Enzyme Source Research Articles

Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex

Phycocyanobilin (PCB) is widely used in healthcare, food processing, and cosmetics. Escherichia coli is the common engineered bacterium used to produce PCB. However, it still suffers from low production level, precursor deficiency, and low catalytic efficiency. In this study, a highly efficient PCB-producing strain was created. First, chassis strains and enzyme sources were screened, and copy numbers were optimized, affording a PCB titer of 9.1 mg/L. Most importantly, the rate-limiting steps of the PCB biosynthetic pathway were determined, and the supply of precursors necessary for PCB synthesis was increased from endogenous sources, affording a titer of 21.4 mg/L. Then, the key enzymes for PCB synthesis, HO1 and PcyA, were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD, and 23.5 mg/L of PCB was obtained. Finally, the basic conditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB. The final titer of PCB reached 147.0 mg/L, which is the highest reported titer of PCB so far. This research provided the foundation for the industrial production of PCB and its derivatives.

Amanita sp. from subtropical region of Saudi Arabia as a source of chitinase enzyme and its antifungal activity

Mushroom products have been used as a biotechnological tool for many applications. Particularly, thermostable chitinase plays a vital role in biowaste management and biological control. In the present investigation, Amanita sp. was recorded in the subtropical region of Saudi Arabia, therefore, it was utilized for chitinase production using substrates chitin and dead fungal mycelia (DFM). Compared with the DFM, chitin was more suitable for chitinase activity at different temperatures and pH. Amanita sp. produced chitinase up to 70 °C, but the optimum was 50 °C. The chitinase activity was 4.98, 3.5, and 0.9 U.mg-1 with the use of chitin, while it was 4.6, 3.1, and 0.6 U.mg-1 with the use of DFM at 50, 60, and 70 °C, respectively. Chitinase activity was stable up to 60 °C, then it began to decrease at 70 °C. The chitinase activity was better at pH 4 and 5 than pH 8 and 9. The antifungal effect of the produced chitinase at 50 °C was more effective than at 60 °C. For instance, the Alternaria alternata colony radius was 3.50 cm and 2.26 cm at 50 °C while it was 4.35 cm and 4.13 cm at 60 °C when using DFM and chitin, respectively.

Marine-derived fungi as biocatalysts.

Marine microorganisms account for over 90% of ocean biomass and their diversity is believed to be the result of their ability to adapt to extreme conditions of the marine environment. Biotransformations are used to produce a wide range of high-added value materials, and marine-derived fungi have proven to be a source of new enzymes, even for activities not previously discovered. This review focuses on biotransformations by fungi from marine environments, including bioremediation, from the standpoint of the chemical structure of the substrate, and covers up to September 2022.

Salmon oyster mushroom: growth characteristics and protease production on a laboratory scale

Pleurotus have an easy cultivation and are excellent sources of proteolytic enzymes. The aimed was evaluate the mycelial growth and extracellular protease activity of Pleurotus ostreatoroseus. The mushroom was grown on potato dextrose agar (BDA) with yeast extract (YE) 0.5% (w/v). The radial mycelial growth velocity (RCV) and mycelial density occurred on different media containing 0.5% (w/v) YE: Potato Dextrose agar (BDA), GYP agar (glucose, yeast extract and peptone), malt extract agar (MEA), purple yam extract agar (EIR), torn yam extract agar (CEIN), aria extract agar (EA). Proteolytic activity was determined by the gelose block method. In mycelial growth (VCM) is used: cupuaçu exocarp (CC), açaí (Sac), pineapple (CsAb) and sawdust (SER), supplemented with rice bran (FA) or wheat bran (FT) were used. Proteases were extracted in sterile distilled water under 180 rpm agitation at 30 °C. Significant RCV was observed in MEA+YE (12.32 ± 0.10 mm/day). However, GYP agar showed strong mycelial density. Significant substrate mixture with strong mycelial density occurred on Sac+FA (0.61 ± 0.09 cm/day). The tested media were efficient in the production of extracellular proteases by this mushroom.

Improving chestnut physicochemical properties through fermentation – Development of chestnut Amazake

The increased awareness of population regarding the impact of consumption habits is leading to interest in new, innovative, diversified and health promoting foods. In this work, two new amazake fermented products were developed with chestnut (Castanea sativa Mill.), using rice or chestnut koji as source of glycolytic enzymes. The analysis of the amazakes evolution showed improvements in chestnuts physicochemical characteristics. The fermented products presented higher values of soluble protein, sugars, starches, antioxidant capacity, and similar values of ascorbic acid for chestnut koji amazake. The adhesiveness increased, which is related to the enhanced concentrations of sugars and starches. The evolution into less structured products was observed in the firmness followed by a consistent decrease of the viscoelastic moduli.The developed chestnut amazakes can represent a suitable alternative to traditional amazake, creating an opportunity for valorisation of chestnut industrial by-products, as new, tasty, and nutritive fermented products with potential functional characteristics.

Is the Phytotoxic Effect of Digestive Fluid of Nepenthes x ventrata on Tomato Related to Reactive Oxygen Species?

The digestive fluid of pitcher plants is a rich source of enzymes and secondary metabolites, but its impact on higher plant growth and development remains unknown. The aim of the study was to determine the phytotoxicity of the digestive fluid of the pitcher plant (Nepenthes x ventrata) on the germination of tomato (Solanum lycopersicum L.) seeds, elongation growth and cell viability of roots of tomato seedlings. The digestive fluid was collected from pitchers before feeding and four days after feeding; the pH and electrical conductivity of the fluid were determined. Undiluted and 50% fluids were used in the study. An inhibition of germination of tomato seeds, by around 30% and 55%, was respectively observed in 50% and 100% digestive fluids collected before and after feeding. Digestive fluid did not affect the root growth of tomato seedlings; a slight (6%) inhibition was only observed after the application of 100% digestive fluid from an unfed trap. The roots of the tomato seedlings treated with undiluted fluid were characterized by reduced cell viability. Reactive oxygen species (H2O2 and O2•-) were mainly localized in the root apex regardless of the used phytotoxic cocktail, and did not differ in comparison to control plants.

Biochemical and Kinetic Study for the Partial Purified Thioredoxin Reductase from Healthy Human Serum

The research included the separation of thioredoxin reductase (TrxR) from human blood serum using different biochemical techniques. Two proteinous peaks had been isolated by gel filtration using sephadex)G-50) and three proteinous peaks isolated from sephadex (G-100) that produced by ammonium sulphate precipitation (65%). The approximately molecular weight of the isolated protein as a source of enzyme using gel filtration chromatography (G-100) was (118059 + 925) Dalton.
 The results showed two peaks of enzyme when using ion exchange-type DEAE-cellulose and when using electrophoresis technique type SDS-PAGE indicated the presence of two bands from peak of separation column type sephadex G-100 and one band of each peak, resulting from the ion exchange process with molecular weights of 58231 +490 Dalton.
 The results showed that the optimum conditions of purified enzyme from human serum was at (200 µg/ml) of protein as a source of the enzyme using (0.4 mol/l) citric acid-Na2HPO4 buffer solution at pH (7) act for (25) minutes at (40°C). Using Line Weaver-Burk plot, the values of maximum velocity (Vmax) and Michaelis constant (Km) were found to be (0.182 µmol/ min) and (0.013 mol/l) respectively using 5,5-- dithio bis (2-nitrobenzoic acid) (DNTB) as a substrate. Finally, this was, also, involved the study of the effect of some chemicals and drugs on the enzyme activity. The results showed that gundinium - HCl is uncompetitive inhibitor, while bromoacetic acid is noncompetitive inhibitor and zinc sulphate is a competitive inhibitor for the enzyme at different concentrations of inhibitors, but dexathamazone was an activator to the enzyme.

Dynamics and thermodynamics of bimolecular reactions at heterogeneous biological interfaces.

The behaviors of biomolecules at heterogeneous biological aqueous-nonaqueous interfaces markedly differ from those in bulk water. We found that various biomolecular reactions, including protein unfolding, protein-ligan binding, chlorophyll demetallation, redox reactions, and crystallization, are accelerated by a factor of 103 or higher at the interface of micron-sized water droplets (microdroplets) (PNAS, 2015; QRevPhys, 2015 and 2017). The FRET analysis revealed that molecules are highly localized at the nanoscale regime near the water surface, which is in part responsible for the observed accelerated dynamics (JPCB, 2020). Besides, the thermodynamics of biomolecular reactions are also altered near the water surface. We found that molecules are in highly ordered states near the aqueous-nonaqueous interface (Sci Adv, 2020, Sci Rep, 2020). This minimized entropy lowers entropic barriers so that thermodynamically unfavorable reactions such as phosphorylation and RNA formation can proceed without any enzyme or ATP energy source at room temperature (PNAS 2017, 2018). Moreover, the charge exchange between water molecules and biomolecules spontaneously occurs at the water interface, leading to the oxidation of water molecules and the reduction of biomolecules in the Krebs cycle (JACS, 2019; Nat Commun, 2018; PNAS, 2018, 2019; Chem Sci, 2019). Vibrational Stark spectroscopy coupled with stimulated Raman excited fluorescence microscopy showed the presence of a strong intrinsic interfacial electric field formed at the surface of the microdroplet (JPCL, 2020). This strong electric field, combined with the partial desolvation effect, can be responsible for the unusual behaviors of biomolecules at the aqueous-nonaqueous interface (Annu Rev Phys Chem, 2020). These findings suggest that the dynamics and thermodynamics of biomolecular reactions occurring in cells with various aqueous-nonaqueous interfaces and compartments may significantly differ from those in bulk water.

황칠나무 추출물의 고초균 발효물로 제조된 가쓰오부시 단백가수분해물의 Lactobacillus plantarum 발효를 통한 고농도 GABA 생산

To develop a multi-functional ingredient, the bioconversion of katsuobushi protein was optimized using Bacillus subtilis HA and Lactobacillus plantarum KS2020. The Dendropanax morbiferus extract (DME) culture with protease activity (102 unit/mL) was prepared by B. subtilis with 2% glucose and 1% skim milk through one day of alkaline fermentation. Katsuobushi protein was effectively hydrolyzed by the DME culture at 60°C for 3 hours, resulting in a tyrosine content of 156.85 mg%. Subsequently, a second lactic acid fermentation was carried out with 10% monosodium glutamate (MSG) using L. plantarum KS2020 to produce higher levels of GABA. Following co-cultivation for three days, DME exhibited a pH of 8.3 (0% acidity). After seven days, the viable cell count of L. plantarum increased to 9.33 CFU/mL, but viable Bacillus cells were not detected. Taken together, a multi-functional ingredient with enriched GABA, peptides, probiotics, and umami flavor was developed through lactic acid fermentation using hydrolyzed katsuobushi protein. These results indicate that katsuobushi protein could be used as a byproduct to produce a palatable protein hydrolysate using alkaline-fermented DME culture as a proteolytic enzyme source.

Fish By-Products: A Source of Enzymes to Generate Circular Bioactive Hydrolysates.

Fish viscera are usually discarded as waste, causing environmental problems, or as low-value by-products. This study describes a self-sufficient and zero waste approach to obtain enzymes and protein hydrolysates from fish by-products. Firstly, recovery steps of viscera enzymatic extract were applied, and the resulting raw extract was stable at a pH range of 8-9 and at temperatures between 40 and 50 °C. The application of the extracted enzymes and alcalase on fish by-products hydrolysis was also determined. The selected conditions for the enzymatic hydrolysis were 10% (E/S) for 6 h using viscera enzymatic extract and 3% (E/S) for 2 h using alcalase. Fish protein hydrolysates (FPH) proved to have a notable antioxidant capacity with similar activity, ~11 mg ascorbic acid/g dry extract (ABTS assay) and ~150 mg Trolox/g dry extract (ORAC assay). FPH were also able to inhibit the angiotensin-converting enzyme, however, alcalase hydrolysates revealed a higher antihypertensive potential, IC50 of 101 µg of protein/mL. In general, FPH obtained by both enzymes systems maintained these bioactivities after the passage throughout a simulated gastrointestinal tract. The hydrolysates also displayed important technological properties, namely oil absorption capacity (~1 g oil/g sample) and emulsifying property (~40%). Therefore, it will be conceivable to use fish by-products based on a circular economy approach to generate added value compounds for animal and human nutrition.

Differences in Carbon Sequestration Ability of Diverse Tartary Buckwheat Genotypes in Barren Soil Caused by Microbial Action.

Planting plants to increase soil carbon input has been widely used to achieve carbon neutrality goals. Tartary buckwheat not only has good barren tolerance but is also rich in nutrients and very suitable for planting in barren areas. However, the effects of different genotypes of Tartary buckwheat roots and rhizosphere microorganisms on soil carbon input are still unclear. In this study, ozone sterilization was used to distinguish the sources of soil organic acids and C-transforming enzymes, and the contribution of root and rhizosphere microorganisms to soil carbon storage during the growth period of two genotypes of tartary buckwheat was studied separately to screen suitable varieties. Through the analysis of the experimental results, the conclusions are as follows: (1) The roots of Diqing tartary buckwheat have stronger carbon sequestration ability in a barren environment than Heifeng, and the microorganisms in Diqing tartary buckwheat soil will also increase soil carbon input. Therefore, Diqing tartary buckwheat is more suitable for carbon sequestration than Heifeng tartary buckwheat in barren soil areas. (2) In the absence of microorganisms, the rhizosphere soil of tartary buckwheat can regulate the storage of soil organic carbon by secreting extracellular enzymes and organic acids. (3) The structural equation model showed that to promote carbon sequestration, Heifeng tartary buckwheat needed to inhibit microbial action when planted in the barren area of Loess Plateau, while Diqing tartary buckwheat needed to use microbial-promoting agents. Adaptive strategies should focus more on cultivar selection to retain carbon in soil and to assure the tolerance of fineness in the future.

The Genues Trichoderma: Ecologicological Diversity, Taxonomic Classification and Its Agricultural, Human Health, Industrial and Environmental Applications

The genus Trichoderma is a diverse group of free-living fungi in the family Hypocreaceae (class Ascomycetes), that are living at different ecosystems in a wide range of climatic zones and that can be found all over the globe. The common ecological nich of the genus Trichoderma includes Natural soils, Decaying wood, Agricultural Habitats, Endophytes and mushroom related substrates. Trichoderma classified base on their morphological and cultural characteristics, conidiophore structure as well as the size of conidia. Now adays, molecularmethods including DNA fingerprinting and sequence analysis of multiple genes (ITS1- 5.8 rDNA-ITS2 and genes encoding translation-elongation factor 1-alpha (tef1) as well as some specific molecular databases have been used for taxonomic classification of this fungi. Trichodermahave been used agriculture biological control of plant diseases and as plant growth promoter, in industry as sources of cellulolytic and hemicellulolytic enzymes used in different industries, in human health producer of secondary metabolites that have clinical importance and in enviromt they are as a potential biodegrader of toxic compounds and soil bioremediation.Therefore, this review article presented the ecological diversiy, taxonomic classification and application of genus Trichoderma.

17β-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection.

The 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization.

Characterization and bioactive component analysis of filamentous bacterium XJ-16

Actinomycetes, which can produce a variety of bioactive compounds in the metabolic process, is one of the important sources of novel drugs, enzymes, anti-tumor drugs and enzyme inhibitors. It has been the focus of researchers to find and develop Actinomycetes with special characters. Strain XJ-16 is a blue alkali-resistant filamentous bacterium with high antimicrobial activity isolated from saline-alkali land of Xinjiang. Based on the classification, the enzyme production, metabolite antibacterial activity, and antibacterial substance isolation of XJ-16 were explored. which showed that XJ-16 belongs to the blue group of Streptomyces sp, and it can secrete cellulase, lipase, urease, protease, catalase and oxidase during metabolism. In addition, the bacteriostatic substance secreted by the strain XJ-16 showed inhibitory effects against both Gram-positive and Gram-negative bacteria, as well as the yeast Candida albicans. Then it was found that the bacteriostasis produced by XJ-16 has strong tolerance to acid, weak tolerance to alkali, and easy to be inactivated. After tested by HPLC, the retention time of antimicrobial substance was 13.261 min. This study provides new research ideas and theoretical support for searching for new antibacterial compounds and further developing the blue alkaline Actinomycete XJ-16.

Исследование особенностей культивирования молочнокислых бактерий в подсырной сыворотке и уфпермеате для получения β-галактозидаз

Lactic acid microorganisms are potential sources of the β-galactosidase enzyme, which can be used for the hydrolysis of lactose in the production of low- and lactose-free food products, as well as a catalyst in the biosynthesis of lactulose, galactooligosaccharides, and other valuable food additives. The need of lactic acid bacteria for the presence in the cultivation medium of additional sources of carbon, nitrogen, and other components significantly increases the cost of bacterial β-galactosidases. Lactose-containing raw materials, in particular whey and its UV permeate, are a promising and fairly cheap analogue of expensive complex media for the cultivation of lactic acid microorganisms. In this work, the features of cultivation of various types of lactic acid bacteria (Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus LGG, Lactococcus lactis ssp.) in different types of secondary milk raw materials (whey and UV permeate) were studied.. It has been shown that cheese whey is a more favorable medium for their growth than UV permeates. The highest concentration of lactic acid was obtained in experiments with L. acidophilus. To accelerate the growth of lactic acid bacteria in the permeate; it was proposed to use the addition of 2% peptone.

Bacillus cucumis: A New Source of L-Methioninase Enzyme with Potential for Cancer Therapy

Bacillus cucumis: A New Source of L-Methioninase Enzyme with Potential for Cancer Therapy

Activity of hydrolytic lipase enzyme synthesised by Aspergillus terreus isolated from petroleum-contaminated soil

Lipase enzyme-mediated processes have biological significance and tremendous potential in various industrial and technological areas due to the enzymatic properties and broad specificity for a wide spectrum of substrates. This research involved the production of hydrolytic lipase enzyme by Aspergillus terreus extracted from petroleum-contaminated soil and optimising the environmental parameters that may affect lipase production. Hydrolytic lipase enzyme activity of the isolated Aspergillus terreus from petroleum-contaminated soil was undertaken in submerged fermentation. Dry cell weight, lipase enzyme pellets, and lipase enzyme concentration by measuring the percentage transmission by spectrophotometry were measured to determine the effects of various abiotic factors (pH, temperature, carbon and nitrogen sources, lipid substances and vegetable oils) affecting cell growth and lipase enzyme production by Aspergillus terreus on modified lipase production media. The results showed a precipitate on Tween 20 and a bright pink-fluorescent halo on the rhodamine B agar plate. After temperature adjustment to 35 °C, statistical analysis showed that Aspergillus terreus produced a dry cell weight of 3.14 g/L and a lipase enzyme pellet of 0.33 g with a spectrophotometric transmission of 0.03%. The sucrose substrate displayed a dry cell weight of 3.11 g/L and a lipase enzyme pellet of 1.15 g. Spectrophotometric analysis showed that sucrose, glucose and peptone had a similarity in transmission at p = 0.321. The addition of Neem oil improved enzyme yield as a dry cell weight of 3.02 g/L and a lipase enzyme pellet of 0.95 g was obtained. Statistically, sunflower and tributyrin similarly affected enzyme production by showing a spectrophotometric transmission at p = 0.024. This study has indicated that Aspergillus terreus could be employed as a reliable source of lipase enzymes for industrial processes due to its stability across different abiotic factors.

Potential of mushroom bioactive compounds for use in pharmacology

Mushrooms contain many different biologically active compounds. The immunomodulatory action of mushroom β-glucans has been shown. Native liquids of mushroom cultivation can be sources of different valuable enzymes with significant potential for use in medicine and pharmacology.

Biodegradation and reduction of toxicity of Azo Trypan Blue dye by Amazonian strains of gasteroid fungi (Basidiomycota).

Amazonian strains of Cyathus spp. and Geastrum spp. were studied for the ability to discolor the trypan blue azo dye and reduce its toxicity. Discoloration of trypan blue dye (0.05%) was evaluated in solid and aqueous medium over different periods. The reduction of dye toxicity after treatment was assessed by seed germination and the development of lettuce seedlings (Lactuca sativa L.) and toxicity test in Artemia salina (L.) larvae. All evaluated strains showed the potential to reduce the color intensity of trypan blue dye. Cyathus strains reached 96% discoloration, and C. albinus and C. limbatus also reduced dye toxicity. Geastrum strains showed a high efficiency degree in color reduction, reaching 98% discoloration, however, the by-products generated during the process presented toxicity and require further investigation. For the first time, Amazonian strains of gasteroid fungi degrading trypan blue are reported, some even reducing its toxicity. Thus, making them promising sources of enzymes of interest to bioremediation scenarios involving synthetic dyes.

Isolation and Screening of Amylase Producing Bacteria from Soil and Production of Amylase

Amylase falls under class of hydrolytic enzymes catalysing breakdown of starch into monosaccharide units. There are various biological sources of enzymes viz. plants, fungi and bacteria. But the preferred one is microbial source due to ease of bulk production and stability of enzymes to work at extreme environmental conditions. This study reports isolation of amylase producing bacteria from sorghum cultivated farm soil and using them for production of amylase enzyme. During the course of the study three isolates viz. AP- 1, AP-2 and AP-3 were obtained which were identified as Bacillus cereus, Bacillus alvei and Bacillus licheniformis, respectively on the basis of morphological and biochemical characters. Amongst three isolates highest yield was obtained from Bacillus alvei which was 17.6 U/ml. Optimum conditions of pH, temperature and substrate concentration were determined for enzymatic activity. Crude enzyme samples obtained from all three isolates found to work best at 1.5% starch concentration and slightly acidic to neutral pH. The enzyme produced by B. alvei and B. cereus was found to be thermostable working best at 50 oC. In view of applications and demands of amylases in various fields further extension of study is needed for qualitative and quantitative enhancements