Phenotypic and Genetic Characterization of Bacillus Species Exhibiting Strong Proteolytic Activity Isolated from Terasi,An Indonesian Fermented Seafood Product

In this study, two bacilli strains namely S2-3 and S4-5, isolated from Terasi, a traditional fermented seafood product of Indonesia, were studied in terms of their phenotypic and genotypic properties. Both strains are of great interests due to their high proteolytic activity. Initially, they were subjected to morphological determination and a series of biochemical tests. These bacteria were Gram-positive, endospore-forming bacilli. Based on 16S rRNA gene sequence analysis, the identities of the strains S2-3 and S4-5 were confirmed as Bacillus thuringiensis and B. subtilis, respectively. Additionally, the two strains were also evaluated for their antibiogram profiles. It was found that they were susceptible to chloramphenicol, erythromycin, kanamycin, tetracycline and vancomycin and resistant to ampicillin and intermediately susceptible to bacitracin.

ANGIOTENSIN Ⅱ-INDUCED CELLULAR HYPERTROPHY: POTENTIAL ROLE OF THE PROTEOLYTIC ACTIVITY IN CULTURED PROXIMAL TUBULE CELLS (LLC-PK1)

In a number of renal disease tubular epithelial cells often display hypertrophy rather than hyperplasia. This hypertrophy, characterized by an increase in protein conten and cell size, as well as an accumulation of extracellular matrix, is a key process which may lead subsequently to tubulointerstitial fibrosis and end-stage renal failure.

Differential subcellular distribution renders HAI-2 a less effective protease inhibitor than HAI-1 in the control of extracellular matriptase proteolytic activity

The integral membrane,Kunitz-type serine protease inhibitors HAI-1 and HAI-2,can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency.High levels of extracellular matriptase proteolytic activity have,however,been observed in some neoplastic B-cells with high levels of endogenous HAI-2,indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level.The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here.Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells,which naturally express matriptase with very low levels of HAI-2 and no HAI-1,nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme.With increasing HAI-1 expression,cellular matriptase-HAI-1 complex increased,and extracellular active matriptase decreased proportionally.Increasing HAI-2 expression,however,resulted in cellular matriptase-HAI-2 complex levels reaching a plateau,while extracellular active matriptase remained high.In contrast to this differential effect,both HAI-1 and HAI-2,even at very low levels,were shown to promote the expression and cell-surface translocation of endogenous matriptase.The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2.The HAIs,therefore,resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity.

Effect of proteolytic starter culture isolated from Chinese Dong fermented pork(Nanx Wudl)on microbiological,biochemical and organoleptic attributes in dry fermented sausages

The effect of a proteolytic starter culture isolated from Nanx Wudl,on microbiological,biochemical and organoleptic attributes of dry fermented sausages was investigated during processing.Based on preliminary screening,the combination of Staphylococcus xylosus SX16 and Lactobacillus plantarum CMRC6,showing excellent proteolytic activity in vitro,was selected as the multi-strain starter(starter LS).For comparison,the single-strain starter culture of L.plantarum CMRC6(starter LB)and non-inoculated control were also tested.During fermentation,lactic acid bacteria and staphylococci dominated the microbiota and suppressed the Enterobacteriaceae growth in LS-inoculated sausages.The addition of LS starter accelerated acidification and proteolysis during ripening,improving the contents of total free amino acids and several essential free amino acids(Phe,Ile and Leu).Volatile compounds analysis revealed that LS-fermented sausage showed the highest abundance of 3-methyl-1-butanol,probably due to the inoculated S.xylosus.The inoculation of LS starter improved the sensory properties of sausages,especially the flavor attribute.Therefore,S.xylosus SX16 and L.plantarum CMRC6 are promising candidates for inclusion as multi-strain starters in the manufacture of gourmet fermented dry sausage.

Heterogenous Expression of Synechocystis sp. PCC 6803 Deg Proteases and Their Possible Roles on Phycobiliprotein Degradation in vitro

The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA （degP, slr1204）, hhoA （degQ, sll1679） and hhoB （degS, sll1427）. This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and phycobiliproteins in vitro, which implicated that they were proteases possibly involved in phycobiliprotein degradation in vivo.

Partial Purification and Characterization of Protease from Abrus precatorius Linn. (Fabaceae) from Cameroon

Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.

The combined effect of ultrasound treatment and leek (Allium ampeloprasum) extract on the quality properties of beef

One of the most important physicochemical properties of meat is its tenderness.The impact of Leek (Allium ampeloprasum ) extract and ultrasound on meat quality and molecular weight distribution of Longissimus lumborum muscles was studied.Samples have been treated with ultrasound (100 and 300 W during 10,20,and 30 min) and Leek-derived exogenous proteases (16.9 and 33.8U/g) simultaneously and separately.The combined effect of enzyme and ultrasound in 10 and 20 min increased the pH value.It was also increased the proteolytic activity of meat.Regarding meat quality,the filtering residues,cooking loss,shear force,and hardness were reduced;water-binding capacity,emulsion capacity,and emulsion stability were improved.The electrophoretic pattern of myofibrillar proteins showed the muscle fibers were severely degraded in the combined treatments.It can be concluded that this process could be used as an alternative to the chemical tenderizing agents in the meat industry.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins(SREBPs)are the key transcriptional regulators of lipid metabolism.The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi,where it is sequentially cleaved by site-1 protease(S1P)and site-2 protease and releases a nuclear form to modulate gene expression.To search for new genes regulating cholesterol metabolism,we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease(POSH),encoded by C120RF49,is critically involved in the SREBP signaling.Ablation of POSH decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes.POSH binds S1P,which is synthesized as an inactive protease(form A)and becomes fully mature via a two-step autocatalytic process involving forms B/B and C/C.POSH promotes the generation of the functional S1P-C/C from S1P-B/B(canonical cleavage)and,notably,from S1P-A directly(non-canonical cleavage)as well.This POSH-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6,CREB3 family members and the a/p-subunit precursor of N-acetylglucosamine-1-phospho-transferase.Together,we demonstrate that POSH is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis,unfolded protein response,lipoprotein metabolism and lysosome biogenesis.