A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10−3 mM s−1), and Kcat/Km (763.92 mM−1 s−1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

Flavonoids as Human Intestinal α-Glucosidase Inhibitors

Certain flavonoids can influence glucose metabolism by inhibiting enzymes involved in carbohydrate digestion and suppressing intestinal glucose absorption. In this study, four structurally-related flavonols (quercetin, kaempferol, quercetagetin and galangin) were evaluated individually for their ability to inhibit human α-glucosidases (sucrase, maltase and isomaltase), and were compared with the antidiabetic drug acarbose and the flavan-3-ol(−)-epigallocatechin-3-gallate (EGCG). Cell-free extracts from human intestinal Caco-2/TC7 cells were used as the enzyme source and products were quantified chromatographically with high accuracy, precision and sensitivity. Acarbose inhibited sucrase, maltase and isomaltase with IC50 values of 1.65, 13.9 and 39.1 µM, respectively. A similar inhibition pattern, but with comparatively higher values, was observed with EGCG. Of the flavonols, quercetagetin was the strongest inhibitor of α-glucosidases, with inhibition constants approaching those of acarbose, followed by galangin and kaempferol, while the weakest were quercetin and EGCG. The varied inhibitory effects of flavonols against human α-glucosidases depend on their structures, the enzyme source and substrates employed. The flavonols were more effective than EGCG, but less so than acarbose, and so may be useful in regulating sugar digestion and postprandial glycaemia without the side effects associated with acarbose treatment.

An Apodictic Review on Recent Approaches in Enzyme Technology

Enzymes are the most powerful biochemical moieties, predominantly the working tools in all living systems. Many studies have revealed the usage of various enzymes even in the pre-historical periods. Enzymes are known to be the extremely active biocatalyst that is widely involved in many metabolisms. Living systems explore these biomolecules for their metabolism and are exhaustively explored for various industrial and clinical applications. Due to the increasing need for enzyme-based products, various recent research focuses on exploring distinct enzymes & enzyme sources with relatively enhanced characteristics. The elegant motive of this review is to enable the readers and enzyme researchers to compend the basics of enzymes, explore the enormous recent clinical & industrial applications of enzymes like amylase, cellulase, protease, lipase, and esterase. And also, the review highly emphasizes the various enzyme source and their enriched properties like enzyme activity, annotated by recent research works carried out by various research teams across the globe. The review also accentuates the recent advancements in production technologies and high throughput activity prediction assays for the above-mentioned industrially important enzymes.

Lignolytic Enzyme Activity of Isolated Bacteria from Termite (Coptotermes Sp.) and Milkfish (Chanos chanos Forsskal, 1775) Guts

Bacteria BSR 2, Pseudomonas alcaligenes (BSR 3), Brevibacillus parabrevis (BSR 8), Brevibacillus sp. (BSR 9), isolated from termite gut and Bacillus licheniformis (BSA B1) isolated from milkfish gut have been known to possess celluloytic activity. However, their lignolytic ability has not been known. This study aimed to determine the lignolytic ability of bacteria isolated from termit (Coptotermes sp.) and milkfish (Chanos chanos Forsskal, 1775) guts and their enzymes characterization. The qualitative test was done through the spot test method, while quantitative assay was performed spectrophotometrically at 335 nm to calculate vanillin concentration. The isolates were grown in Lignin Mineral Medium, then the optical density (OD620) were measured every 24 hours for 5 days using spectrophotometer to determine their growth profile and the best isolation time of the lignolytic enzyme. Based on results, the best lignolytic enzyme isolation time for strains Bacillus licheniformis (BSA B1) and BSR 2 were 5 days, yielding lignolytic enzyme activity of 0.961 ± 0.168 U/mg and 2.176 ± 0.088 U/mg respectively,  while strains Pseudomonas alcaligenes (BSR 3), Brevibacillus parabrevis (BSR 8), and Brevibacillus sp. (BSR 9) were 4 days, yielding of 1.206 ± 0.045 U/mg, 1.162 ± 0.191 U/mg, and 0.896 ± 0.108 U/mg, respectively. The strain BSR 2 showed the highest lignolytic activity compared to other strains. The optimum temperature for lignolytic enzyme activity of BSR 2 was 30 ℃ and the optimum pH was 7. The lignolytic enzyme activity showed that these bacterial isolates can be a chance to be used as new alternative lignolytic enzyme source in commercial bioconversion process.

An overview of the solid state fermentation in the production of fungal protease enzymes

Enzymes are among the most important products obtained for human needs through microbial sources. A large number of industrial processes in the area of industrial, environmental and food technology utilize enzymes at some stage or another; Solid State Fermentation (SSF) holds tremendous potential for the production of enzymes, especially in those processes where the crude fermented product may be used directly as the enzyme source. Fungal proteases are used in many industrial processes for the production of foods and metabolites, production of enzymes from fungi offered many advantages which include low cost and high productivity. Hence because of the higher yielding capacity of SSF and the demand for proteases it highly imperative to search for novel microorganisms from possible environment and subject them to SSF for protease investigation to add up to the nation need of the enzymes and boast economy.

Production of protein hydrolysates using marine catfish Bagre panamensis muscle or casein as substrates: Effect of enzymatic source and degree of hydrolysis on antioxidant and biochemical properties.

Protein hydrolysates from fishery by-products, have resulted to be nutraceutical ingredients with potential to be applied in human nutrition; however, critical quality attributes are dependent of some process parameters such as enzyme source and degree of hydrolysis. This study reports the biochemical properties and antioxidant activity of protein hydrolysates at 10, 20 and 30% degree of hydrolysis (DH), prepared from sea catfish muscle and casein by treatment with alcalase (ALC) and a semi-purified protease extract (SPE) from intestinal tissues of sea catfish (Bagre panamensis). With SPE, the DH was reached faster than ALC regardless the protein substrate used. Sea catfish muscle (MUSC) hydrolysate made with SPE at 30% DH showed the highest antioxidant activity (DPPH: 118.8 µmoles TE/mg; ABTS: EC50 of 1.5 mg/mL). In FRAP assay, the MUSC hydrolysates produced with SPE or ALC at 20% DH showed the higher activity (0.38 and 0.40 µmoles TE/mg, respectively). MUSC hydrolysates made with SPE, contained the highest proportion of peptides with MW < 1.35 kDa, high protein content (72 to 78%,) and almost 50% of amino acids were essential. These results, suggest that intestinal proteases and muscle of marine catfish represents a potential source to elaborate antioxidant protein hydrolysates. Our results promote the full utilization of this fish species and offering a biotechnological strategy to the management and valorization of its byproducts.

Influence of temperature and germination time on diastatic power of blue and red maize (Zea mays L.) malts

Maize endosperm consists of about 70 % starch, which makes it an excellent substrate for fermentation. However, due to its low diastatic power, it is used in brewing as an adjunct, mainly. In order to include both red and blue maize as an enzyme source in the brewing process, the effect of temperature and time germination on the diastatic power of malts was studied. The research consisted in a completely randomized three-factor experimental design where the involved factors were colour of maize (blue and red), germination temperature (15, 20, and 25 °C), and germination time (3, 4, 5, 6, 7, 8, and 9 days). The response variables were germination percentage, acrospire length, malting yield, and diastatic power. Data was analysed through Analysis of variance and Comparison Multiple Tukey’s Test. Results showed that both temperature and germination time encouraged the acrospire length, which had a negative effect on malting yield. Regarding to diastatic power, it maintained an increase from third to seventh germination day, at the three tested temperatures. Additionally, as the germination temperature increased, the diastatic power also increased. The highest diastatic power for blue and red maize malts were 39 and 42 °L, respectively, and it was reached when these malts were germinated for 7 days at 25 °C. It was concluded that, by germinating both blue and red maize under the resulting optimum conditions, the obtained malts would be capable of converting their own starch.