Enzyme Fraction Research Articles

Broad Distribution of TPI-GAPDH Fusion Proteins among Eukaryotes: Evidence for Glycolytic Reactions in the Mitochondrion?

Glycolysis is a central metabolic pathway in eukaryotic and prokaryotic cells. In eukaryotes, the textbook view is that glycolysis occurs in the cytosol. However, fusion proteins comprised of two glycolytic enzymes, triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were found in members of the stramenopiles (diatoms and oomycetes) and shown to possess amino-terminal mitochondrial targeting signals. Here we show that mitochondrial TPI-GAPDH fusion protein genes are widely spread across the known diversity of stramenopiles, including non-photosynthetic species (Bicosoeca sp. and Blastocystis hominis). We also show that TPI-GAPDH fusion genes exist in three cercozoan taxa (Paulinella chromatophora, Thaumatomastix sp. and Mataza hastifera) and an apusozoan protist, Thecamonas trahens. Interestingly, subcellular localization predictions for other glycolytic enzymes in stramenopiles and a cercozoan show that a significant fraction of the glycolytic enzymes in these species have mitochondrial-targeted isoforms. These results suggest that part of the glycolytic pathway occurs inside mitochondria in these organisms, broadening our knowledge of the diversity of mitochondrial metabolism of protists.

Characterization and purification of the crude Trematosphaeria mangrovei laccase enzyme

Objectives The aim of this work was to study the purification and characterization of the crude extracellular laccase produced by the marine-derived fungus Trematosphaeria mangrovei. Methods The general properties of the crude laccase enzyme produced by T. mangrovei were investigated. These include the effect of temperature, pH, thermal and pH stabilities, and enzyme and substrate concentrations on the laccase activity. Partial purification of the T. mangrovei laccase enzyme was carried out by fractional precipitation with ammonium sulphate, ethanol and acetone. Further purification was carried out on a Sephadex G-100 column. Results and conclusion The results obtained showed that the crude enzyme reached its maximal activity at 35πC, pH 4.5, at an enzyme concentration of 5.429 mg protein/reaction mixture and at a substrate concentration of 40 mmol/l 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). The enzyme was stable for 60 min at 35πC and retained about 80–90% of its activity after treatment for 60 min from 40 to 50πC. The enzyme showed maximum stability (100%) at pH 4.5 and 91.6% at pH 4.0 after 60 min. Fractional precipitation of the fungal extracellular T. mangrovei laccase enzyme with different methods showed that the enzyme fraction precipitated at 60% acetone was the most favourable enzyme fraction; it showed 4.84 purification fold. Laccase obtained from the 50–60% acetone fraction was purified by Sephadex G-100. The final preparation thus obtained reached 31.47-fold that of the culture filtrate (1466.49 U/mg protein) and showed a single band on native polyacrylamide gel electrophoresis.

Mechanistic Study of 17α-Ethinylestradiol Biodegradation by Pleurotus ostreatus: Tracking of Extracelullar and Intracelullar Degradation Mechanisms

The white rot fungus Pleurotus ostreatus is able to completely remove the synthetic hormone 17α-ethinylestradiol (EE2, 200 μg in 20 mL) from a liquid complex or mineral medium in 3 or 14 days, respectively. Its efficiency has also been documented in the removal of estrogenic activity that correlated with the EE2 degradation. A set of in vitro experiments using various cellular and enzyme fractions has been performed and the results showed that EE2 was degraded by isolated laccase (about 90% within 24 h). The degradation was also tested with concentrated extracellular liquid where degradation reached 50% mainly due to the laccase activity; however, after a supplementation with H₂O₂ and Mn²⁺, residual manganese-dependent peroxidase activities (40 times lower than Lac) raised the degradation to 100%. Moreover, the intracellular fraction and also laccase-like activity associated with fungal mycelium were found to be efficient in the degradation too. Isolated microsomal proteins appeared to also be involved in the process. The degradation was completely suppressed in the presence of cytochrome P-450 inhibitors, piperonylbutoxide and carbon monoxide, indicating a role of this monooxygenase in the degradation process. Attention was also paid to monitoring of changes in the estrogenic activity during these particular in vitro experiments when mainly degradations related to ligninolytic enzymes were found to decrease the estrogenic activity with EE2 removal proportionally. Several novel metabolites of EE2 were detected using different chromatographic method with mass spectrometric techniques (LC-MS, GC-MS) including also [¹³C]-labeled substrates. The results document the involvement of various different simultaneous mechanisms in the EE2 degradation by P. ostreatus by both the ligninolytic system and the eukaryotic machinery of cytochromes P-450.

Evidence of structural changes of an enzymatic extract entrapped into alginate beads

In this work, we analyzed the structural changes of araujiain entrapped into alginate beads. Araujiain is an enzymatic preparation containing three known enzymatic fractions with each fraction individually presenting a similar catalytic performance. Fluorescence and infrared spectroscopy, thermal analysis and residual catalytic activity studies were carried out. A small red shift in the spectrum of araujiain was observed after the entrapment process. Changes in the polarity around the tryptophan (Trp) residues were associated with an enzyme conformational change. From the Fourier transform infrared spectroscopy (FTIR) analysis, it was demonstrated that interactions between the enzyme extract and Ca alginate caused different structural behavior in araujiain. According to the diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) study, it was possible to conclude that a secondary structure with a high α-helical character was responsible for the highest activity of entrapped araujiain. Finally, from thermal analysis measurements, it was proved that entrapment of araujiain augments the thermal stability of both the enzyme extract and Ca alginate, indicating a possible interaction between enzyme extract and its support.

Partitioning of alkaline protease from Bacillus licheniformis (ATCC 21424) using PEG–K2HPO4 aqueous two-phase system

An aqueous two-phase system (ATPS) containing polyethylene glycol (PEG) and potassium phosphate (K2HPO4) was used for partitioning and partial purification of alkaline protease produced by Bacillus licheniformis ATCC 21424. The effect of PEG molar mass, pH and concentration of NaCl salt addition on partition coefficient, yield and purification factor (PF) for ATPS were studied. The highest partition coefficient (5.3) was achieved in an ATPS of 25% (w/w) PEG 10,000 (g/mol) and 10% (w/w) NaCl at pH 9. Cation and anion exchange chromatography was employed for purification of alkaline protease from protein mixture and the yield and PF for enzyme fractions were obtained to make a comparison between ATPS and ion exchange chromatography. Although, the results of the study indicated that using ATPS leads to lower PF than ion-exchange chromatography, but considering its simplicity and high yield adding up lower investments make ATPS a promising purification option in industrial scale.

Characterization of the non-coagulating enzyme fraction of different milk-clotting preparations

Proteolytic milk-clotting enzymes are extracted from various sources (animals, plants, fungi) and processed according to various methods that are specific to each manufacturer or cheese-maker. Chemical composition and polypeptide patterns of 24 milk-clotting preparations from animal and fungal sources: 10 commercial rennets, 9 artisanal calf rennets, 2 recombinant chymosin preparations and 3 microbial preparations, were compared in order to identify differences according to both their manufacturing process and their source. The preparation from Cryphonectria parasitica had the highest ammonia and small peptide content. Commercial rennets and preparations from Rhizomucor miehei had the highest NaCl and pH values while artisanal rennets had the lowest and recombinant chymosins were intermediate. In comparison with the other commercial preparations, commercial rennets had the highest variability in chemical composition and their polypeptide profiles showed numerous protein bands ranging from 15 kDa to 197 kDa, like artisanal rennets. Protein composition of commercial rennets revealed the presence of bovine serum albumin, either native or degraded, and degraded chymosin. The results indicated that the source of coagulating enzymes and the conditions applied for enzyme extraction led to specific chemical compositions, polypeptide patterns and protein composition which are described in this article.

Beneficial Effect Administration of Vitamin C in Amelioration of Lead Hepatotoxicity

Previous human and experimental studies have demonstrated that lead exposure may modify the metabolism of lipid. Oxidative stress with subsequent lipid peroxidation has been postulated as one mechanism for lead toxicity. The protective action of vitamins C against lead affects lipid hydroperoxide level and liver functions in male rats has been studied. Experiments were performed on male waster rats with body weights of 120-160 g. Male wistar rats were exposed to 3 g/l lead acetate in drinking water for 5 weeks and treated thereafter with vitamin C (500 mg/kg, orally) for 28 days. One day after the feeding was over, venous blood samples, under chloroform anesthesia, were collected. The animals were killed by exsanguinations and the liver was excise for determination the metal content and histopathological changes. Similarly, the tissue lipid (lipid peroxidation) and the enzyme fraction (superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (ALP), acid phosphatase (ACP) and glutathione (GSH) were also measured in the liver. Metal content in blood and liver was determined by means of atomic absorption spectrophotometry. Administration of lead acetate (3 g/l) in drinking water for 5 weeks induced a significant increase in the levels of hepatic ALP, ACP and lipid peroxidation. Lead acetate exposure also produced detrimental effects on the redox status of the liver indicated by a significant decline in the levels of liver antioxidants such SOD, CAT and GSH. Further, there was a significant increase in the levels of lead in blood and liver of animals exposed to lead. However, oral administration of vitamin C at dose level of 500 mg/kg body weight reduced the alterations in the previous parameters. Histological examination of the liver also revealed pathophysiological changes in lead acetate-exposed group and treatment with vitamin C improved liver histology. The result of this study strongly indicate that vitamin C has got a potent antioxidant action against lead acetate induced hepatic damage in rats.

The novel ADAMs-like microbial metalloendopeptidase

Heterologous gene expression of extracellular minor metalloendopeptidase of Bacillus pumilus 3-19 in protease-deficient B. subtilis strain has been studied. The fraction of enzyme in total pool of B. pumilus 3-19 secreted proteases composes less than 8%. The enzyme was isolated from culture liquid of recombinant strain, its primary structure was determined, physicochemical properties were investigated. It was concluded that secreted metallo endopeptidase of B. pumilus 3-19 represents the first prokaryotic homolog of eukaryotic adamalysin/reprolysin protein family.

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.

Development of Fecal Microbial Enzyme Mix for Mutagenicity Assay of Natural Products

Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.

Specific Domains in Yeast Translation Initiation Factor eIF4G Strongly Bias RNA Unwinding Activity of the eIF4F Complex toward Duplexes with 5′-Overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

Immobilization of lipase B from Candida antarctica on porous styrene–divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.

Production of β-(1,3)-glucanases by Trichoderma harzianum Rifai: Optimization and Application to Produce Gluco-oligosaccharides from Paramylon and Pustulan

β-(1→3)-Glucanases were produced by Trichoderma harzianum Rifai PAMB-86 cultivated on botryosphaeran in a bench-fermenter and optimised by the response surface method. Maximal enzyme titres occurred at 5 days, initial pH 5.5 and aeration of 1.5vvm. β-(1→3)-The β-glucanolytic enzyme complex produced by T. harzianum Rifai PAMB86 was fractionated by gel filtration into 2 fractions (F-I, F-II), and employed to produce gluco-oligosaccharides from algal paramylon ((1→3)-β-D-glucan) and lichen pustulan ((1→6)-β-D-glucan). Both enzymes attacked paramylon to the extent of ~15-20% in 30 min releasing glucose and laminaribiose as major end-products, and laminarioligosaccharides of degree of polymerization (DP) ≥3. Only F-I degraded pustulan resulting in ~2% degradation at 30 min, with glucose, gentiobiose and gentio-oligosaccharides of DP ≥4 as major products. The difference in the nature of the hydrolysis products can be explained by the substrate specificities of each enzyme fraction, and the structural differences of the β-D-glucans attacked.

A <i>β</i>-Glucosidase Activity Potentially Involved in Cell Division and Wall Development of <i>Phyllostachys</i> Bamboo Suspension Cells

We propose a novel Madake (Phyllostachys bambusoides) bamboo suspension culture model for investigation of key enzyme(s) activity involved in growth/differentiation. Sedimented Cell Volume (SCV) and fresh weight (FW) of the suspension cultured cells reached 34% (v/v) and 8.7 g in 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium while only 7% (v/v) SCV and 1.9 g FW of the cells in 10 μM gibberellic acid (GA3)-containing medium in 14 days. Proportion of mitotically active cells (S to G2/M phases) at a log phase was identified as 29.5% in the former cells with tiny cytoplasmic features while 5.4% in the latter cells with elongation, wall thickening, and lignification by using flow cytometry and laser scanning microscopic analysis. The total β-glucosidase (BGL) activity under the 2,4-D condition increased from 4.8 U in day 2 to 26.2 U in day 14 (ca. 5.5-fold) while a slight reduction, from 4.4 U in day 2 to 2.1 U in day 14 (ca. 0.5-fold), occurred when cell division was suppressed under the GA3 condition. Ratio of the BGL activity of the soluble fractions to the membrane-associated fractions varied depending of the culture condition. The ratio was stable (2 to 8) during the culture period under the 2,4-D condition. Interestingly, the activity of the soluble enzyme fractions increased up to ca. 65% under the GA3 condition in inverse proportion to the membrane-associated enzymes. All together, it was strongly suggested that the detected specificity/variability of BGL activity is potentially involved in cell division and lignification in Madake bamboo cells.

Degradation of Metsulfuron Methyl by Agaricus blazei Murrill Spent Compost Enzymes

ABSTRACT Metsulfuron methyl (MM) is an herbicide used in cereal crops. The white rot mushroom Agaricus blazei Murrill is an important edible and medicinal mushroom reported to be a major laccase producer, a lignin-degrading enzyme with low substrate specificity. A search for assaying the potential use of A. blazei spent mushroom compost (SMC) as a remediation tool for cleaning MM polluted soils was carried out. A phytotoxic dose of this herbicide was separately incubated with two enzyme preparations obtained from the SMC after the second mushroom fruiting flush; the phytotoxicity of the resulting reaction mixtures was then assayed by using a plantlet growing test with Brassica napus L. Thus, the crude enzyme SMC extract preparation (I) or the partially purified enzyme SMC extract (II) and their dilutions, 1:10 and 1:100, were mixed with MM (5 × 10−3 ppm final concentration) and incubated at 25°C for 24, 48, 72, and 96 h. Plantlets separately exposed for 72 and 96 h to the resulting reaction mixtures between MM and those enzyme preparations showed a highly significant increase in their hypocotyl length with respect to plantlets exposed to MM alone. It was thus demonstrated the ability that complex enzyme fractions present in A. blazei SMC have to degrade MM during the right incubation time to compounds with no or lower phytotoxicity than this herbicide.

Active Site Quantification of an ω-Transaminase by Performing a Half Transamination Reaction

Measurement of the active enzyme fraction in a given enzyme preparation is a requirement for accurate kinetic measurements and activity comparisons of, for example, engineered mutants. omega-Transa ...

Characterization and purification of the crude laccase enzyme produced by the marine-derived fungus Trematosphaeria mangrovei

The extracellular laccase produced by the marine-derived fungus Trematosphaeria mangrovei was purified and characterized. The crude enzyme reached its maximal activity at 35°C, pH 4.5, enzyme concentration of 5.429mg protein/reaction mixture and substrate concentration of 40mM ABTS. The enzyme was stable for 60min at 35°C and retained about 80 to 90% of its activity after treatment for 60min at 40°C up to 50°C. And the enzyme activity showed the highest stability after 60min exposure at pH 4.5. Also at pH 4 the enzyme retained about 91.60% of its activity after 60min exposure. Fractional precipitation of the fungal extracellular T. mangrovei laccase enzyme with different methods showed that, the enzyme fraction precipitated at 60% acetone was the most favourable enzyme fraction, exhibited 4.84 purification fold. Purification of laccase on sephadex G-100 column showed that, the fraction number 8 of laccase component was the most active has the highest specific activity (1466.49 U/mg protein) and revealed 6.5 purification fold (purification fold 31.47 compared to the culture filtrate). Native polyacrylamide gel electrophoresis (PAGE) of fraction 8 showed a separated single band similar to that of standard enzyme indicating its purity and homogeneity, and different from that of partial purified enzyme.

Endo-β-1,3-galactanase from Winter Mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.

In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 2. In vitro protein digestibility of common and alternative feed ingredients

Using enzyme fractions isolated from the pyloric caeca of farmed Atlantic cod, the in vitro degree of protein hydrolysis (DH) of numerous conventional and novel feed ingredients were measured by a closed-system pH-Stat assay. Regression equations describing the relationship between in vivo apparent protein digestibility (ADC) and in vitro protein DH were used to predict in vitro protein ADC. The equations resulted in good correlation (< 4 percentage points difference) between ‘measured’ and ‘predicted’ protein ADC in the majority of cases ( r = 0.90-0.99; R 2 = 0.88-0.99), while some ingredients were either over- or under-estimated (6–7 percentage points) which appears related to high ash or chitin content ( r = 0.75; R 2 = 0.61) and may indicate the need for an acid pre-hydrolysis phase and full account of non-protein nitrogen (NPN) content. The ‘predicted’ in vitro protein ADC were above 95% for wheat gluten meal, soy protein concentrate, soy protein isolate and whole krill meal; relatively high (85-95%) for soybean meal, white lupin meal, herring meal, anchovy meal, canola protein concentrate, pea protein concentrate and poultry by-product meal; mid-range (75-85%) for crab meal, shrimp meal and canola meal; and low (< 75%) for hydrolyzed feather meal and flaxseed meal. Further research is needed on the development of a two-stage hydrolysis assay for gadoids involving an acid (gastric) pre-digestion step prior to this assay to further increase agreement between in vivo protein ADC and in vitro protein DH.

In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 1. Development of the method

The method described here involves the extraction and partial purification of an enzyme fraction from the dissected pyloric caeca of commercially farmed Atlantic cod, Gadus morhua (1 kg fish) and the development of a pH-Stat method to predict protein digestibility. The various extraction and partial purification steps successfully concentrated the alkaline serine protease enzymes, trypsin (> 4-fold) and chymotrypsin (> 12-fold). It was found that the enzyme fractions produced in the manner described in this study were completely stable for up to 8 months when stored at − 20 °C and at least 10 months when stored − 80 °C after which significant loss of enzyme activity can occur, although the degree of protein hydrolysis (DH) of casein was unaffected after 12 months. It is recommended that enzyme fractions produced in a similar manner should be stored at − 80 °C and used within 8–10 months. The most suitable substrate concentration [S] to use for closed-system in vitro pH-Stat DH assays was established using a standard purified protein source (vitamin-free casein) with four [S] (0.25, 0.5, 0.75 and 1 mg N/mL protein suspension solution). No significant differences (P > 0.05) were found in the DH values between the [S] tested. The DH curve for casein at a [S] of 0.5 mg N/mL showed a rapid increase initially before leveling off at maximum DH (26%) which was achieved within a moderate duration of the assay (5–6 h). The closed-system pH-Stat assay with a [S] of 0.5 mg N/mL and minimum assay duration of 8 h is recommended for further investigation of conventional and novel feed ingredients for gadoid diets.