Enzyme Preparation Research Articles

Sprayed Aqueous Microdroplets for Spontaneous Synthesis of Functional Microgels.

The development of sustainable synthesis route to produce functional and bioactive polymer colloids has attracted much attention. Most strategies are based on the polymerization of monomers or crosslinking of prepolymers by enzyme- or cell-mediated reactions or specific catalysts in confined emulsions. Herein, a facile solution spray method was developed for spontaneous synthesis of microgels without use of confined emulsion, additional initiators/catalysts and deoxygenation, which addresses the challenges in traditional microgel synthesis. The polarization of air-water interface of the microdroplets can spontaneously split hydroxide ions in water to produce hydroxyl radicals, thereby initiating polymerization and crosslinking in air environment. This synthesis strategy is applicable to a variety of monomers and enables the fabrication of microgels with tunable chemical structures and variable sizes. Importantly, the synthesis route also allows for the preparation of enzyme- or drug-loaded microgels via the in-situ encapsulation, which also display high enzymatic activity and stimuli-triggered drug release. Therefore, this work not only is of great significance to macromolecular science and microdroplet chemistry, but also may bring new insights into cellular biochemistry and even prebiotic chemistry due to the prevalence of microdroplets in the environment.

Heterologous and High Production of Ergothioneine in Bacillus licheniformis by Using Genes from Anaerobic Bacteria.

This study aimed to utilize genetically engineered Bacillus licheniformis for the production of ergothioneine (EGT). Given the value of EGT and the application of Bacillus licheniformis in enzyme preparation production, we cloned the key enzymes (EanA and EanB) from Chlorbium limicola. Through gene alignment, new ergothioneine synthase genes (EanAN and EanBN) were identified and then expressed in Bacillus licheniformis to construct strains. Additionally, we investigated the factors influencing the yield of EGT and made a comparison with Escherichia coli. The relevant genes were cloned and transferred into Bacillus licheniformis. Fermentation experiments were conducted under different conditions for yield analysis, and the stability of this bacterium was also evaluated simultaneously. The constructed strains were capable of producing EGT. Specifically, the yield of the EanANBN strain reached (643.8 ± 135) mg/L, and its stability was suitable for continuous production. Genetically engineered Bacillus licheniformis demonstrates potential in the industrial-scale production of EGT. Compared with Escherichia coli, it has advantages, thus opening up new possibilities for the application and market supply of EGT.

Enzymatic Modification of Apple Pomace and Its Application in Conjunction with Probiotics for Jelly Candy Production

This study aimed to evaluate the applicational possibilities of enzymatically modified apple pomace (AP) in conjunction with probiotics as value-added ingredients for the production of jelly candies. AP was enzymatically modified with Pectinex® Ultra Tropical, Viscozyme® L, and Celluclast® 1.5 L (Novozyme A/S, Bagsværd, Denmark), and the soluble and insoluble dietary fibre content was determined using the Megazyme kit (Megazyme International Ireland Ltd., Wicklow, Ireland), reducing sugar content using the 3,5-dinitrosalicylic acid assay. The technological properties of the modified AP, such as its swelling capacity, water-retention capacity, oil-retention capacity, bulk density, and static and thermal emulsion stability, were evaluated. Enzymatically modified AP hydrolysed with Celluclast® 1.5 L was used for the production of jelly candies supplemented with Bifidobacterium animalis DSM 20105. The survival of probiotics in the jelly candies during in vitro digestion, the viability of probiotics during candy storage, and candy quality characteristics were analysed. Enzymatically modified AP had different carbohydrate compositions and technological properties, depending on the enzyme preparation used. Although the viability of probiotics in the jelly candies decreased during storage, a significantly higher viability of B. animalis was determined in jelly candies supplemented with hydrolysed AP compared with control candies made without AP after digestion in the saline, gastric, and intestine phases. This study shows that Celluclast® 1.5 L can be used for increasing the soluble dietary fibre in AP (18.4%), which can be further applied, in conjunction with B. animalis, for added-value jelly candy production.

Enzymatic and microbial preparations of neohesperidose from naringin and naringin present in orange (citrus sinensis) and mousami (citrus limetta) peel powders

Enzymatic and microbial preparations of neohesperidose from naringin and naringin present in orange (citrus sinensis) and mousami (citrus limetta) peel powders

The Regulatory Role of Pancreatic Enzymes in the Maintenance of Small Intestinal Structure and Enterocyte Turnover with Special Reference to Alpha Amylase.

The aim of this study was to elucidate the impact of porcine pancreatic enzymes (Creon® pancrelipase) in comparison to microbial-derived alpha amylase (MD amylase) on the small intestine wall structure, mucosal glycogen accumulation, and enterocyte turnover. The impact of enzyme supplementation on the small intestine was explored in 18 pigs with surgically induced exocrine pancreatic insufficiency (EPI). Four healthy pigs served as the control group. EPI led to reduced villus length, crypt depth, and thickness of the mucosa and muscularis layers compared to those of healthy pigs. All these changes appeared to be reversible after enzyme supplementation. Brush border thickness was decreased in EPI and increased with both enzyme preparations, with MD amylase treatment leading to the highest values in the proximal jejunum. No EPI-induced changes were observed in the goblet cell (GC) population, but significant increases in GC number and area were observed following MD amylase treatment. Glycogen accumulation within the duodenal mucosa was significantly increased in EPI pigs. EPI was also shown to significantly increase apoptotic activity and decrease proliferative activity in comparison to healthy animals, while both enzyme preparations resulted in the complete recovery of both proliferative and apoptotic activity in all investigated intestinal segments. Creon® influenced the morphology of the small intestine. However, supplementation of exogenous microbial amylase alone also affected gut morphology in a similar way to that of the complex host pancreatic enzymes offered orally. These data indicate that in addition to their role in digestion of nutrients in EPI, intraluminal pancreatic enzymes, especially amylase, contribute to gut health through maintenance of the intestinal wall architecture and physiological enterocyte turnover.

ГИДРОЛИЗУЮЩАЯ АКТИВНОСТЬ ЦГТаз БАКТЕРИЙ РОДА PAENIBACILLUS ПО ОТНОШЕНИЮ К α- И β-ГЛИКАНАМ

The cell wall of micromycetes is crucial for the shape and function of cells, as well as for their protection from environmental influences and contains up to 80-90 % of polysaccharides with different types of bonds. Cyclodextrin glucanotransferase microorganisms were tested and the following strains were selected Paenibacillus ehimensis IB-739, Paenibacillus illinoisensis IB-1087, Paenibacillus sp. IB-663, Paenibacillus macerans IB-I4. These strains were able to hydrolyze homopolysaccharides (starch, carboxyme-thylcellulose) and heteropolysaccharides (glucomannan, galactomannan). Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme capable of catalyzing various types of reactions: cyclization, binding, disproportionation and hydrolysis. It was found that strains and their preparations of CGTase, in addition to cyclizing activity, have a hydrolyzing ability against α- and β-glicans. The method of cluster analysis for strains revealed substrate preferences during hydrolysis in favor of α glicans (P. ehimensis IB-739, Paenibacillus sp. IB-663) or β-glicans (P. illinoisensis IB-1087, P. macerans IB-I4). The advantage of the preparative form of the CGTase enzyme when exposed to the components of the fungal cell wall (α-glicans and β-glicans) in comparison with the intact effect of cellular hydrolases is shown. The culture of P. macerans IB-I4, whose CGTase equally hydrolyzes all the presented polymers, turned out to be the most active in terms of the degree of destruction of glicans. The prospect of using CGTase enzyme preparations as agents in the fight against phytopathogenic fungi is discussed.

Determining patterns of lactose hydrolysis in liquid concentrates of demineralized whey

The object of this study was the process of milk sugar hydrolysis in liquid whey concentrates. The problem solved was the intensification of the lactose hydrolysis reaction in liquid whey concentrates through the use of a combination of leavening and enzyme preparations. Patterns in the lactose hydrolysis process in liquid concentrates of demineralized whey were studied. It was established that the use of an enzyme preparation for liquid whey concentrates does not make it possible to achieve a degree of lactose hydrolysis higher than 75–77 % within 10 h. The simultaneous use of enzyme and leavening preparations for 6 hours ensures the conversion of more than 95 % of lactose for concentrates with a mass fraction of dry substances of 10–30 % and more than 90 % for a 40 % concentrate. The rational duration of lactose hydrolysis in demineralized whey concentrates for the combination of preparations is 6 hours for 10 and 20 % concentrates, and 8 h for 30 and 40 % concentrates, which enables lactose hydrolysis at the level of 96.8–100 %. The rheological properties of concentrates with a solids content of 30 and 40 % indicate that these systems have a high ability to restore the structure. The dynamics of monosaccharide formation during hydrolysis are similar for 10 and 20 % concentrates, in which galactose slightly predominates the systems. Data on the increase in glucose content in fermented 30 and 40 % concentrates contradict the known data regarding the consumption of glucose by the acidophilic bacillus and its conversion to galactose. This may indicate suppression of the activity of the acidophilic bacillus under conditions of increased osmotic pressure in the concentrates. The results of the work could be used in the technology of whey ice cream, as well as dairy products that require adjustment of the chemical composition, primarily in terms of protein and lactose content

Economic justification for the use of new generation enzyme preparations in feeding broiler chickens

Economic justification for the use of new generation enzyme preparations in feeding broiler chickens

МЕТОДЫ ПОВЫШЕНИЯ ЭФФЕКТИВНОСТИ ВЫХОДА СОКА ИЗ ЯГОД ОБЛЕПИХИ

The purpose of the study is a comparative analysis of various pretreatment methods to improve the efficiency of juice yield from wild sea buckthorn berries. Tasks: to determine the yield of components from sea buckthorn berries – juice, seeds, oil with various pre-treatment methods; select enzyme preparations to increase the efficiency of sea buckthorn juice yield. The object of the study is the fruits of wild-growing sea buckthorn, harvested in the period of technological maturity in the Tomsk Region. The fruits of wild-growing sea buckthorn corresponded to GOST 33823-2-16. To determine the juice yield, sea buckthorn berries were pressed in a juicer. In order to increase the yield of juice, blanching and enzymatic processing methods were used. Sea buckthorn berries were placed in a combi steamer for 15 min at a temperature of 50 °C, and the juice yield was determined. Enzymatic treatment was carried out using enzyme preparations of pectolytic and cellulolytic action. Enzyme preparations were used – pectinase and cellulase. Pectinase (polygalacturonase) was used with an activity of 35 U/g, optimal pH 3.7–4.3, cellulase with an activity of 4000 u/ml, optimum pH 3.5–4.5. It has been established that blanching of berry raw materials increases the juice yield by 1.00 % compared with direct extraction of juice from sea buckthorn berries without preliminary pretreatment. Enzymatic treatment was carried out with pectolytic and cellulolytic enzymes at a temperature of 40 °C and a treatment time of 60 minutes. Treatment with enzymes of pectolytic action provided the highest yield of juice from sea buckthorn berries and amounted to 79.89 % when using the enzyme at a concentration of 0.1 %. The use of a cellulolytic enzyme ensured the highest yield of juice from sea buckthorn berries 74.64 % at an enzyme concentration of 0.15 %.

Биосинтез лактулозы с использованием ферментов лактозосбраживающих микромицет и бактерий

Lactulose is a well-studied prebiotic popular in medicine and the food industry. β-Galactosidases offer an alternative to traditional chemical methods of lactulose production. The article describes lactulose biosynthesis with crude enzyme preparations of β-galactosidases obtained by separate and joint cultivation of lactose-fermenting yeasts and lactic acid bacteria in whey permeate. The research featured the following producers of β-galactosidases: lactose-fermenting yeasts of Kluyveromyces lactis VKM Y-1333 and Y-1339, Kluyveromyces marxianus VKM Y-459 and Y-1338, and viscous strains of lactic acid bacteria of Lactobacillus acidophilus and Streptococcus thermophilis. The β-galactosidase activity was measured using the method of o-nitrophenyl-β-D-galactopyranoside hydrolysis. The method of high-performance liquid chromatography made it possible to reveal the carbohydrate composition. The lactulose formation by yeast enzymes demonstrated the following pattern: its concentration increased rapidly during the first 30 min of reaction with subsequent decrease or stabilization. Some combined unpurified enzyme preparations of yeasts and lactic acid microorganisms were more active than β-galactosidases obtained by separate cultivation. The patterns of lactulose formation depended on both the yeast strain and the type of lactic acid bacteria. The highest yields of lactulose belonged to the samples that combined β-galactosidases of all yeast strains with Streptococcus thermophilis. Co-cultivation of yeast and lactic acid bacteria in some combinations produced combined crude enzyme preparations with higher activity and greater lactulose yields than separate yeast cultivation. These results can help to obtain purified enzyme preparations of β-galactosidases and lactulose-containing products from secondary dairy raw materials.

Transcription Factors ClrB and XlnR and Their Effect on the Transcription of Cellulase Genes in the Filamentous Fungus Penicillium verruculosum.

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars.

Purification and physico-chemical properties of Bacillus atrophaeus protease with elastolytic and fibrinogenolytic activity

Microbial proteases, among which proteases capable of cleaving elastin, fibrin, fibrinogen, and collagen, have been a matter of interest to researchers due to their significant biotechnological potential along with low production cost. We previously showed that Bacillus atrophaeus 08 synthesizes an extracellular protease complex that exhibits high elastolytic, fibrinogenolytic, fibrinolytic activity, and minor caseinolytic and collagenase activity. The aim of the work was to isolate and purify the Bacillus atrophaeus 08 protease from the culture liquid supernatant and to study the physicochemical properties and substrate specificity of enzyme preparation. Precipitation with ammonium sulfate of 90% saturation, gel-permeation and ion-exchange chromatography were used in the experiment. According to the data obtained, the yield of the purified enzyme with a molecular weight of about 30 kDa was 6%, its elastase activity increased 30 times (420 U/mg protein), and fibrinogenolytic activity 31.8 times (350 U/mg protein). In addition, it also exhibited fibrinolytic (35.3 U/mg protein), minor caseinolytic activity (1.2 U/mg protein) and no collagenase activity. The optimum of elastin hydrolysis was at 37°C, pH 3.0 and 9.0-10.0, the optimum for fibrinogen hydrolysis was 12°C, pH 4.0. SDS-PAAG electrophoresis showed that the Bβ-chain of fibrinogen was almost not cleaved even after 1 h of incubation with the enzyme, while the Aα-chain disappeared already at the 30th min with the production of fragments with M.W. of about 30-45 kDa. The activity of the studied enzyme preparation towards fibrin was much lower than towards fibrinogen. Keywords: Bacillus atrophaeus 08, elastolytic and fibrinogenolytic activity, pH and thermooptimum, protease, substrate specificity

Solar Energy-Promoted Bisphenol A Degradation with Immobilized Laccase in an Fe3O4-Embedded Metal-Organic Framework.

Bisphenol A (BPA) is a well-recognized endocrine-disrupting chemical that poses risks to both human health and the environment. Laccase can effectively biodegrade bisphenol A, but the low environmental temperature (∼25 °C) restricts the biodegradation efficiency. In this study, the enzyme laccase and Fe3O4 with solar-thermal conversion capability were coimmobilized into zeolitic imidazolate framework-8 (Lac@ZIF-8-Fe3O4) to facilitate efficient biodegradation of bisphenol A under simulated solar irradiation. Compared to free laccase, Lac@ZIF-8-Fe3O4 exhibited high activity recovery (115.5%), an ∼39% increased catalytic constant, more effective bisphenol A biodegradation (up to 24-fold) at extensive bisphenol A concentrations (5-100 mg/L), excellent thermal stability (50 °C, 12 h), acid-tolerance (pH 3), and storage ability in 10 days. Simulated solar irradiation (1 kW/m2) increased the temperature of Lac@ZIF-8-Fe3O4 solution (10 μg laccase/mL) from 25 to 42.5 °C within 15 min, resulting in 96.4% biodegradation of bisphenol A within 60 min, nearly double the biodegradation efficiency under dark condition (55.9%). Furthermore, Lac@ZIF-8-Fe3O4 maintained 99.0% biodegradation efficiency even after 12 recycles of use under simulated solar irradiation (5 mg/L bisphenol A, 80 min/cycle). This work has thus offered efficient biocatalysis for integrating solar-energy promotion and enzymatic catalysis in treating environmental BPA pollutants. Further, the experimental findings benefited from the development of more sustainable and high-performance immobilized enzyme preparations for pollutant treatment via solar-thermal promotions.

The effects of different doses of compound enzyme preparations on the production performance, meat quality and rumen microorganisms of yak were studied by metagenomics and transcriptomics.

Yak (Bos grunniens) is a large ruminant endemic to the Tibetan plateau. The addition of enzyme complexes to feed can significantly improve their growth performance. Therefore, studying the effects of ruminant compound enzyme preparations dosage on yak rumen microorganisms and production performance is crucial to promoting the development of the yak industry. This study aimed to determine the effects of feeding yaks with different doses of ruminant enzyme compounds on the performance, meat quality, and rumen microorganisms of yaks. Three kinds of experimental diets with doses of 0.5 g/kg (LE group), 1 g/kg (ME group), and 2 g/kg (HE group) were selected to determine the growth index, meat quality, serum biochemical indexes, rumen fluid pH and other indexes of the three experimental groups. Metagenomics studies were used to investigate the differences in rumen microbial composition and function among yak groups, and transcriptome sequencing of the longest dorsal muscle was performed to reveal the expression of differential genes among different groups. It was determined that the levels of dietary enzyme complexes significantly affected growth performance, rumen fluid pH, and serum biochemical indices. At the phylum level, the dominant phylum in all three treatment groups was Bacteroidota, Bacillota, Kiritimatiellota, and Pseudomonadota. At the genus level, Prevotella, Methanobrevibacter, Oscillibacter. Fibrobacter showed statistically significant differences in abundance (p < 0.05). CAZymes family analysis revealed significant differences in GHs, CTs, and CEs among the three groups. Genome-wide differential gene expression in the longest muscle of the yak back was analyzed by RNA-seq between the three experimental groups. Some DEGs were found to be enriched in the ECM, PI3K-Akt, PPAR, and protein digestion and absorption receptor pathways. Combined metagenomics and transcriptomics analyses revealed that some microorganisms were significantly associated with the genes COL11A1, POSTN, and PTHLH, which are involved in growth metabolism. In summary, this study investigated the effects and interrelationships of ruminant complex enzymes on yak performance, meat quality, and rumen environment. The results of this study provide a scientific basis for adding ruminant enzymes to yaks.

Free amino acids accelerate the time-dependent inactivation of rat liver nucleotide pyrophosphatase/phosphodiesterase Enpp3 elicited by EDTA

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

Semi-rational design in simultaneous improvement of thermostability and activity of β-1,3-glucanase from Alkalihalobacillus clausii KSM[sbnd]K16

Endo-β-1,3-glucanase (β-1,3-GA) is a key enzyme capable of acting on the β-1,3-glycosidic bond of β-1,3-glucan, resulting in the production of β-1,3-gluco-oligosaccharides with higher water solubility. Higher temperatures are beneficial for curdlan hydrolysis; however, low enzymatic activity and thermal stability limit their applicability. In this study, a mutant library of Endo-β-1,3-glucanase (AC-GA) derived from Alkalihalobacillus clausii KSM-K16 was constructed by a semi-rational design using amino-acid-based multiple sequence alignment and protein structure-based computer-aided engineering. The best combination mutant (S52T/M120L) was screened through ordered recombination mutations, which showed a 24.88 % increase in specific enzyme activity over the wild-type. The melting temperature (Tm) value, an enzyme protein denaturation temperature, was raised to 82.99 °C from 78.60 of the wild type. In comparison, the Km for hydrolysis of curdlan by S52T/M120L was reduced by 12.1 %, while the kcat was increased by 59.39 %, thus leading to a higher catalytic efficiency (kcat/Km, 227.73 vs 125.46 mL·s−1·mg−1). Molecular dynamics (MD) simulations showed that mutations resulted in a reduction in the overall flexibility of the enzyme, an increase in rigidity, and a more stable structure. An increase in the hydrophobic network at the entrance of the substrate increases the accessibility of the substrate to the enzyme, resulting in increased enzyme activity. High-efficiency mutants have potential industrial applications in the enzymatic preparation of β-1,3-gluco-oligosaccharides.

Tyrosinases: a family of copper-containing metalloenzymes

Tyrosinases (TYRs) are a family of copper-containing metalloenzymes that are present in all domains of life. TYRs catalyze the reactions that start the biosynthesis of melanin, the main pigment of the animal kingdom, and are also involved in the formation of the bright colors seen on the caps of mushrooms and in the petals of flowers. TYRs catalyze the ortho-hydroxylation and oxidation of phenols and the oxidation of catechols to the respective o-quinones. They only need molecular oxygen to do that, and the products of TYRs—o-quinones—are highly reactive and will usually react with the next available nucleophile. This reactivity can be harnessed for pharmaceutical applications as well as in environmental and food biotechnology. The majority of both basic and applied research on TYRs utilizes “mushroom tyrosinase”, a crude enzyme preparation derived from button mushroom (Agaricus bisporus) fruiting bodies. Access to pure TYR preparations comes almost exclusively from the production of recombinant TYRs as the purification of these enzymes from the natural source is usually very laborious and plagued by low yields. In this text an introduction into the biochemistry of the enzyme TYR will be given, followed by an overview of available structural data of TYRs, the current model for the catalytic mechanism, a survey of reports on the recombinant production of this important metalloenzyme family, and a review of the applications of TYRs for the synthesis of catechols, as biosensors, in bioremediation, for the cross-linking of proteins and medical hydrogels as well as for melanoma treatment.Graphical

Effects of Bacterial Enzyme Cooperative Fermentation Diet on Growth Performance, Blood Biochemical Indices, and Fecal Microflora of Growing–Finishing Pigs

This research utilized Fourier transform infrared (FTIR) spectroscopy to analyze and discuss the molecular structure of pig diets, aiming to provide new insights into the application of fermented feeds in livestock and poultry production. Moreover, the impacts of the fermented diet on growth performance, apparent digestibility, blood biochemical indices, and fecal microorganisms at different stages of pig fattening were also explored. Forty-eight pigs (Duroc × Landrace × Large white three-way hybrid) with a mean body weight of 16.55 ± 3.88 kg were randomly divided into three groups with four replicates per group and four pigs per replicate. The control group was fed a basal diet. The pigs in the fermented diet group (T1) were fed Pediococcus acidilactici (PA), Lactobacillus reuteri (LR), and Bacillus velezensis (BS) (ratio of 1:1:1) at a 6% inoculation dose. The pigs in the cooperative fermentation group (T2) were fed 6% PA, LR, BS, and a 0.2% compound enzyme preparation. The T1 and T2 diets were fermented with 45% water at 33 °C for 48 h. The pre-feeding period lasted 7 days, and the experimental period lasted 84 days. The experimental results showed that the bacterial enzyme cooperation fermentation process significantly increased the contents of crude protein, calcium, and phosphorus in the diet; increased the area of amide Ⅰ region; increased the apparent digestibility of neutral detergent fiber and phosphorus; significantly increased average daily gain; and decreased the feed-to-gain ratio in the late fattening and growth period. During the whole experiment, the serum concentrations of total protein and immunoglobulin A were significantly increased, the serum concentrations of superoxide dismutase and glucose were decreased, and the diversity and richness of fecal microorganisms were increased. These results show that the bacterial enzyme cooperative fermentation diet can improve the apparent digestibility of nutrients and improve overall health by increasing the area of amide Ⅰ region.

Enzyme Engineering: Performance Optimization, Novel Sources, and Applications in the Food Industry.

This review summarizes the latest progress in enzyme preparation, including enzyme design and modification technology, exploration of new enzyme sources, and application of enzyme preparation in food processing, detection, and preservation. The directed evolution technology improved the stability and catalytic efficiency of enzymes, while enzyme immobilization technology enhanced reusability and industrial applicability. Extremozymes and biomimetic enzymes exhibit excellent performance under harsh conditions. In food processing, enzyme preparation can improve food quality and flavor. In food detection, enzymes combined with immune detection and biosensors realize rapid detection of allergens, pollutants, and pesticide residues. In food preservation, enzymes enhance food quality by extending shelf life and inhibiting microbial growth. In the future, enzyme engineering will be combined with computer-aided design, artificial intelligence, and new material technology to promote intelligent enzyme design and multifunctional enzyme preparation development and help the technological upgrading and sustainable development of the food industry and green chemistry.

Effect of enzyme preparation Hemicell® HT on the metabolism of young geese

Effect of enzyme preparation Hemicell® HT on the metabolism of young geese