab′)2 produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab′)2/l serum. This novel, high yield method for processing serum to highly pure F(ab′)2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.

Carotenoprotein from shells of Pacific white shrimp (Litopenaeus vannamei) was extracted with the aid of proteases from hepatopancreas of the same species at various levels
(5–30 units/g shrimp shell) for different times (0–180 min). Recovery of carotenoprotein increased with increasing protease levels and hydrolysis times, but mainly reached the plateau at 120 min of hydrolysis. Carotenoprotein contained astaxanthin and astaxanthindiester as major carotenoids. Carotenoids extracted from carotenoprotein showed increasing
ABTS, DPPH radical scavenging activities, FRAP and metal chelating activity when the concentrations increased up to 5 mg/ml (po0.05). Carotenoprotein consisted of 73.58% protein, 21.87% lipids and 2.63% ash contents. It was rich in essential amino acids and Asp/Asn and Glu/Gln were found as dominant amino acids. Therefore, carotenoprotein from shell of Pacific white shrimp could be successfully extracted using shrimp hepatopancreas proteases and could serve as the value-added nutritive food ingredients or as the animal feed

In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO4) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100 U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680 U/mL). Additionally, a new extracellular 51 kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70 °C and pH 10. Its half-life times at 70 and 80 °C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5 L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5 L; which offers an interesting potential for its application in the laundry detergent industry.

Microbial  proteases have  wide industrial  applications  and  proteases  of  the lactic  acid bacteria  (LAB) have  received  special  attention  because  of  their  importance  in  the  food  and  dairy  industry.  Of  all  the  LAB,  the proteolytic  system  of  the  pediococci  is  the  least  studied.  Therefore,  this  study  was  aimed  at  characterizing  and purifying  the  protease  produced  by  Pediococcus  acidilactici  grown  in  de  Man,  Rogosa  and  Sharpe  (MRS)  broth. Casein concentration of 2% (w/v) and 2.5 ml of the crude enzyme were optimal for the activity of the protease. The crude protease had temperature and pH optima of 28 oC and 4.0 respectively thus indicating that the enzyme is a mesophilic  and  acidic  protease.  Purification  of  the  enzyme  by  gel  filtration  chromatography  on  Sephadex  G75 following  ammonium  sulphate  precipitation  gave  2.26  fold  increase  in  purification  with  specific  activity  of  46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced purification fold (1.24  -  1.59) with specific activity ranging between 25.39  -  32.54 units/mg protein. The protease showed a characteristic band on SDS-PAGE and  the  molecular  weight  was  estimated  between  45  and  66  kDa.  The  potential  applications  of  the  protease  are discussed.

The aim of this research was to study the effect of the dual treatment of sorghum decortication and protease addition during liquefaction with -amylase, on the concentration of free amino nitrogen (FAN), fusel alcohols and ethanol during yeast fermentation. A bifactorial experiment was designed to revise the differences among grains (maize, whole and 9.7% decorticated sorghum) and the effectiveness of protease addition during liquefaction. The decorticated sorghum was more susceptible to protein hydrolysis compared to the whole kernel sorghum due to its lower fiber content. The protease improved the levels of FAN approximately to 60% and 30% in the maize and sorghum mashes, respectively. The maize mash contained the highest amount of FAN followed by the decorticated and whole sorghum mashes. The protease treatment improved the fusel alcohol concentration in both sorghum beers and did not affect ethanol concentration in the maize mash. Both sorghum decortication and protease addition during liquefaction are therefore recommended treatments to obtain mashes with a higher FAN level. The decorticated protease-treated sorghum mashes yielded higher amounts of ethanol compared to the maize treatments and produced the maximum ethanol after only 20 h of fermentation. This research proved that a positive synergistic effect on FAN concentration, fusel alcohols and bioethanol yields can be achieved through the proposed proceeding of sorghum decortication and protease addition.

The present study focuses on fungal extracellular enzymes as factors affecting fungal parasitism on the cotton leafworm, Spodoptera littoralis, especially with some new strains of entomopathogenic fungi such as Beauveria bassiana 1513, B. bassiana 3286, Paecilomyces fumosoroseus 2734 and Nomuraea rileyi 1972. Our results showed a significant increase in mortality 24 h after injection of fungi and tended to increase until pupation. The 6 th instar larvae were more susceptible to N. rileyi 1972, followed by B. bassiana 3286, and then P. fumosoroseus 2734. The larvae were least susceptible to B. bassiana 1513. At a dose of 9000 bl/insect, the median lethal times of the tested fungal isolates were between 36.5-51 h. Fungi were able to produce detectable amounts of proteases, chitinases and lipases. The activity of proteases was strong along with weak activities of chitinases and lipases. Interestingly, there appeared to be no clear correlation between pathogenicity and activity of extracellular enzymes. The extracellular enzymes always act as a unit, and everyone alone can not permit to the fungus penetration into the host. So, virulence may be the result of a set of enzymes acting together.

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50
C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell roliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Industrial enzymes are produced by various organisms using a wide variety of substrates. The present study was carried out to investigate the production of protease by Aspergillus niger using fish scale as the growth media. Fish scale, the chief waste material of fish processing industries, was enzymatically hydrolysed by protease produced by A. niger. Two mutated strains of A. niger, namely, AB30 and AB60 were also used apart from the wild type A. niger strain. Trichloroacetic acid buffer was used to maintain the optimum moisture content of the substrate. No separate nutrient sources apart from the fish scales were used. This proves the cost effectiveness and efficiency of this process. The enzyme activity was analyzed from the 5 th day and it was further analyzed for three consecutive days and the results were plotted and analyzed.

Peranan hevein dalam lateks pohon karet telah mendorong penelitian-penelitian tentang keberadaan enzim proteolitik yang memiliki peranan dalam pembentukan hevein. Tujuan dari penelitian yang dilakukan adalah untuk mengisolasi dan menentukan karakter protease yang berasal dari BSL lateks karet klon PR-255 dan WR-101. Penelitian dimulai dengan mengambil lateks segar dan sentrifugasi untuk memperoleh tiga fraksi utama. Fraksi bawah yang mengandung partikel lutoid dikumpulkan dan dilakukan proses beku-cair untuk memecah membrane lutoid, larutannya kemudian disentrifugasi dan supernatant dikeringkan dengan menggunakan alat freeze dryer. Pemurnian protease dari BSL dilakukan dengan tahap-tahap pengendapan dengan kejenuhan 0-100% garam ammonium sulfat, kromatografi filtrasi gel dengan Sephadex G25, kromatografi penukar kation CMC CM32 dan kromatografi penukar anion DEAE DE 32. Kedua protease yang berhasil diisolasi memiliki aktivitas optimum pada pH 7,5 dan waktu inkunasi selama 30 menit. Sedangkan suhu optimum pada 55°C untuk klon PR-255 dan 50°C untuk WR-101. Protease tersebut memiliki berat molekul sebesar 67 kDa. Penentuan jenis protease dengan menggunakan inhibitor spesifik menunjukkan bahwa kedua protease tersebut tergolong protease sistein.

Biotechnology has almost unlimited potential to change our lives in very exciting ways. Many of the chemical reactions that produce these products can be fully optimized by performing them at extremes of temperature, pressure, salinity, and pH for efficient and cost-effective outcomes. Fortunately, there are many organisms (extremophiles) that thrive in extreme environments found in nature and offer an excellent source of replacement enzymes in lieu of mesophilic ones currently used in these processes. In this review, I discuss the current uses and some potential new applications of extremophiles and their products, including enzymes, in biotechnology.

Protease producing bacterium was isolated from soil and identified as Pseudomonas aeruginosa by ribotyping.  An extracellular protease was extracted and characterized from the culture supernatant of P. aeruginosa. The optimum pH, temperature and thermal stability of protease were found to be 8.5, 60 0C and 50 0C respectively. The carbon sources of media were optimized for protease production by response surface methodology and found to be casein [0.72%], yeast extract [0.4%] and glucose [0.5%] in minerals. Highest protease activity under these conditions was 932.6±2.3 U/ml as compared to that of 232.2±1.5 U/ml in basal reference media.  In order to identify the associated proteins involved in the expression of protease, total proteins were extracted from P. aeruginosa grown in optimized media for protease production [test] and LB media [control] and subjected to two dimensional [2D] gel electrophoresis.  From comparative analysis of proteome profile, two proteins, namely SC1 and SC2, were expressed only in the cells grown in LB medium.  Result obtained from MALDI TOF indicated SC1 as hypothetical protein and SC2 as two component response regulator PhoP.  These proteins might have a significant negative regulation on protease production.

The production of alkaline protease by Bacillus licheniformis (MTCC 1483) and Bacillus subtilis of indigenous isolates using cheapest sources-groundnut cake (N 2 Source) and wheat bran (C source) was studied. The proteolytic activity was found maximum at 72 hr of the culture for both the bacterial types but the pH of the medium for maximal enzyme production varied as 9 for Bacillus licheniformis and as 8 for Bacillus subtilis. Thus investigations of this study reveal the possibility of employing cheapest nutrient source for microbial proteases that can be used on a commercial scale.

The unhairing stage of leather processing is associated with the production of significant amounts of solid and
liquid wastes. The use of enzymes to replace the polluting sulphides currently used for unhairing is a viable
alternative. Various proteases from different Bacillus cereus strains as well as many other bacterial strains
have been used successfully for the unhairing of skins. However, no previous work has assessed the use of the
crude alkaline protease extract from Bacillus cereus strain 1-p, a novel Bacillus cereus strain obtained from the
shores of Lake Bogoria - a soda lake in Kenya – in the unhairing of goatskins. This study, therefore, evaluates
the potential of the protease extract from the Bacillus cereus strain 1-p to unhair goatskins.
Optimum variables for unhairing using the protease were investigated. Complete unhairing was achieved within
12 hours at 27°C and pH 12 using the crude enzyme. The period and temperature required for complete unhairing
were significantly lower than that of other enzymatic unhairing techniques. Compared to the leather unhaired
with sulphide, the leather unhaired with the enzyme did not only show superior organoleptic properties but
also recorded comparable or superior physical properties, namely tensile strength (26.94 N/mm2), percentage
elongation (76.29%), tear strength (43.59 N/mm), and distension at grain crack and burst (7.9 mm and 8.2
mm respectively). The wastewater from the enzymatic unhairing process recorded a significant reduction
in biochemical oxygen demand (78%), chemical oxygen demand (83%), and the wastewater volume (50%)
compared to the process that uses sulphide. It was concluded that the use of the crude protease extract from
the Bacillus cereus strain 1-p in unhairing goatskins is feasible.

The current studies were aimed at to investigate role of pH, dissolved oxygen for production protease in bioreactor by alkalophilic bacterium and application of saw dust for its purification. The production of proteolytic enzyme by Bacillus subtilis IC-5 started as pH of medium falls to 9 and reached to maximum at pH 7 i.e., 4400 Uml-1. Likewise dissolved oxygen decreased in the medium as the protease production progresses. Saw dust was successively utilized for partial purification of protease. The partial purification of protease increased the specific activity to5.3 fold. The optimum pH and temperature for purified activity was 11 and 70 0 C, respectively. The purified enzyme was stable up to pH 12 and 80 o C.

Trypsin from hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies including diethylaminoethyl sepharose and soybean trypsin inhibitor sepharose 4B columns. Trypsin was purified to 50.4-fold with a yield of 13.7%. Based on native-polyacrylamide gel electrophoresis (PAGE), the purified trypsin showed a single band. Trypsin had a molecular weight of 24 kDa as estimated by sodium dodecyl sulphate-PAGE. The optimal pH and temperature for α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) hydrolysis were 8.0 and 60C, respectively. Trypsin was stable to heat treatment up to 60C and over a pH range of 7.0–11.0. The activity was strongly inhibited by soybean N-ρ-tosyl-L-lysine chloromethyl ketone. Purified trypsin had Michaelis–Menten constant (Km) and catalytic constant (kcat) of 1.60 mM and 3.33 s−1, respectively, when BAPNA was used as the substrate. Trypsin with high kcat indicated its high capacity of hydrolysis and it could serve as a promising protease.

There is a wealth of new data regarding zinc proteases present in many bacterial species, many of which cause serious disease, such as Listeria spp, Vibrio spp., Pseudomonas aeruginosa, Legionella pneumophila, Streptomyces spp., Clostridium spp., Enterococcus spp., etc. Many of these pathogens developed resistance to the clinically used antibiotics, which normally target bacterial cell wall biosynthesis or affect protein synthesis on ribosomes. Zinc-containing proteases play critical functions in these organisms, related to colonization and evasion of host immune defenses, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. However, only in the last decade such targets started to be investigated in detail. Thus, there are a lot of data available regarding the cloning, purification and biochemical characterization of many such zinc proteases, from both gram-positive as well as gram-negative pathogens. But for the moment, few potent and specific inhibitors for bacterial such proteases have been reported except for Clostridium histolyticum collagenase, botulinum and tetanus neurotoxin and anthrax lethal factor. No inhibitors of the critically important and ubiquitous IgA-specific metallopeptidases or AAA proteases have been reported to date, although such compounds would presumably constitute a new class of highly effective antibiotics. The clostridial neurotoxins, tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT, of which seven distinct serotypes are known, BoNT/A-G), together with the anthrax lethal factor (LF) are zinc proteases and among the most toxic compounds known in Nature. The substrate of TeNT is a 120-residue protein anchored to the membrane of cell vesicles, which has been termed VAMP, whereas BoNTs/B, D, F and G cleave specifically VAMP at different single peptide bonds. BoNTs also possess two other intracellular targets in addition to VAMP: SNAP-25 (25 kDa synaptosomal-associated protein) and syntaxin, both of which are cleaved between Gln#Arg (by BoNT/A), Lys#Ala (by BoNT/C), Arg#Ala (by BoNT/C) or Gln#Lys (different neurotoxins) residues. The three proteases contain a Zn(II) ion coordinated by two His and a Glu residue, similarly to many other zinc proteases. Many different peptidomimetic and non-peptide inhibitors were discovered for the three enzymes, after the report of their X-ray crystal structures, alone or complexed to substrates/inhibitors. Such compounds, presently in clinical development, may be effective in case of terroristic attacks in which such toxins are used or to treat diseases provoked by these bacteria which are widespread in nature. Furthermore, BoNT A has many applications in medicine, for the management of dystonias, strabismus, facial wrinkling, brow position, as well as palmar and axillary hyperhidrosis. More recently, BoNT A was also shown to be useful in the symptomatic therapy of pain, together with disorders in which muscular hyperactivity plays a prominent role, such as for example glandular hypersecretion, lower urinary tract dysfunction, prostatic disorders and vaginismus in women suffering with pelvic floor muscle tension.

Cellulase and protease producing microorganisms were isolated from the gut of Archachatina marginata (Giant African snail). The microorganisms isolated were Bacillus subtilis, Staphylococcus aureus, Streptococcus casseliflavus, Streptococcus faecalis, Aspergillus flavus, Rhizopus sp, Fusarium sp. and Aspergillus niger. When the isolates were tested on skimmed milk agar (1%) and Carboxymethyl Cellulase, B. subtilis and A. niger were the best producing microorganisms for both cellulase and protease enzyme. For cellulase production, B. subtilis revealed its highest biomass yield after 24 hours of incubation with activity of 0.62mg/ml/secˉ4 at 80℃ and pH of 5. Aspergillus niger showed its highest biomass yield after 5 days with an activity of 0.74mg/ml/secˉ4 and an optimum temperature of 60℃ and the optimum pH was 5. For protease production, B. subtilis was grown for 30 hours; its highest biomass yield was after 18 hours with an enzyme activity of 2.95µg/ml/secˉ4 and an optimum temperature of 60℃, while A. niger was grown for 7 days and showed highest yield after 6 days with enzymatic activity of 3.14µg/ml/secˉ4, and an optimum temperature of 50℃. The optimum pH was at 7 and 8 for B. subtilis and A. niger respectively. The results from this study suggest that B. subtilis and A. niger can be harnessed for the production of cellulase and protease.

Bacterial thermozymes based on hot spring research, with a wide range of applications across different industrial, biomedical and agricultural sectors, have grown tremendously in the last few years. However, there are few reports in this field concerning the Ethiopian context, although the country abounds in hot springs. The aim of this project is to assess the potential for isolation, screening and characterization of prospective thermophilic bacteria from local hot springs in the Koye Gore farmland of Addis Ababa City for the production of thermo tolerant hydrolytic enzymes such as amylase and protease. Various characterization tools and techniques have been used to elucidate the biochemical characteristics of the thermo tolerant microbe and the use of their enzymes in their crude state. Three bacterial isolates were found to be positive for gram staining, catalase, starch and casein hydrolysis tests. Producers of potential amylase and protease enzymes for prospective biotechnological applications of extracted enzymes in the production of glucose from wheat flour hydrolysis and protein (albumin) as detergent-inclusion assessed. This study examined the thermo tolerant bacteria with multi-enzyme producers and confirmed their biotechnological application with their respective substrate.

DegS peptidase acts as a chaperone at low temperatures but switches to a peptidase (heat shock protein) at higher temperatures. Molecular chaperones and peptidases control the folded state of proteins by recognizing hydrophobic stretches of polypeptide that become exposed by misfolding or unfolding. They then bind these hydrophobic substrates to prevent aggregation or assist in protein refolding. If attempts at refolding fail, then irreversibly damaged proteins are degraded by peptidases such as DegS.

Antioxidative activities of hydrolysates from skin of seabass (Lates calcarifer) with different degrees of hydrolysis (DH: 10–40%) prepared using an ammonium sulphate precipitated fraction (ASPF) from Pacific white shrimp hepatopancreas and commercial Alcalase were compared. The hydrolysate prepared using ASPF or Alcalase had increases in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidative power (FRAP) as DH increased (P < 0.05). When the hydrolysate prepared using ASPF with 40% DH was subjected to a gastrointestinal model system (GIMs), ABTS radical scavenging activity and chelating activity increased, especially in the duodenal condition. The hydrolysate
(500–2000 mg/L) could inhibit lipid oxidation in a lecithin liposome system in a dose dependent manner. Based on gel filtration using a Sephadex G-15 column, peptide with
a molecular weight of 364 Da showed the strongest ABTS radical scavenging activity. Therefore, the extract from hepatopancreas could be used to increase DH of hydrolysate from seabass skin.

Phomopsis azadirachtae is the causal agent of destructive die-back disease of neem. The molecular mechanism of pathogenicity is not clear. Pectinases, cellulases, hemicellulases and ligninases have been extensively studied because of their plant cell wall degrading nature. Hence, this study describes the screening and assay of extracellular enzymes produced by the pathogen. The pathogenicity enzymes screened were: laccase, polygalacturonase, xylanase, amylase, cellulase, protease and lipase. A total of fifteen isolates of Phomopsis azadirachtae were from different agroclimatic regions of Karnataka, Tamil Nadu and Andhra Pradesh from diseased samples. The isolates were subjected to screening of the above mentioned enzymes on solid media supplemented with respective substrates. All the isolates were found to produce polygalacturonase, laccase, protease and xylanase. Polygalacturonase and xylanase were further assayed in order to quantify the enzyme produced in all the isolates. In addition to pathogenicity enzymes, isolates were also found to produce some industrially important enzymes such as L-asparaginase and urease.

Compositions and yield of lipids extracted from hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) subjected to autolysis at 60 C for different times (0, 30, 60, 90 120 and 150 min) were investigated. Extraction yield increased from 7.4% to 8.8% as autolysis time increased from 0 to 150 min. Coincidental increase in total carotenoid content was obtained with increasing autolysis time (p < 0.05). The increases in thiobarbituric acid-reactive substances (TBARS) and p-anisidine value (AV) of lipids were noticeable when autolysis time increased (p < 0.05). However, no changes in free fatty acid (FFA) content were observed within the first 60 min of autolysis (p > 0.05), but subsequently increased up to 150 min. (p < 0.05). No differences in fatty acid profiles of lipids extracted from hepatopancreas without and with 60 min prior autolysis were observed. Lipids extracted contained docosahexaenoic acid (DHA; C22:6(n  3)) as the most abundant fatty acid, followed by eicosapentaenoic acid (EPA; C20:5(n  3)). Therefore, prior autolysis at 60 C for 60 min increased the extraction yield without negative effect on lipid quality.

Se evaluó el efecto de cuatro fuentes de nitrógeno sobre la actividad enzimática de proteasas alcalinas, secretadas por una cepa nativa de Bacillus sp Alcalofílico, cultivada a diferentes concentraciones de LMF (Licor de Maíz Fermentado). El crecimiento de la cepa no es afectado por los pH de inoculación de 7,0; 8,5 y 9,5; en contraste con la actividad enzimática y producción de proteína verdadera, que tuvieron sus mejores resultados a pH inicial de 8,5. A este pH se evaluaron dos fuentes de nitrógeno orgánico (extracto de levadura y peptona) y dos inorgánicos (NH4Cl y NaNO3

PACS: 87.17.Uv

The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) d...

Background: Rhamnolipids are hydrophilic glycolipids, often classified as biosurfactants. They are produced by different bacterial species. Rhamnolipids are extensively studied in biological research because of their interesting features like antimicrobial, antifungal, and antiviral activities. Materials and Methods: To study the antimicrobial effect of rhamnolipid, we conducted a crosssectional study on the eight different pathogenic bacterial strains from November 2019 to June 2020. These bacterial strains were isolated from the organic compost. Both disk diffusion and broth microdilution methods were used to evaluate the inhibitory effects of rhamnolipids on these pathogenic bacteria. Also, protease and amylase enzyme activities were evaluated in these eight bacterial isolates. Results: For Bacillus stearothermophilus, within the area of 31.5 mm, no growth was observed, hence proving the inhibitory effect of rhamnolipid. After calculating the Minimum Inhibitory (MIC) and Minimum Lethal Concentrations (MLC) for each bacterial strain, it has been found that the studied bacteria were more susceptible to rhamnolipids than most of the antibiotics. Strains were also quantified for their enzymatic activity of proteases and amylases. The bacterial strains of Bacillus stearothermophilus, Brevibacillus brevis, Bacillus licheniformis, and Bordetella petrii showed maximum protease activity. Whereas Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes, and Pseudomonas aeruginosa showed high amylase activity. Conclusion: Rhamnolipids can be used as a potential antimicrobial agent for treatment of infections.

Background: The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitiza-tion. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease — actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). Methods: Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. Results: Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. Conclusion: Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. General significance: In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.

The parasitic life cycle requires the successful execution of several difficult tasks, including invasion across the host integuments, procurement of nutrients for growth and development, and evasion of host immune defenses. Virulence factors are the unique molecular attributes of the pathogen used to facilitate the colonization and infection of the host and contribute to the production of disease.  Proteolytic enzymes are essential virulence factors for prokaryotic and eukaryote parasites during all stages of the infectious process.  This review investigates the hypotheses that, by blocking the actions of the proteinases of the infectious cycle, proteinase inhibitors of the host have important roles in limiting parasitic infection.  Of particular interest are proteinase inhibitors with a broad reactive capacity for diverse proteinases.  It is suggested that these are especially suited to recognize and inactivate the range of proteinases that an animal is likely to encounter during multiple cycles of infection by different parasites.

Проводилось изучение фосфогидролазной и протеолитической активности белков вакуолей и тонопласта методами электрофореза и зимографии. Установлено, что с вакуолярной мембраной связан целый ряд кислых и щелочных фосфогидролаз, кислая фосфатаза, а также щелочная пирофосфатаза, присутствующая в двух изоформах. Кроме того, с белками тонопласта связана щелочная протеолитическая активность, отличная от кислой протеолитической активности, связанной с белками вакуолярного сока. Щелочная протеолитическая активность проявляется тиоловыми эндопептидазами, часть которых являются металлозависимыми. Выявлена способность этих ферментов к расщеплению собственных мембранных белков, что позволяет сделать предположение о вероятной роли ферментов не только в выполнении литической функции, но и в регуляции обновления белков на границе цитоплазмы и тонопласта, а через это в регуляции физиологических функций тонопласта.

Due to the urgent need of drugs to control the COVID-19 pandemic, repositioning of already marketed drugs could be a fast and convenient option  to identify agents to aid in controlling and treating COVID-19. This work presented a computational work regarding homology modeling and molecular docking of repurposing drugs related to the SARS-CoV-2. We have created a homology model of the cell surface transmembrane protease serine 2 protein (TMPRSS2) in order to investigate and analyze the interactions of already known small-molecules. This study indicates the most active inhibitors, poceprevir, simeprevir and neoandrgrapholide, that can be used further to search for better TMPRSS2 inhibitors.  Moreover,  we analyzed  the most important atomistic connections between these compounds and the modeled protein pockets. This study will focus on TMPRSS2-targeted drugs by comparing the binding mode of approved and experimentally used TMRSS2 inhibitors with other agents with TMPRSS2 inhibitory a...

This paper presents some interesting results on the physic-chemical and morphological properties of the
enzymatic degradation of prickly pear seed vegetable fiber (PPS) enhanced Polyhydroxy(butyrate-co-vale
rate) (PHBV) biocomposite. It is shown that the b-glucanase enzyme at pH = 5,5 accelerated the degradation of PHBV/PPS biocomposite compared to PHBV as well as compared to a-amylase (pH = 7) and protease (pH = 8) enzymes, which is confirmed by the significant weight loss in b-glucanase. The Fourier
transform infrared spectroscopy analysis FTIR analysis allowed seeing the decrease of carbonyl group
and thus its hydrolysis and degradation by ester chain cleavage. Morphological analysis is evaluated
by scanning electron microscope (SEM); this analysis also confirms the enzymatic degradation by the
presence of small holes on the surface of PHBV/PPS biocomposite immersed in the enzymatic solution
of b-glucanase. In addition, the effect of the incorporation of the PPS fiber plays an important role in this
degradation

COVID-19 adalah penyakit pernapasan akibat virus yang menyebabkan keadaan darurat kesehatan global dan diumumkan sebagai penyakit pandemi oleh Organisasi Kesehatan Dunia. Kurangnya molekul obat tertentu atau strategi pengobatan untuk melawan penyakit ini membuatnya semakin parah. Oleh karena itu, molekul obat yang efektif sangat dibutuhkan untuk melawan COVID-19. Protease utama (Mpro) dari SARS CoV 2, komponen kunci dari replikasi virus ini, dianggap sebagai target utama pengembangan obat anti-COVID-19. Tujuan penelitian ini untuk mengetahui apakah senyawa yang ada di dalam tumbuhan Camellia sinensis berpotensi sebagai obat COVID-19. Jenis penelitian adalah in silico menggunakan metode molecular docking. Hasil studi ini menunjukkan bahwa dari 8 senyawa aktif, CG (Catechin gallat) dan ECG (Epicatechin gallate) menunjukkan potensi untuk diteliti lebih lanjut, karena potensi aktivitasnya lebih kuat dibandingkan dengan senyawa pembanding Remdesivir, dengan score masing-masing-111,407 dan-111.391. Selain itu juga melakukan visualisasi residu setiap senyawa yang ada pada tumbuhan Camellia sinensis dengan Reseptor SARS-CoV-2 menggunakan Webserver PLIP. Sebanyak 5 senyawa aktif dari tanaman Camellia sinensis disaring melalui penapisan berdasarkan parameter Lipinski's Rule of Five dan prediksi ADMET menggunakan pkCSM. Potensi inhibisi SARS-CoV-2 oleh Catechin gallat dan Epicatechin gallat dari penelitian ini memiliki potensi sebagai alternative pengganti remdesivir dalam menghambat main protease SARS-CoV-2 atau berpotensi sebagai kandidat obat anti-COVID-19.

Serratia marcescens is a Gram negative bacterium (Enterobacteriaceae) often associated with infection of insects. In order to find pathogenic bacteria with the potential to control scarab larvae, several bacterial strains were isolated from the hemocoel of diseased Phyllophaga spp (Coleoptera:Scarabaeidae) larvae collected from cornfields in Mexico. Five isolates were identified as Serratia marcescens by 16S rRNA gene sequencing and biochemical tests. Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality. No insecticidal activity was observed for Spodoptera frugiperda larvae (Lepidoptera: Noctuidae) by oral inoculation. S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay. Heat treated

The term fermentation is derived from the Latin word for “forever” to boil thus
describing the appearance of the action of yeast on extracts of fruit or malted grain. The
boiling experience is due to the production of CO  bubbles caused by the anaerobic
2
catabolism of the sugars present in the extract. Pasteur applied the term “Fermentation” to
those anaerobic reactions through which microorganisms obtained energy for growth in
the absence of oxygen. Today fermentation has much broader meaning. It applies to both
aerobic and anaerobic metabolic activities of the microorganisms where in specific
chemical changes are brought about by an organic in substrate.

A variety of substances such as alcohols, organic acids, amino acids, vitamins,
antibiotics, enzymes, single cell proteins, hormones etc., are produced through
fermentations by employing different microorganisms. The enzymatic yield obtained
from fermentation, cost of their production and downstream processing cost determines
the final cost of the enzyme. To develop a viable industrial process, lowering production
cost and increasing enzyme productivity are very important. Selection of best
fermentation techniques and optimization of culture conditions contribute much in
achieving enzyme productivity. Currently, enzyme production by microorganisms can be
achieved using submerged fermentation or solid state fermentation.

This book is aimed at helping new comers to know about upstream and
downstream processing of enzymes and also it helps to understand the fermentation
techniques and avoid obvious mistakes and pitfalls we have all made. It has been written
for researchers, scientists, students, academicians coming from various disciplines of
Chemistry, Biology, Engineering and industry personnel as well. The content in this book
is quite readable and thorough in its presentation of the subject provides the readers a
broad coverage and insight into the area. To meet the above objectives this book covers
following topics: chapter-1 contains introduction, mechanism of action, sources and
classification of proteolytic enzymes. This chapter also deals with properties and
applications of alkaline proteases. Chapter 2-5 contains practical aspects on isolation,
screening and characterization of protease producing microorganisms, protease
production by submerged fermentation, optimization by Plackett-burman design,
Response surface methodology, solid-state fermentation, enzyme recovery and
purification procedures, characterization of purified enzyme, estimation of kinetic
parameters, evaluation of industrial applications. Chapter-6 deals with various
fermentation strategies to improve the yield. The present book has comprised of six
chapters presenting an overview of the research being carried out in the laboratory in the field
of fermentation technology.