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.     

Comparative characterization of proteolytic enzymatic preparations by the extent of hydrolysis of 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 comparison to that induced by bacterial preparations. The amino acid composition of these hydrolysates was studied. Amyloprotooryzin, a preparation fromAspergillus 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.     

Incorporation of C14 from amino acids and peptides into protein by Clostridium perfringens type D

Hauschild, Andreas H. W. (University of Toronto, Toronto, Ontario, Canada). Incorporation of C(14) from amino acids and peptides into protein by Clostridium perfringens type D. J. Bacteriol. 90:1569-1574. 1965.-Uptake of C(14) from C(14)-labeled amino acids and peptides by Clostridium perfringens was measured in culture media containing acid or papain hydrolysates of C(14)-labeled Chlorella protein. Between 2 and 4 hr of growth, the rate of C(14) uptake from peptides was higher than from free amino acids. Peptides extracted from cells with hot ethyl alcohol contained six to nine times more C(14) after 4 hr of growth with C(14)-labeled peptides than with C(14)-labeled amino acids. Incorporation of C(14)-labeled glycine, serine, threonine, alanine, and proline into both cellular and exocellular protein was two to five times higher when these were supplied as components of dialyzable peptides rather than as free amino acids.     

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。     

The evaluation of the temperature effect and hydrolysis conditions for amino acid analysis

A systematic investigation of the optimal temperature and hydrolysis time for amino acid analysis has been carried out under various conditions. It is found that some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes and 110 C/24-72 hour hydrolysis can be achieved without loss of accuracy and performance in amino acid analyses of proteins and peptides. The effects of hydrolysis temperature and heating time on the recoveries of various labile and hydrophobic amino acids are exemplified in the hydrolysis of oxidized ribonuclease A, lysozyme and lens crystallin. The method provides a rapid processing of multiple samples within hours instead of days with the potential for the total automation of amino acid analysis starting from the preparation of protein hydrolysates.     

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.     

Hydrolysis of Soybean Proteins with Kamchatka Crab Hepatopancreas Enzyme Complex

To perform hydrolysis with the enzyme complex from the hepatopancreas of the Kamchatka crab, a protein mixture was isolated from soybean meal by extraction at alkaline pH values. Extractable low-molecular impurities were removed by ultrafiltration and precipitation of proteins with alcohol. The amino acid composition of the obtained protein extract turned out to be similar to the composition of the fish meal traditionally used in the production of fish feeds. Analysis of the products of fermentolysis by DDS-electrophoresis, HPLC, and mass spectrometry showed a high degree of hydrolysis of soybean proteins. Depending on the time of fermentolysis, the hydrolysates contained up to 60% (18 h of hydrolysis) of free amino acids (the fraction of the weight of the hydrolyzed protein mixture) and short peptides (2–20 amino acid residues).     

蛋白水解物制备工艺及其在生物技术领域中的应用研究进展

蛋白水解物是蛋白质经酶、酸、碱和发酵等方法水解得到的混合物,由于其含有丰富的氨基酸和多肽,并为细胞生长提供多种营养成分、贴壁因子及生长因子类似物等,从而在生物技术领域中得到了广泛的应用。本文综述了蛋白水解物制备工艺、在生物技术领域中的应用及其在细胞培养中对细胞增殖、代谢的影响,以期为蛋白水解物的开发和应用提供参考。     

Peptide Utilization by Amino Acid Auxotrophs of Neurospora crassa

The ability of auxotrophs of Neurospora crassa to grow on certain tripeptides, despite the presence of excess competing amino acids, suggests it has an oligopeptide transport system. In general, dipeptides did not support growth except in those instances where extracellular hydrolysis occurred, or where the dipeptide appeared to be accumulated by an uptake system which is sensitive to inhibition by free amino acids. Considerable intracellular peptidase activity toward a large number of peptides was demonstrated, including a number of peptides which could not be utilized for growth. The intracellular peptidase activity was shown to be selective for amino acid composition and sequence (N-terminal or C-terminal) within the peptide; glycine-containing peptides were particularly poor substrates for peptidase activity. Only a small amount of extracellular peptidase activity could be detected.     

Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.     

Amino acid and peptide ratio in protein hydrolysates for parenteral feeding

Using the method of amino acid analysis and routine methods of protein biochemistry, the ratio of amino acids and peptides in acid and enzyme protein hydrolyzates was determined. Depending on the production procedure, the hydrolyzates under study contained various amounts of free amino acids and peptides in which the number of amino acid residues varied from 2 to 7. Additional hydrolysis of these preparations by leucine aminopeptidase led to a decrease in the peptide content and to an increase in the amino acid content. This may have a beneficial effect on the quality of protein hydrolyzates.     

Enzymatic generation of peptides from potato proteins by selected proteases and characterization of their structural properties

The use of low grade starting material for the generation of peptides with bioactivity properties is of interest. The proteins from the potato starch industry byproduct is a promising source, as several health benefits may be associated with their hydrolysates. The efficiency of selected proteases (Novo Pro‐D, Alcalase, Flavourzyme, and Papain), exhibiting different substrate specificities and cleavage action modes, to hydrolyze potato protein isolate (patatin and protease inhibitors) was investigated. Novo Pro‐D resulted in the lowest degree of hydrolysis, whereas Alcalase, Flavourzyme, and Papain exhibited a high catalytic efficiency for the hydrolysis of potato proteins. The degree of hydrolysis behaved in a concentration dependent manner. However, the end‐product profile (peptides and free amino acids) was dependent not only on the protease specificity, its cleavage action mode (endo/exo) and the availability of the intermediate substrates but also on the contribution of the protease inhibitors to the reaction kinetics through their inhibitory effects. Indeed, the dependence of the exo‐activity on the catalytic efficiency of the endo‐action of protease was shown to be significant. Papain generated more unique peptide sequences with homology assessment matching several potato proteins when compared with Flavourzyme. This can be attributed to the high exo‐peptidase activity of Flavourzyme resulting in the generation of shorter peptides which were difficult to match. Flavourzyme produced more peptides originated from patatin fraction, whereas Papain resulted in the release of more peptides corresponding to the protease inhibitor fractions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:420–429, 2016     

Plant protein hydrolysates as fish fry feed in aquaculture. Hydrolysis of rapeseed proteins by an enzyme complex from king crab hepatopancreas

Rapeseed proteins were processed by an enzyme complex isolated from king crab hepatopancreas in order to obtain a hydrolysate for use as fish fry feed. The amino acid composition of the obtained protein preparation was close to the amino acid composition of fishmeal traditionally used in the production of fish feed. SDS-PAGE, HPLC, and mass spectrometric analysis of the products of enzymatic hydrolysis of rapeseed proteins showed that the proteins were hydrolyzed to a high degree. The composition of the hydrolysates depended on the hydrolysis time and included free amino acids (27% of the total weight of the protein mix after 3 h of hydrolysis and 56% after 21 h of hydrolysis), short peptides (2 to 20 amino acid residues), and small amounts of protein fragments with a molecular weight of approximately 14 kDa, as shown by by SDS-PAG electrophoresis.     

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

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.     

Influence of the rapeseed protein hydrolysis process on CHO cell growth

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.     

The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

Quantitation of aspartate and glutamate in HPLC analysis of phenylthiocarbamyl amino acids

In the quantitation of amino acids by precolumn derivatization with phenylisothiocyanate, the yields of N'-phenylthiocarbamyl (PTC)-aspartate and PTC-glutamate from protein hydrolysates are often suboptimal, particularly in analyses following rapid hydrolysis at 160 degrees C. In this paper we show that these losses are due to the presence of materials extracted from the glass container during hydrolysis. In the presence of these extracts, the repeated drying and neutralization steps which precede phenylthiocarbamylation result in samples not fully solubilized by the presently used derivatizing mixtures. Thus the coupling yields for the acidic residues are highly variable. A coupling buffer with the composition 35% H2O, 30% acetonitrile, 25% pyridine, and 10% triethylamine (v/v/v/v) is an efficient solvent for all amino acids in hydrolysates and permits consistent, quantitative derivatization of all amino acids, including aspartate and glutamate.