Hours Of Hydrolysis Research Articles

Avaliação de variáveis que influenciam a hidrólise enzimática da caseína do leite de cabra Moxotó

O objetivo deste trabalho foi avaliar os efeitos da temperatura, pH, relação enzima:substrato (E:S) e tempo de reação sobre a hidrólise enzimática da caseína do leite de cabra Moxotó, com uso de diferentes enzimas proteolíticas. A hidrólise enzimática da caseína caprina foi realizada com uso de tripsina, pepsina e papaína. Para cada enzima, foram utilizados dois níveis de cada variável, na avaliação de seus efeitos sobre a hidrólise da caseína, em um fatorial completo 2(4). Os produtos da hidrólise foram visualizados em eletroforese SDS-PAGE. O melhor valor do grau de hidrólise com a enzima papaína foi obtido em pH 6,5, E:S de 1:150 e 5 horas de hidrólise a 50ºC (28,17%); com a tripsina, em pH 8,5, E:S de 1:150 e 5 horas a 40ºC (29,55%); e com a pepsina, em pH 3,0, E:S de 1:100 e 5 horas a 40ºC (38,27%). A hidrólise da caseína caprina é influenciada positivamente pelo pH e tempo de hidrólise, com o uso da pepsina. Foram observadas interações significativas antagônicas entre pH e relação E:S, com o uso da tripsina; e entre pH, temperatura e tempo de hidrólise, com o uso da papaína. A pepsina apresenta elevada eficiência na hidrólise de αs1, β e κ-caseína, evidenciada por hidrolisados com massa molar abaixo de 14,4 kDa.

Pré-hidrólise enzimática da gordura de efluente da indústria de pescado objetivando o aumento da produção de metano

A aplicação de preparados enzimáticos sólidos (PES) ricos em lipases foi avaliada no tratamento anaeróbio de efluente de indústria de conservas de pescado. O PES foi produzido pelo fungo Penicillium simplicissimum por fermentação em meio sólido (FMS) de resíduo industrial, sendo empregado na hidrólise de gorduras presentes no efluente a fim de viabilizar a utilização de metano como fonte de energia. O efluente contendo 1500 mg O&G.L-1 foi hidrolisado com 0,2, 0,5 e 1,0% (m/v) de PES a 30 °C por até 18 horas. O efeito do pré-tratamento enzimático dos O&G não foi significativo com relação à remoção de DQO, pois, independente das condições adotadas, obtiveram-se valores de 91 a 95%. Por outro lado, a produção específica de metano apresentou valores que variaram com a adição do PES e o tempo de hidrólise. Em experimentos controle (sem adição de PES), a produção específica de metano aumentou com o tempo de incubação, atingindo um máximo com 18 horas (138 mL CH4.g-1 DQOinicial). No entanto, valores mais elevados de produção específica de metano foram obtidos com 0,5 e 1,0% de PES, destacando-se a hidrólise com 0,5% de PES e 8 horas de hidrólise, com 216 mL CH4.g-1 DQOinicial. Quando se compara o experimento controle 0 horas (efluente bruto) com o efluente hidrolisado com 0,5% de PES durante 8 horas, um aumento de 2,7 vezes na produção específica de metano foi observado, indicando que a matéria orgânica foi mais facilmente assimilada pelos microrganismos anaeróbios nesta última condição.

Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of ?-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35°C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70°C and 4.5, respectively. Amylase activity was stable for 1 h at 60°C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (? and ß), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an ?-amylase and a glucoamylase.

KINETICS OF ENZYMATIC HYDROLYSIS OF STEAM-EXPLODED COTTON GIN WASTE

The hydrolytic kinetics of cotton gin waste (CGW) at various initial concentrations of two enzymes, Novozymes NS50052 and Spezyme AO3117, was investigated. The experiments showed that the concentrations of reducing sugars increased with increasing initial enzyme concentration for the two enzymes. The reducing sugars produced per gram of Novozymes were higher than those produced by Spezyme. However, the reducing sugars produced per FPU of Novozymes were lower than those produced by Spezyme. The concentrations of reducing sugars reached 6.41 g/L and 4.93 g/L after seven hours of hydrolysis at the initial Novozymes enzyme loading of 12.3 filter paper unit (FPU)/g substrate and Spezyme loading of 3.68 FPU/g substrate, respectively. A two-parameter model based on enzyme deactivation and its analytical expression have been derived. The model was used to fit the experimental data of the hydrolytic kinetics of CGW. The enzyme deactivation rate constants in this model decreased as the initial enzyme concentration for the two enzymes increased. The initial product-formation rates were used to determine the maximum hydrolytic rate constant Vm, the half-saturation constant K, and the rate constant k2 of sugar formation for the two enzymes. In addition, by fitting the kinetic data into a series expression of the partial differential equation of diffusion, the average diffusivities of Novozyme and Spezyme enzymes on the CGW were estimated to be 7.14 × 10−17 m2/s and 5.58 × 10−17 m2/s, respectively.

The optimization of conditions for the production of acid‐hydrolysed winged bean and soybean proteins with reduction of 3‐monochloropropane‐1,2‐diol (3‐MCPD)

SummaryResponse Surface Methodology (RSM) was used to establish the optimum time and temperature for production of acid‐hydrolysed winged bean protein (aHWBP) and acid‐hydrolysed soybean protein (aHSBP). Seven hours of hydrolysis at 125 °C was the optimum condition for producing aHWBP, whereas it was 5 h of hydrolysis at 125 °C for production of aHSBP. Although aHWBP and aHSBP produced using these conditions had favourable sensory qualities, they were found to have up to 25 mg kg−1 of 3‐monochloropropane‐1,2‐diol (3‐MCPD). This exceeds the maximum level permissible in Commission Regulation (EC) No 466/2001. However, additional alkaline thermal treatment at pH 8.5 for 2 h at 100 °C effectively reduced the 3‐MCPD contents of aHWBP and aHSBP to undetectable levels. aHWBP has a distinctive flavour, which is different from that of aHSBP. The former has higher mean scores for meaty and vegetable attributes but lower mean scores for soy, umami and beany attributes than the latter.

Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.

Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.

Characterization of potentially bioreactive soil organic carbon and nitrogen by acid hydrolysis

Studies determined the potential of acid hydrolysis for estimating the bioreactive fraction of organic carbon in soils (SOC). Three soils (clay loam, silt loam, and sandy loam) were hydrolyzed with 1 M or 6 M HCl under reflux for up to 24 h. Results showed that 1.7 to 3.2 % of SOC could be liberated as CO2 from the acid hydrolysis of soil. This readily hydrolyzed fraction should be a part of the bioreactive SOC. Higher amounts of soluble SOC and N as well as CO2 were released from all soils by 6 M HCl than by 1 M HCl. Soluble SOC and N contents in both 1 M HCl and 6 M HCl hydrolysates of all soils increased rapidly during the initial 2 hours of hydrolysis, and then increased very gradually. The amounts of CO2-C evolved correlated with the amounts of NH4-N released during the acid hydrolysis (r = > 0.88). The ratio of SOC to soluble N was lower in 6 M HCl hydrolysate than in 1 M HCl. Hydrolysis of soil by 1 M HCl for 4 h appeared to be a promising approach for estimating the more bioreactive pools of SOC and N.

Candida and Pseudomonas Lipase‐Catalyzed Hydrolysis of Butteroil in the Absence of Organic Solvents

ABSTRACT Candida rugosa lipase (CRL) and Pseudomonas fluorescein lipase (PFL) were used to hydrolyze a low‐melting point butteroil fraction in the absence of orgaic solvents. CRL and PFL displayed pH activity optima of 6.7 and 7.8, and temperature‐activity optima of 40–50°C and 35°C, respectively. Maximum activity for PFL was observed at 5% (v/v) water content. CRL showed higher specificity towards butyric acid relative to PFL after 3 hours of hydrolysis at 40°C (22.3 mol% vs 7.8 mol%, respectively). Both enzymes displayed, however, high specificities towards palmitic and oleic acids. CRL's inherent specificity towards butyric acid makes it an ideal choice for a biological catalyst to generate useful dairy flavor profiles.

Hydrolysis of κ-casein in solution by chymosin, plasmin, trypsin and Lactobacillus -proteinases

The aim of this study was to examine the enzymatic hydrolysis of κ-casein by isolating and identifying the released peptides. The enzymes employed in the study were chymosin, plasmin and trypsin, as well as a cell-free extract from three Lactobacillus helveticus and nine Lactobacillus casei strains. The findings showed that the bond most sensitive to the proteolytic activity of chymosin was the Phe 105-Met 106. After 24 hours of hydrolysis a few other bonds in the casein macropeptide were also cleaved. Plasmin was found to have weak proteolytic activity under the conditions of this study. When the enzyme-substrate ratio was raised from 1:200 to 1:50, a few peptides were released from the N-terminal region. Trypsin was found to hydrolyze several κ-casein bonds, and peptides were released from almost all regions of the protein. The proteases of Lactobacillus had less effect than chymosin, plasmin or trypsin. The strains could be divided into three categories. L. helveticus strains had activity on bonds in the mid-section and C-terminal region, L. casei strains EB, P3, P8 and A 1 had activity on bonds in the N- and C-terminal regions, while L. casei A5 and M9 had activity only on bonds in the C-terminal region.

Tryptic hydrolysis of caseinomacropeptide in membrane reactor: Preparation of bioactive peptides

Tryptic hydrolysis of caseinomacropeptide (CMP: C-terminal part of k-Casein) was used as a model in membrane reactor to study continuous production and isolation of bioactive peptides from milk proteins. Compared to the batch reactor, productivity of the proposed continuous process was 3 times higher after 3.5 hours of hydrolysis, but only 50% of the substrate is converted at steady state of the system. As shown by reverse-phase HPLC analysis, a good selectivity of the ultrafiltration membrane to various products was also obtained.

Methodology Investigations for the Production of Amino Acid Hydrolysate From Shrimp Waste

Various hydrolysis reaction conditions were examined in order to produce high quality hydrolysate from shrimp waste for its possible use in animal feed and some food products. In a first set of experiments, shrimp waste was hydrolysed with varying concentrations of HCl and samples taken after various hydrolysis times and tested for ninhydrin positive substances (NPS). Twelve hours of hydrolysis with 5N HCl gave maximum NPS yields and was, therefore, chosen as a suitable hydrolysing agent for subsequent hydrolysis trials.In a second set of experiments, shrimp waste samples of varying weights were hydrolysed with 5N HCl for various times and tested for NPS and total amino acids. On a dry weight basis, amino acid contents of all 12h hydrolysates varied from 29 to 35%. Maximum yields varying from 32 to 37% were achieved when the hydrolysis time was increased to 24h. Prolonging hydrolysis time to 48h reduced amino acid yields by 15–20 percent. The amino acid contents decreased with an increase in sample weight. Of the total amino acid contents approximately 40–42% were essential amino acids with considerable amounts of lysine, valine, leucine and threonine. The remaining 58–60% non-essential amino acid fraction was dominated by glutamic and aspartic acids.

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.

Quantitative zytochemische Untersuchungen der Veränderungen des Nukleoproteins von Spermien bei Fertilitätsstörungen des Mannes

Introduction There is some evidence that fluctuations of the Feulgen-DNA content of spermatozoa associated with male infertility do not represent true differences in DNA content. It is rather the changes of the nuclear proteins modifying the stoichiometry of the Feulgen reaction which seem to be responsible for these facts ( Gledhill et al., 1966 ). These phenomena should be more precisely defined, as proposed by Sandritter et al. (1965) by investigating the kinetics of the “Feulgen hydrolysis curve” of the spermatozoa. Material and Methods Ejaculated spermatozoa were fixed immediately in 96% ethanol, hydrolyzed, as proposed by Bachmann (1968) at 39.5°C in 0.75 N HCL for 2, 3.5 and 5 hours, and stained with Schiff's reagent ( Graumann, 1953 ) according to the Feulgen procedure. The total dye content of the stained spermatozoa was determined using a type GN 2 scanning integrating microdensitometer (Barr & Stroud, Glasgow, Scotland). The nuclei of 50 sperm heads of each slide were measured at 570 nm, the mean value and the variation of the measurements calculated. The significance of this variation between the spermatozoa of fertile and infertile men was checked by an F-test. Furthermore, the hydrolysis curves of spermatozoa of fertiles and infertiles were compared. Fertility and infertility were identified by standard andrological examinations. Results and Discussion The hydrolysis curve of spermatozoa shows a fertility-linked significant difference as can be demonstrated by Figure 1. The mean values of dye content in arbitrary units (AE) of the spermatozoa of fertile male are lower and the curve does not reach a maximum after 5 hours of hydrolysis, indicating a different stoichiometry of the Feulgen reaction. The variations (s 2) of our cytophotometric measurements also show a difference between the populations of fertile and infertiles spermatozoa. The latter show a significant higher scattering of the cytophotometrically determined dye content after different hydrolysis times. The results of these calculations are summarized in Figure 2 and Table 1, insignificant values being drawn with heavy-faced typs. All these findings are considered as fertility-linked disturbances in synthesis and structure of acid nuclear proteins and the so-called (acid-insoluble) “residual proteins” of the sperm heads. Considering data of Bahr and Wied (1971) this control of protein synthesis seems to be lax under physiological conditions with respect to quantity but not to quality. This means that the macromolecular components of sperm cells are assembled without regard to strict proportionality. It now seems obvious that under pathological conditions linked to infertility, which is expressed by the abnormal histological pattern of spermiogenesis (Fig. 3), the lack of control of protein synthesis may increase. This simple investigation of the structure of nucleoproteins of the sperm head enables us to investigate the complex problems of male infertility.

The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. 1. Studies of snake venom phosphodiesterase.

Snake venom phosphodiesterase induces about fifteen exonucleolytic cleavages for each endonucleolytic cleavage during the first hour of hydrolysis of wheat embryo ribosomal RNA, under the conditions of hydrolysis used in this present investigation. The polynucleotide chains in the ribosomal RNA preparation have an average degree of polymerization in the neighborhood of 1300 nucleotide residues, and there is a mean of between 5 and 10 endonucleolytic breaks per chain during this first hour of phosphodiesterase-induced hydrolysis. The cleavages occur widely throughout most of the polynucleotide chains in the ribosomal RNA preparation, as judged by the sharp decrease in mean sedimentation rate which accompanies a limited degree (about 10%) of exonucleolysis of the RNA. Studies of phosphodiesterase-induced endonucleolysis of wheat embryo soluble RNA are reported, but because of the much lower initial degree of polymerization (about 80 nucleotide residues per polynucleotide chain), the results of endonucleolysis are less pronounced in terms of the proportional increment in chain termini and the proportional decrease of mean sedimentation rate. The endonucleolysis of RNA is discussed in terms of the minor nucleotide components in both ribosomal and soluble RNA, and particular reference is made to pseudouridylate which has been found in relatively high proportion among the chain termini after limited hydrolysis with venom phosphodiesterase.Purified venom phosphodiesterase preparations, devoid of ribonuclease or 5′-nucleotidase contamination, were found to convert nucleoside 2′(3′),5′-diphosphates to 5′-nucleotides under conditions which had virtually no effect on nucleoside 3′-phosphates or nucleoside 5′-phosphates. The possibility that this reaction may be catalyzed by the venom phosphodiesterase itself is discussed.

Lipolytic enzyme content of rat tissues.

Summary1. Studies have been made of the concentration in the tissues of the rat of enzyme that catalyzes the hydrolysis of olive oil and tributyrin. 2. Serum liberated from an emulsified olive oil substrate an average of 6.6 micromoles of fatty acid per ml during the first hour of hydrolysis. 3. Liver, kidney and spleen were found to contain 3 to 6 times as much enzyme that splits olive oil as observed in serum. Pancreas showed 6000 times as much enzyme that splits olive oil as found in serum. Brain yielded low and questionable values. 4. In analyses with emulsified tributyrin as substrate, tissue extracts liberated 4 to 100 times as much fatty acid as was split from olive oil under the same conditions.

Serum lipase determination using a one-hour period of hyrolysis

1. 1. Optimum conditions have been determined for the measurement of serum lipase with respect to emulsifying agent, buffer, pH of hydrolyzate, and time of hydrolysis. 2. 2. The procedure developed makes use of the first hour of hydrolysis of triglyceride. 3. 3. Normal human serum lipase values range from 2 to 12 units per milliliter. A unit is the amount of enzyme that catalyzes the liberation of 1 μmole of fatty acid from triolein or olive oil in 1 hr per milliliter of serum. 4. 4. In acute pancreatitis markedly elevated serum lipase values were observed when determined with olive oil as substrate.

Electron Microscopic and Electron Diffraction Studies on Hydrous Thorium Oxide

Hydrous thorium oxide has been prepared by the addition of ammonium hydroxide to solutions of thorium chloride (Series A) and thorium nitrate (Series B), followed by dialysis for varying periods of time at room temperature; and by hydrolysis at 100° of thorium chloride solution (Series C) and thorium nitrate solution (Series D), followed by dialysis at 100° for varying periods of time. Samples from Series A and B were found by electron diffraction to be essentially amorphous, the diffraction pattern consisting of a few broad bands, in agreement with earlier work. Primary particles in the range 8–20 Å were observed on electron micrographs, the size increasing slowly with time of dialysis and aging, Samples from Series C and D likewise were found to be essentially amorphous at first, but the electron diffraction patterns became sharper and became identical with that from normal cubic ThO2 after several hours of hydrolysis and aging at 100°. Electron micrographs show a growth in crystal size to 200–400 Å as the hydrolysis and aging proceeds. A very small percentage of plate-like particles have been detected by both electron microscopy and electron diffraction in samples for Series C (hydrolysis of thorium chloride at 100°). The number of such plate-like particles increases but slightly with prolonged aging. The plates exhibit a hexagonal crossgrating single crystal type of electron diffraction pattern. They have not been detected in samples prepared from the nitrate. Because of the infrequent occurrence of the plates, attempts to secure such plates isolated from the bulk of the small crystals of hydrous thorium oxide were unsuccessful. Their chemical composition is not known. Future work will involve hydrolysis in a high pressure bomb, with the view of producing the plate-like material in larger amounts, and with the view of eliminating the possibility that the plates result from a recurring impurity.

HYDROLYSIS OF I131-THYROPROTEIN BY PANCREATIC ENZYMES

Until about 5 years ago, treatment with hot alkali was the most widely used method for hydrolyzing the iodoprotein of thyroid tissue. By such a procedure, Kendall first isolated thyroxine from the thyroid glands of oxen (1). Proteolytic enzymes have also been used for this purpose with varying degrees of success, since the early work of Hutchison (a), Oswald (3), and Harington (4). More recently, the usefulness of enzymic hydrolysis has been extended considerably by combining this method for fragmenting the thyroid protein with the very sensitive techniques of chromatography and radioisotopic labeling for the isolation and identification of the products of hydrolysis. Although this combinat,ion of procedures has been used in several laboratories (5-la), a thorough study of the hydrolysis with proteases has not been reported. In our investigation of this procedure we found, in agreement with Roche et al. (7), that the iodotyrosines are released quite rapidly from the thyroprotein, whereas the release of thyroxine proceeds more slowly. In addition, we found that the yield of thyroxine can be increased by performing the hydrolysis in the presence of certain metal cations (Mn++, Mg++, Ca++). These findings are described below, along with a procedure for the analysis of 1131-labeled thyroid tissue. This procedure requires 10 to 20 hours of hydrolysis, yields approximately 95 per cent recovery of 1131 protein in the form of 1131-amino acids, and results in little release of inorganic I131.

The amino acid composition of tobacco mosaic virus protein

The first complete analysis of the tobacco mosaic virus protein after it has been freed from nucleic acid is reported. Analyses after 24 and 48 hours of hydrolysis indicate marked decomposition of serine and threonine during hydrolysis and the values for these amino acids in the protein have been obtained by extrapolation to zero time. The corrected analytical values yield integral numbers for most of the amino acids and account for almost all the weight of the protein. The analyses indicate a minimal peptide chain molecular weight of 18,270 composed of the following 164 amino acid residues: Asp 19 Glu 17 Pro 8 Ser 18 Thr 17 Gly 6 Ala 15 Val 14 Leu 13 Isol 9 Tyr 4 Trypt 2 Phe 8 CySH 1 Lys 2 Arg 11 (-CONH 2) 20. From the above composition the calculated value for the partial specific volume ( V ) of the protein is 0.731.