Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Enzymatic Pretreatment with Laccases from Lentinus sajor-caju Induces Structural Modification in Lignin and Enhances the Digestibility of Tropical Forage Grass (Panicum maximum) Grown under Future Climate Conditions.

Since laccase acts specifically in lignin, the major contributor to biomass recalcitrance, this biocatalyst represents an important alternative to the pretreatment of lignocellulosic biomass. Therefore, this study investigates the laccase pretreatment and climate change effects on the hydrolytic performance of Panicum maximum. Through a Trop-T-FACE system, P. maximum grew under current (Control (C)) and future climate conditions: elevated temperature (2 °C more than the ambient canopy temperature) combined with elevated atmospheric CO2 concentration(600 µmol mol-1), name as eT+eC. Pretreatment using a laccase-rich crude extract from Lentinus sajor caju was optimized through statistical strategies, resulting in an increase in the sugar yield of P. maximum biomass (up to 57%) comparing to non-treated biomass and enabling hydrolysis at higher solid loading, achieving up to 26 g L-1. These increments are related to lignin removal (up to 46%) and lignin hydrophilization catalyzed by laccase. Results from SEM, CLSM, FTIR, and GC-MS supported the laccase-catalyzed lignin removal. Moreover, laccase mitigates climate effects, and no significant differences in hydrolytic potential were found between C and eT+eC groups. This study shows that crude laccase pretreatment is a potential and sustainable method for biorefinery solutions and helped establish P. maximum as a promising energy crop.

Co-immobilization of amylases in porous crosslinked gelatin matrices by different reticulations approaches.

The aim of this study was to carry out the co-immobilization of α-amylase and glucoamylase in crosslinked gelatin porous supports. For this, two methods of co-immobilization were proposed based on the crosslinking with glutaraldehyde (Ggta) or CaCl2 in presence of alginate (Gcal). The supports characterization revealed a porous microstructure with good interaction between its components according to the FTIR analysis and thermal properties. Optimal pH and temperature of the Gcal co-immobilized enzymes were determined at 60 °C and pH 6.0, present an enzymatic activity of 120 µmol·mL·min-1. Moreover, both supports were reused for up to 8 hydrolysis cycles. In addition, co-immobilized enzymes were more efficient than free enzymes in starch saccharification of starch in the long term. These results reveal that the co-immobilization of amylases in gelatinous supports is a promising approach in enzymatic chain reactions.

Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment.

The production of bioactive peptides from organic by-waste materials is in line with current trends devoted to guaranteeing environmental protection and a circular economy. The objectives of this study were i) to optimize the conditions for obtaining bioactive hydrolysates from chicken combs and wattles using Alcalase, ii) to identify the resulting peptides using LC-ESI-MS2 and iii) to evaluate their chelating and antioxidant activities. The hydrolysate obtained using a ratio of enzyme to substrate of 5% (w/w) and 240 min of hydrolysis showed excellent Fe2+ chelating and antioxidant capacities, reducing Fe3+ and inhibiting 2, 2'-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The mapping of ion distribution showed that a high degree of hydrolysis led to the production of peptides with m/z ≤ 400, suggesting low mass peptides or peptides with multiple charge precursor ions. The peptides derived from the proteins of cartilage like Collagen alpha-2(I), Collagen alpha-1(I), Collagen alpha-1(III) and elastin contributed to generation of bioactive compounds. Hydrolysates from chicken waste materials could be regarded as candidates to be used as ingredients to design processed foods with functional properties.

Histopathological and Reproductive Evaluation in Male Rats Fed Jatropha curcas Seed Cake with or without Alkaline Hydrolysis and Subjected to Heat Treatment.

Jatropha curcas cake, a by-product of biodiesel production, is rich in protein and has potential to be used in livestock feed; however, the presence of antinutritional factors and phorbol esters limits its use. Thus, this study investigated toxicological and reproductive effects in male Wistar rats after subchronic exposure to J. curcas cake subjected to detoxification procedures. Rats were divided into seven groups (n = 10) and treated for 60 days. The control group received commercial feed, while experimental groups received a diet containing 5% J. curcas cake nonhydrolyzed or hydrolyzed with 5 M NaOH. The cakes were unwashed or washed with ethanol or water and were autoclaved at 121°C for 30 minutes. Alkaline hydrolysis combined with ethanol washing decreased the phorbol ester concentration in the cake by 98%. Histopathological findings included diffuse degeneration of the liver and edema around the pulmonary vessels in the nonhydrolyzed groups. In addition, nontreated females mated with males of nonhydrolyzed unwashed group showed a decreased number of live fetuses and an increased placental weight. There were no signs of toxicity in rats given hydrolyzed cakes washed and unwashed, indicating that alkaline hydrolysis associated with heat treatment is an efficient method for detoxification of the J. curcas cake.

The anti-Trichomonas vaginalis phloroglucinol derivative isoaustrobrasilol B modulates extracellular nucleotide hydrolysis.

Trichomonas vaginalis causes trichomoniasis, a neglected sexually transmitted disease. Due to severe health consequences and treatment failure, new therapeutic alternatives are crucial. Phloroglucinols from southern Brazilian Hypericum species demonstrated anti-T. vaginalis and anti-Leishmania amazonensis activities. The modulation of biochemical pathways involved in the control of inflammatory response by ectonucleotidases, NTPDase, and ecto-5'-nucleotidase represents new targets for combating protozoa. This study investigated the activity of phloroglucinol derivatives of Hypericum species from southern Brazil against T. vaginalis as well as its ability on modulating parasite ectonucleotidases and, consequently, immune parameters through ATP and adenosine effects. Phloroglucinol derivatives screening revealed activity for isoaustrobrasilol B (IC50 38 µm) with no hemolytic activity. Although the most active compound induced cytotoxicity against a mammalian cell lineage, the in vivo model evidenced absence of toxicity. Isoaustrobrasilol B significantly inhibited NTPDase and ecto-5'-nucleotidase activities, and the immune modulation attributed to extracellular nucleotide accumulation was evaluated. The production of ROS and IL-6 by T. vaginalis-stimulated neutrophils was not affected by the treatment. Conversely, IL-8 levels were significantly enhanced. The associative mechanism of trophozoites death and ectonucleotidases modulation by isoaustrobrasilol B may increase the susceptibility of T. vaginalis to host innate immune cell like neutrophils consequently, contributing to parasite clearance.

Biochemical Characterization of ß-Lactamases from Mycobacterium abscessus Complex and Genetic Environment of the ß-Lactamase-Encoding Gene.

The objectives of this study were to determine the kinetic parameters of purified recombinant BlaMab and BlaMmas by spectrophotometry, analyze the genetic environment of the blaMab and blaMmas genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that BlaMab and BlaMmas have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. BlaMmas hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. BlaMab and BlaMmas showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A ß-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the blaMab and blaMmas promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of ß-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.

Biohydrogen and methane production via a two-step process using an acid pretreated native microalgae consortium.

A native microalgae consortium treated under thermal-acidic hydrolysis was used to produce hydrogen and methane in a two-step sequential process. Different acid concentrations were tested, generating hydrogen and methane yields of up to 45mLH2gVS-1 and 432mLCH4gVS-1, respectively. The hydrogen production step solubilized the particulate COD (chemical oxygen demand) up to 30%, creating considerable amounts of volatile fatty acids (up to 10gCODL-1). It was observed that lower acid concentration presented higher hydrogen and methane production potential. The results revealed that thermal acid hydrolysis of a native microalgae consortium is a simple but effective strategy for producing hydrogen and methane in the sequential process. In addition to COD removal (50-70%), this method resulted in an energy recovery of up to 15.9kJ per g of volatile solids of microalgae biomass, one of the highest reported.

Ozonolysis combined with ultrasound as a pretreatment of sugarcane bagasse: Effect on the enzymatic saccharification and the physical and chemical characteristics of the substrate.

Sugarcane bagasse (SCB) was treated in three stages using ozone oxidation (O), washing in an alkaline medium (B) and ultrasonic irradiation (U). The impact of each pretreatment stage on the physical structure of the SCB was evaluated by its chemical composition, using an infrared technique (FTIR-ATR), and using thermogravimetric analysis (TGA/DTG). The pretreatment sequence O, B, U provided a significant reduction of lignin and hemicellulose, which was confirmed by changes in the absorption bands corresponding to these compounds, when observed using infrared. Thermogravimetric analysis confirmed an increased thermal stability in the treated sample due to the removal of hemicellulose and extractives during the pretreatment. This pretreatment released 391mg glucose/g from treated SCB after the enzymatic hydrolysis, corresponding to a yield of 94% of the cellulose available.

Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom.

Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders.

Enhancement of Penicillium echinulatum glycoside hydrolase enzyme complex.

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and ß-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and ß-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance ß-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and ß-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance ß-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed ß-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-ß-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with ß-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.

Methionine Exposure Alters Glutamate Uptake and Adenine Nucleotide Hydrolysis in the Zebrafish Brain.

Hypermethioninemic patients may exhibit different neurological dysfunctions, and the mechanisms underlying these pathologies remain obscure. Glutamate and ATP are important excitatory neurotransmitters co-released at synaptic clefts, and whose activities are intrinsically related. Adenosine-the final product of ATP breakdown-is also an important neuromodulator. Here, we investigated the effects of long-term (7-day) exposure to 1.5 or 3 mM methionine (Met) on glutamate uptake in brain tissues (telencephalon, optic tectum, and cerebellum) and on ATP, ADP, and AMP catabolism by ecto-nucleotidases found in brain membrane samples, using a zebrafish model. Also, we evaluated the expression of ecto-nucleotidase (ntdp1, ntdp2mg, ntdp2mq, ntdp2mv, ntdp3, and nt5e) and adenosine receptor (adora1, adora2aa, adora2ab, adora2b) genes in the brain of zebrafish exposed to Met. In animals exposed to 3.0 mM Met, glutamate uptake in the telencephalon decreased significantly. Also, ATP and ADP (but not AMP) catabolism decreased significantly at both Met concentrations tested. The messenger RNA (mRNA) levels of ntpd genes and of the adenosine receptors adora1 and adora2aa increased significantly after Met exposure. In contrast, adora2ab mRNA levels decreased after Met exposure. Our data suggest that glutamate and ATP accumulate at synaptic clefts after Met exposure, with potential detrimental effects to the nervous system. This phenomenon might explain, at least in part, the increased susceptibility of hypermethioninemic patients to neurological symptoms.

Chitooligosaccharides as novel ingredients of fermented foods.

Chitooligosaccharides (COSs) have been clinically evaluated for their immunostimulating effects after oral intake. Similar to dietary supplements, prebiotics and biopreservatives, these water-soluble bioactives are easily incorporated into dairy products and beverages. Notwithstanding, the use of COS in fermented foods would be limited by its antimicrobial properties. In order to study the interaction with yoghurts as a model of fermented food, the effects of COS on chemical composition, viability, morphology and metabolism of lactic acid bacteria, fatty acid profiles and conjugated linoleic acid (CLA) were assessed over 28 days and after chemical digestion. There were no significant differences between the nutritional composition of controls and yoghurts supplemented with concentrations up to 0.1% w/w of COS. However, the acidification of milk decreased at 0.5% (p < 0.05) and the formation of yoghurt failed at 3.0%, without affecting viable counts. Lipid hydrolysis of yoghurts supplemented with 0.1% COS was not affected by chemical digestion. No significant differences were found between CLA percentages of controls and supplemented yoghurts after digestion. Although the nutritional composition, fatty acids and viable counts were not significantly modified after COS supplementation, the present study shows that COS diminishes bacterial acidification at concentrations higher than 0.1%, thus limiting the amounts that could be added to yoghurt.

Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK).

Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.

Chemical composition and in vitro digestibility of fresh sugarcane hydrolysed with calcium oxide (CaO) / Composição bromatológica e digestibilidade in vitro da cana-deaçúcar in natura hidrolisada com óxido de cálcio (CaO)

The objective was to evaluate the chemical composition and in vitro digestibility of sugarcane hydrolysed with increasing doses of calcium oxide and varying air exposure times. A completely randomised, split plot design was used; the doses were allocated to the plots, and the air exposure times were allocated to the subplots, with four repetitions. The data underwent analysis of variance and were laid out according to the effect of the treatment on the components of polynomial regressions, and evaluated at the 5% probability. The increase in the dosage negatively affected the quantities of neutral-detergent fibre (NDF), acid-detergent fibre (ADF), lignin (LIG), total carbohydrates (TC), cellulose (CEL), crude protein (CP), and ether extract (EE); and positively affected the quantities of non-fibrous carbohydrates (NFC) and mineral matter (MM). The addition of calcium oxide improved the in vitro digestible dry matter (IVDMD) coefficients and was able to keep up to 72 hours. The in vitro digestibility of the neutral-detergent fibre (IVDNDF) and of the acid-detergent fibre (IVDADF) coefficients decreased when calcium oxide was added. Calcium oxide has the ability to hydrolyse the fibrous fraction and conserve chopped sugarcane. Doses of 0.5 and 1.0% lime exhibited similar results to those achieved at higher doses; therefore, higher doses are not required in the hydrolyses of sugar...(AU)

Objetivou-se avaliar a composição bromatológica e a digestibilidade in vitro da cana-de-açúcar hidrolisada com doses crescentes de óxido de cálcio e diferentes tempos de exposição ao ar. Utilizouse o delineamento inteiramente casualizado, em esquema de parcelas subdivididas, sendo as doses alocadas nas parcelas e os tempos de exposição ao ar nas subparcelas, com quatro repetições. Os dados foram submetidos à análise de variância e desdobrados quanto ao efeito de tratamento nos componentes de regressões polinomiais, e avaliados a 5% de probabilidade. A elevação das doses influenciou negativamente os teores de FDN, FDA, LIG, CT, CEL, PB e EE, e positivamente nos teores de CNF e MM. A utilização do aditivo melhorou os coeficientes de DIVMS e foi capaz de manter até 72 horas. Houve decréscimo nos coeficientes de DIVFDN e DIVFDA com a adição de óxido de cálcio. O óxido de cálcio tem capacidade de hidrolisar a fração fibrosa como também conservar a cana-de-açúcar já picada. As doses 0,5 e 1,0% de cal, apresentaram resultados semelhantes às doses superiores, não havendo necessidade de utilização de doses mais elevadas na hidrolise da cana-de-açúcar. Com o decorrer do tempo ocorre deterioração da cana-de-açúcar, porém esta é menor quando tratada com óxido de cálcio.(AU)

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance.

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k(cat)). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.

Partial Molecular Characterization of Arctium minus Aspartylendopeptidase and Preparation of Bioactive Peptides by Whey Protein Hydrolysis.

In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity.

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.

Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and ß-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB).