Comparative characteristics of proteolytic enzyme preparations by degree of hydrolysis of a microbial protein

Proteolytic enzymatic preparations obtained from fungi and bacteria were compared by their ability to hydrolyze yeast protein. Fungal preparations were more effective. There was a more than twofold increase in the level of amine nitrogen in cell biomass hydrolysates in of cell biomass comparison to that induced by bacterial preparations. The amino acid composition of these hydrolysates was studied. Amyloprotooryzin, a preparation from Aspergillus oryzae 387, displayed the highest potency in profound protein hydrolysis: the concentration of free amino acids released was 34.7% in comparison to 20.6% induced by amyloryzin and 10.5% by protosubtilin.     

Enzyme preparations for research on protein hydrolysates and amino acid mixtures not containing peptides

Some properties and the hydrolysing ability of two novel enzyme preparations, a proteolytic preparation "C" from Acremonium chrysogenum (Cephalosporium acremonium) and a peptidase preparation (the producer from the family Pseudomanadaceae), are described. The preparations can be used for obtaining protein hydrolysates with different ratios of free amino acids and peptides. The protein hydrolysis with the preparation "C" enables one to obtain hydrolysates containing 13-18% of free amino acids. The further treatment of the hydrolysates with the peptidase preparation results either in complete hydrolysis of the remained peptide fractions or in obtainment of solutions containing from 60 to 85% of free amino acids and low-molecular weight peptides.     

Antioxidant Peptides from the Protein Hydrolysates of <Emphasis Type="Italic">Conus betulinus</Emphasis>

The current study aims at the isolation and characterization of the peptides, believed to have antioxidant activity, from Conus betulinus by using different types of enzymes. The body and viscera of C. betulinus were treated with three enzymes viz. trypsin, pepsin and papain to obtain peptide hydrolysates. The activities of the hydrolysates were analyzed by DPPH and hydroxyl radical assay by using electron spin resonance (ESR) device. Active hydrolysates were purified using ion exchange chromatography followed by gel filtration chromatography. The activity of the separated fractions was analyzed by ESR; in which the result showed that trypsin hydrolysate of body (28.48 and 76.00%) and viscera (38.45 and 83.00%) respectively have high activity than the other hydrolysates. The HPLC result of purified fraction showed, presence of active amino acids viz., metheonine, cystine, histidine etc. This purified peptide has more antioxidant activity that could reduce the excess free radicals in body in order to prevent free radical induced diseases.     

Proteolysis in vitro as the Way of Modification of the Protein Trophic Component

The protein hydrolysates of two types, fodder and for microbiological medium, have been obtained in vitro, using protein containing wastes of Iceland scallop fishery (WSF) and enzyme preparation from the red king crab hepatopancreas. The degree of protein degradation and composition of obtained hydrolysates were analyzed. Intensive protein hydrolysis was necessary to obtain the microbiological diagnostic cultural media that fit trophic requirements for 12 microbial test-cultures. Addition of the fodder protein hydrolysate to diet of juvenile salmons Salmo salar during mixed feeding produces a positive effect on their survival; the 5 and 20% replacement of fish flour by the fodder hydrolysate results in a reduction of fish mortality by 21 and 57%, respectively. The living weight of chickens increased by 15% after substitution of the 10% fish flour by the fodder WSF hydrolysate in their diet. The results obtained can be explained by a more effective assimilation of partly hydrolyzed proteins in comparison with native ones.     

The nature of the organic nitrogen of soils

Summary Examination of the 6N HC1 hydrolysates from 14 different proteins indicated that a considerable proportion of the total protein nitrogen in the hydrolysates, as determined by the micro-Kjeldahl method, was not accounted for by the NH₄-N and the α amino nitrogen found in the hydrolysates. It seems clear that this hydrolysable unidentified nitrogen (HUN) originates mainly from non-amino nitrogen atoms present in arginine, tryptophan, lysine and proline. These nitrogen atoms do not satisfy the conditions necessary for reaction with ninhydrin. The amounts of each amino acid in a particular protein determine the HUN value which will be obtained for 6N HC1 hydrolysates of that protein. There is good agreement between the HUN values for a wide range of proteins when calculated from the amino acid composition of the protein and when determined experimentally. It is concluded that these findings indicate a considerably higher content of amino acid nitrogen in the organic nitrogen of soils and leaf litter than was previously considered to be the case. It is suggested that the findings support the contention that the organic nitrogen of soils contains leaf protein complexes.     

Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.     

脑蛋白粉的制备及其酶解方法的研究

研究了以鲜猪脑制备蛋白粉并进行酶水解的方法 ;脑蛋白粉质量、不同酶和加酶方法对水解结果的影响 ;分析了水解液中游离氨基酸和低分子肽谱 ,并与国内外类似研究进行了比较。结果表明 :丙酮、乙醚沉淀制得的脑蛋白粉含氮 9.0 %～ 1 1 .5% ,脂肪 2 .5%～ 4 .0 % ,适于酶解 ;水解液含有 1 6种游离氨基酸 ,总量达 3 2 .4 4mg/ml和 4个分子量小于 1万的肽。提出精制脑蛋白粉进行水解、减压浓缩、超滤是提高水解液有效成分的技术措施。     

6 Lignin-protein complex in cell walls of Pinus elliottii: Amino acid constituents

Cell walls of Pinus elliottii callus contain ca 12 % protein. Klason lignin prepared from the walls contained 9 % protein and represented 4.5 % of the wall. The lignin fraction was increased to 22 % of the wall weight by reacting washed cell-wall tissue with coniferyl alcohol and H₂O₂, a reaction catalysed by peroxidase that remained bound to the wall. The augmented lignin preparation yielded 10 % protein. The acid hydrolysate of whole wall tissue included five amino acids at a concentration higher than hydroxyproline. The hydrolysates of both natural and augmented lignin preparations yielded distributions of amino acids in which the concentration of hydroxyproline was higher than that of all other amino acids. The results suggest that polymerizing lignin links covalently with cell-wall glycoprotein, and that the bonds may be formed preferentially with hydroxyproline.     

Enzymatic Hydrolysis of Proteins from Crustaceans of the Barents Sea

Enzymatic preparations from king crab hepatopancreas were shown to be capable, in principle, of producing protein hydrolysates. Hydrolysis of protein-containing waste of deep-water prawn and king crab occurs most successfully at pH 8.0–8.5 and 50–55°C for 5–6 h in the presence of 6 g enzyme per kg substrate. The total chemical composition of the hydrolysates, the molecular weight distributions of proteins and polypeptides, and the contents of free amino acids were studied in dry hydrolysates.     

Radical Scavenging Activity of Seela (<Emphasis Type="Italic">Sphyraena barracuda)</Emphasis> and Ribbon Fish (<Emphasis Type="Italic">Lepturacanthus savala)</Emphasis> Backbone Protein Hydrolysates

We have investigated the antioxidant activity of protein hydrolysates prepared from backbones of two commercially important fishes; seela (Sphyraena barracuda) and ribbon fish (Lepturacanthus savala). Pepsin and trypsin hydrolysates were found more potent to inhibit lipid peroxidation in case of ribbon and seela fish respectively and were further purified by using fast protein liquid chromatography on anion exchange and gel filtration chromatography. The active peaks after gel filtration chromatography of seela fish was able to scavenge 2,2-diphenyl-1-picryhydrazyl and hydroxyl radicals by 61 ± 2.3 and 58.7 ± 2.3% and ribbon fish hydrolysate by 60.0 ± 2.6 and 55.6 ± 1.8% as measured by ESR spectroscopy. And the active fractions showed presence of both essential and non-essential amino acids with high percentages of arginine (11.95 and 12.76%) and lysine (13.49 and 13.89%).     

Amino acid content of leguminous proteins as affected by genetic and nutritional factors. I.

Summary Tetraploid and diploid red clover have been cultivated in greenhouse under aseptic conditions. The plant cultures were given only one source of nitrogen: either they were given nitrate in the nutrient solution or they were inoculated withRhizobium for establishing symbiotic fixation.The amino acid composition of protein from the different series and from different parts of the plants was analysed by paper chromatography.There was a clear arginine reaction from the tetraploid clover material but no arginine at all was found in the diploid clover material.Plants fed on nitrate and those fed on symbiotically fixed nitrogen showed the same amino acid pattern as far as can be judged from the chromatograms. The hydrolysates from inoculated plants showed usually more intense ninhydrine reaction on the chromatograms than hydrolysates from not inoculated plants, although diluted to the same nitrogen content. For this fact we have so far no explanation.     

Enzymatic hydrolysis of proteins from crustaceans of the Barents Sea

Enzymatic preparations from king crab hepatopancreas were shown to be capable, in principle, of producing protein hydrolysates. Hydrolysis of protein-containing waste of deep-water prawn and king crab occurs most successfully at pH 8.0-8.5 and 50-55 degrees C for 5-6 h in the presence of 6 g enzyme per kg substrate. The total chemical composition of the hydrolysates, the molecular weight distributions of proteins and polypeptides, and the contents of free amino acids were studied in dry hydrolysates.     

Effect of the peptide and vitamin composition of casein hydrolysates on the β-galactosidase activity of Kluyveromyces bulgaricus cells

Instead of yeast extracts, casein hydrolysates from several suppliers were added to culture media and analyzed with respect to their ability to stimulate β-galactosidase activity in Kluyveromyces bulgaricus cells. Four enzymatic casein hydrolysates caused a significantly higher stimulation of enzyme activity, while acid casein hydrolysates clearly reduced the enzyme activity. Enzymatic casein hydrolysates, inducing high and low lactase activity, were analyzed with respect to average peptide length (APL), vitamins (niacin, pathothenate) as well as free and bound amino acids. The molecular weights of these casein hydrolysates were estimated by gel filtration. No correlation was found between the degree of enzyme stimulation and the vitamin contents, the APL values and the free amino acid contents of the casein hydrolysates. Casein hydrolysates stimulating the lactase activity were less soluble in water and, in a gel filtration column, they showed three peaks with slightly lower molecular weights than the three peaks seen in hydrolysates which had no effect on activity. APL values of alcoholic precipitates of casein hydrolysates showed an inverse correlation to lactase activity. The molecular weights of alcoholic precipitates of lactase stimulating digests were also lower compared to non-stimulating ones. Alcoholic precipitates with lactase-stimulating activity were more hydrophilic, as was shown by a smaller proportion absorbed in a C18 column and by amino acid analysis. Our results sugest that alcoholic precipitates could probably be important in the lactase stimulation and their composition should be further investigated. The mechanism is nevertheless complex and may be caused by various simultaneous factors.     

Shrimp processing by-products protein hydrolysates: Evaluation of antioxidant activity and application in biomass and proteases production

Protein hydrolysates were prepared from shrimp processing by-products (SPBP) using five proteolytic enzymes: trypsin, Alcalase®, crude enzyme extract from sardinelle (Sardinella aurita) viscera and enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1. The obtained hydrolysates exhibited different degrees of antioxidant activities evaluated through three main tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity, reducing power and β-carotene bleaching assays. Hydrolysates were also tested as nitrogen source for microbial growth and proteases production by Escherichia coli, Saccharomyces cerevisiae, B. mojavensis A21 and B. subtilis A26. The reached results showed that the SPBP protein hydrolysates (SPBPPHs) could be a promising alternative to currently available commercial nitrogen sources of other origins.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Protein hydrolysates from Bluefin Tuna (Thunnus thynnus) heads as influenced by the extent of enzymatic hydrolysis

Functional properties and antioxidant activities of protein hydrolysates from tuna (Thunnus thynnus) heads (THPHs), with different degrees of hydrolysis, obtained by treatment with Bacillus mojavensis A21 alkaline proteases and Alcalase, were investigated. Protein content of all freeze-dried THPHs ranged from 73.74 ± 0.5 to 78.56 ± 1.2%. The THPHs had excellent solubility, compared to untreated tuna head proteins and possessed interfacial properties, which were governed by their concentrations. Similarly, at a degree of hydrolysis (DH) of 12 and 15%, > 90% nitrogen solubility was observed at all experimental pH values tested. The emulsifying activity index (EAI) and emulsion stability index (ESI) of both hydrolysates at different DHs decreased (p < 0.05) with increasing DH. At low DH (5%), hydrolysates exhibited strong emulsifying properties. All THPHs produced by the A21 proteases generally showed higher antioxidative activity than that of the Alcalase protein hydrolysates. The highest DPPH radical-scavenging activity (78 ± 2.1% at 3 mg/mL) was obtained with a DH of 15%. The IC₅₀ value for the β-carotene bleaching assay was 0.5 ± 0.03 mg/mL. Alcalase (DH = 12%) and A21 (DH = 15%) protein hydrolysates contained glutamic acid/glutamine and arginine as the major amino acids, followed by lysine, aspartic acid/ asparagine, histidine, valine, phenylalanine, and leucine. In addition, the THPHs had a high percentage of essential amino acids, which made up 50.52 and 50.47%, of the protein hydrolysates obtained by the Alcalase and A21 proteases, respectively. Therefore, THPHs can be used as a promising source of functional peptides with antioxidant properties.     

Utilization of glucose and amino acids byBacteroides intermedius andBacteroides gingivalis

Growth ofBacteroides intermedius was promoted only moderately by glucose, and the incorporation of¹⁴C-glucose into cells was limited. WithBacteroides gingivalis growth promotion was negligible and glucose incorporation even more restricted. Both species grew prolifically on protein hydrolysates containing peptides, but grew poorly on acid-hydrolyzed casein even when supplemented with amino acids. These results are discussed in relation to the ecological distribution of these species compared to saccharolytic bacteroides.     

New Protein Hydrolysates from Collagen Wastes Used as Peptone for Bacterial Growth

A simple and low-cost procedure was developed for the effective processing of native calf skin and blood wastes to produce protein hydrolysates. The method includes extraction of high–molecular-weight protein from the raw material, followed by enzymatic hydrolysis of the extracted residue. The enzymatic hydrolysis was performed by inexpensive commercial subtilisin DY, produced by Bacillus subtilis strain DY possessing high specific activity. The contents of protein, nitrogen, ash, and amino acids of the obtained hydrolysates were determined and compared with those of the commonly used commercial casein hydrolysate (Fluka Biochemica, Switzerland). The newly obtained calf skin hydrolysate, called Eladin, was found to be suitable as a low-cost alternative peptone in growth media of different microorganisms, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella dublin, and Staphylococcus aureus. The method allows utilization of waste materials by converting them into valuable protein products that could find widespread application in microbiologic practice.     

Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids

Plants are an attractive sourceof renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components, such as aromatics and amino acids, may improve the efficiency of biomass conversion. Pseudomonas putida is a promising host for its ability to metabolize a wide variety of organic compounds. P. putida was engineered to produce methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics. Unexpectedly, P. putida methyl ketone production using Arabidopsis thaliana hydrolysates was enhanced 2–5-fold compared with sugar controls derived from engineered plants that overproduce lignin-related aromatics. This enhancement was more pronounced (~seven-fold increase) with hydrolysates from nonengineered switchgrass. Proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a nine-fold increase in methyl ketone titer (1.1 g/L).