ALKILINE PROTEASE

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40– 70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH 2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, se-quenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

The present investigation includes isolation, enumeration and comparison of the gut microbial flora of Labeo
rohita fed differentially treated Animal Fleshing (ANFL) as a sole protein source in their diets and related increase in
enzyme secretion. ANFL is the proteinaceous tannery solid waste generated during leather processing and inclusion
of ANFL in aqua feed will pave way for the novel alternative cheaper protein source in replacement of fish meal.
Six experimental diets were formulated with differentially processed ANFL. The isolates from the gut extract were
qualitatively screened and quantitatively assayed for amylase, cellulase, lipase and protease activities. The total
culturable bacterial count (10 × 107 CFU/g) as well as the proteolytic bacterial count (27 × 106 CFU/g) was high in the
fish gut fed with diet 5 containing the fermented ANFL. Almost all the bacterial isolates possesses protease activity in
which strains (FF5, CF3and CF4) isolated from the diet 5 (287U) and 7 (282U) exhibited highest activities. Maximum
cellulase (FF2), amylase and lipase (RF6) activity were observed in the strains isolated from the diets 5 and 1 (78U,
186U and 97U). This study proves the existence of diet dependent enzyme producing bacterial community in the
gut of Labeo rohita through SEM analysis and the information generated from the present study might contribute
towards utilization of enzyme producing bacterial isolates as probiotic and in better feed formulations at low cost for
rohu incorporating the tannery solid waste ANFL as a protein source.

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.

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.

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

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.

Aims:  To study protease production from four Bacillus species.
To optimize the production of extracellular protease by testing
various environmental and nutritional factors and its application as
a contact lens cleanser.
Methodology: The assay was carried out in duplicates. Cultures of
Bacillus subtilis, Batcillus licheniformis, Bacillus thermophilus,
and  Bacillus cereus  were used for Protease production and assay.
Sterile Bushnell  –  Hass medium containing 1% casein was used.
The clear zone of casein hydrolysis indicated protease secretion.
Protease assay was done using modified sigma protocol for
enzymatic assay of protease. Effect of incubation time, pH,
temperature and aeration on protease production was studied.
Diameter of colony and zone of clearance was measured and Cx
ratio was calculated. The best protease producer was selected.
Extracted and crude protease enzymes were added to artificial tear
solution prepared with 0.2% lysozyme. Enzyme treatment was
done for 10, 30, 60 and 90 min at optimum temperatures of
respective enzymes. Light  transmission readings were recorded
using visible range spectrophotometer at 285 nm.
Results:  All four species of Bacillus produced protease enzyme.
Bacillus subtilis (Cx ratio=6) was the best protease producer. 37
o
C
temperature, 48 hrs of incubation and pH 7.5 was optimum for
maximum production of protease from Bacillus subtilis. Optimum
temperature and pH for the crude enzyme activity was 40ºC and 8.
The optimum ammonium sulfate fractionation (40% (w/v)
saturation) showed 4.76 fold increase in the specific activity of the
crude extract. Protein solution was degraded by purified enzyme in
30 min whereas crude enzyme required 60 min.
Conclusion:  Protease enzyme extracted from  Bacillus subtilis
showed good activity against artificial tear solution.