Enzyme Biotechnology Research Articles

Discovery of alkaline laccases from basidiomycete fungi through machine learning-based approach

BackgroundLaccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods.ResultsThis study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method.ConclusionsThis study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications.Graphical

The inorganic pyrophosphatases of microorganisms: a structural and functional review

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure–function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.

Komagataella phaffii (Pichia pastoris) as a Powerful Yeast Expression System for Biologics Production

Komagataella phaffii (K. phaffii) (Pichia pastoris), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered significant interest as a platform for the production of recombinant proteins. Numerous benefits include effective secretory expression that facilitates the easy purification of heterologous proteins, high cell density with rapid growth, post-translational changes, and stable gene expression with integration into the genome. In the last thirty years, K. phaffii has also been refined as an adaptable cell factory that can produce hundreds of biomolecules in a laboratory setting and on an industrial scale. Indeed, over 5000 recombinant proteins have been generated so far using the K. phaffii expression method, which makes up 30% of the total cell protein or 80% of the total released protein. K. phaffii has been used to manufacture more than 70 commercial products in addition to over 300 industrial processes that have been granted licenses. Among these are useful enzymes for industrial biotechnology, including xylanase, mannanase, lipase, and phytase. The others are biopharmaceuticals, which include human serum albumin, insulin, hepatitis B surface antigen, and epidermal growth factor. Compared to other expression systems, this yeast is also considered a special host for synthesizing subunit vaccines, which have recently been supplanted by alternative vaccination types, such as inactivated/killed and live attenuated vaccines. Moreover, efficient production of recombinant proteins is achieved through multi-level optimization methods, such as codon bias, gene dosage, promoters, signal peptides, and environmental factors. Therefore, although K. phaffii expression systems are efficient and simple with clearly established process procedures, it is still necessary to determine the ideal conditions since these vary depending on the target protein to ensure the highest recombinant protein generation. This review addresses the K. phaffii expression system, its importance in industrial and biopharmaceutical protein production, and some bioprocessing and genetic modification strategies for efficient protein production. K. phaffii will eventually continue contributing as a potent expression system in research areas and industrial applications.

Harnessing Nature's Toolbox: Protease Enzymes in Biotechnology and Industrial Applications

Harnessing Nature's Toolbox: Protease Enzymes in Biotechnology and Industrial Applications

Antagonistic and enzymatic activities of Bacillus species isolated from the fish gastrointestinal tract as potential probiotics use in Artemia culture.

Probiotics have been used successfully in aquaculture to enhance disease resistance, nutrition, and/or growth of cultured organisms. Six strains of Bacillus were isolated from the intestinal tracts of fish and recognised by conventional biochemical traits. The six isolated strains were Bacillus cereus and Bacillus subtilis using MALDI-TOF-MS technique. The probiotic properties of these Bacillus strains were studied. The tested bacillus strains exhibit antibacterial activity against the different pathogens. The strain S5 gave the important inhibition zones against most pathogens (20.5, 20.33, 23, and 21 mm against Vibrio alginolyticus, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella typhimurium, respectively). According to our results, all Bacillus strains have extracellular components that can stop pathogenic bacteria from growing. The enzymatic characterization showed that the tested strains can produce several biotechnological enzymes such as α-glucosidase, naphtol-AS-BI-Phosphohydrolase, esterase lipase, acid phosphatase, alkaline phosphatase, amylase, lipase, caseinase, and lecithinase. All Bacillus strains were adhesive to polystyrene. The adding Bacillus strains to the Artemia culture exerted significantly greater effects on the survival of Artemia. The challenge test on Artemia culture showed that the protection against pathogenic Vibrio was improved. These findings allow us to recommend the examined strains as prospective probiotic options for the Artemia culture, which will be used as food additives to improve the culture conditions of crustacean larvae and marine fish.

Preparation, characterization of upconverting nanoparticles for uricase immobilization and controlled manipulation of uricase activity by near-infrared light

In recent years, studies on the external stimulation of biotechnological enzyme drugs and their use in the treatment of diseases have gradually increased. Herein, for the first time, the near-infrared (NIR) was used as an external stimulant to manipulate the catalytic activity of the uricase (UOx) enzyme, which is used in the treatment of hyperuricemia, in a controlled manner. For this purpose, NaYF4: Yb3+, Er3+ upconverting nanoparticles (UCNPs) were synthesized by hydrothermal synthesis method and functionalized with diethylaminoethyldextran (DEAE-D) to facilitate UOx immobilization. The obtained materials were characterized in detail by various methods to confirm the preparation of UCNPs and immobilization of UOx. In addition, the biochemical parameters such as optimum pH, optimum temperature, thermal stability, and reusability were preliminarily investigated for free UOx and NaYF4: Yb3+, Er3+/DEAE-D/UOx. Moreover, a sequential experimental method was monitored to assess the effects of NIR excitation intensity, induction distance, and exposure time on the UOx activity. While the optimum pH value was found to be 6.0 for both enzyme forms, the optimum temperature value was recorded as 45 and 50 °C for free UOx and NaYF4: Yb3+, Er3+/DEAE-D/UOx, respectively. The activation energy (Ea) values of free UOx and NaYF4: Yb3+, Er3+/DEAE-D/UOx were calculated to be 7.59 and 2.98 kJ/mol, respectively, implying that the NaYF4: Yb3+, Er3+/DEAE-D/UOx was less temperature sensitive. After thermal incubation for 3 h at 55 °C, the NaYF4: Yb3+, Er3+/DEAE-D/UOx retained 54.68 % of its initial activity, while the free UOx retained 32.94 % of its initial activity at 50 °C. In addition, the findings from the reusability experiments revealed that NaYF4:Yb3+, Er3+/DEAE-D/UOx retained 57.94 % of its initial activity even after 10 reuse cycles. The most striking point in this study was the positive manipulation of UOx activity by NIR. Accordingly, it was observed that when the NIR power was 1500 mW, the UOx activity increased about 2 times compared to the control. Additionally, the UOx activity increased in parallel with the increase in NIR application time and the ideal application distance was 3 cm. In conclusion, this pioneering study provides valuable insights into the controlled manipulation of enzyme activity, showcasing the effectiveness of NIR in enhancing enzyme activity. The outcomes suggest that NIR holds great promise as an efficient, sustainable, and versatile approach applicable to various enzymatic catalysis scenarios.

Evaluation of Heavy Metal Concentrations in Enzyme Embedded Poly Lactic Acid (EPLA) by ICP-OES: Lipase from Candida Rugosa.

Abstract: Enzymes like lipases are essential for catalyzing important reactions in the fields of biotechnology and industrial operations. For quality control and safety evaluations, however, the quantification of these contaminants is crucial because the presence of heavy metals in enzyme preparations can negatively affect their activity and stability. Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), a key component of this investigation, is used to measure the levels of heavy metals in lipase enzyme preparations embedded in polylactic acid (PLA). The lipase enzyme being studied comes from Candida rugosa, which is well-known for its considerable industrial applications. According to preliminary findings, the lipase enzyme is successfully protected from external heavy metal pollutants by the encapsulating technique, maintaining its enzymatic activity and stability. The lipase-PLA composite had only minimal quantities of heavy metals, according to the ICP-OES study. The research's conclusions show great potential for the creation of reliable and contaminant-free lipase enzyme preparations, which will increase their suitability for usage in a variety of industrial processes and encourage the use of biodegradable polymers, supporting environmental sustainability. By highlighting the significance of quality control and safety assessment through the determination of heavy metal concentrations, this research contributes to the larger field of enzyme biotechnology. It also highlights the potential of Candida Rugosa lipase implanted in PLA matrices for eco-friendly and sustainable biocatalytic applications in sectors ranging from food and pharmaceuticals to biofuel generation and other biodegradable polymers.

Exploring the Hot Springs of Golan: A Source of Thermophilic Bacteria and Enzymes with Industrial Promise.

In recent years, there has been a surge in research on extremophiles due to their remarkable ability to survive in harsh environments. Extremophile thermophilic bacteria provide thermostable enzymes for biotechnology and industry. Thermophilic bacteria live in extreme environments like hot springs at 45-80°C. This study screens and isolates thermophilic bacteria and thermozymes from the Golan hot springs in Karakocan, Elazig, Turkey. The study also characterizes thermophilic bacteria and their thermozymes to understand their features and applications better. Golan hot spring water samples at 50°C yielded 12 isolates. GKE 02, 07, 08, and 10 produce amylase, GKE 04, 08, and 11 cellulase, and GKE 06 xylanase. One isolate (GKE 08) displayed both amylolytic and cellulolytic activity on agar plates. GKE 02 had the highest plate assay amylolytic index (2.3) and amylase activity (67.87 U/ml). Plate assay indicates GKE 08 has 1.5 amylolytic index, 1.1 cellulolytic index, 38.57 U/ml amylase, and 6.81 U/ml cellulase. GKE 04 had the greatest cellulolytic index (1.7) and cellulase activity (27.46). GKE 06, the only xylanase producer, has 19.67 U/ml activity and 1.4 plate assay index. The investigation also included determining the optimal pH and temperature conditions for each enzyme. 16S rDNA gene sequencing revealed seven thermozyme-producing bacteria Bacillus, Geobacillus, and Thermomonas. Thermomonas hydrothermalis genome annotation showed glycosyl hydrolase genes for amylolytic and cellulolytic activity. The findings of this study on thermophilic bacteria and thermostable enzyme synthesis in the Golan hot springs are promising, particularly for T. hydrothermalis, which has limited research on its potential as a thermozyme producer.

Elucidation of the catalytic mechanism of a single-metal dependent homing endonuclease using QM and QM/MM approaches: the case study of I-PpoI.

Homing endonucleases (HEs) are highly specific DNA cleaving enzymes, with I-PpoI having been suggested to use a single metal to accelerate phosphodiester bond cleavage. Although an I-PpoI mechanism has been proposed based on experimental structural data, no consensus has been reached regarding the roles of the metal or key active site amino acids. This study uses QM cluster and QM/MM calculations to provide atomic-level details of the I-PpoI catalytic mechanism. Minimal QM cluster and large-scale QM/MM models demonstrate that the experimentally-proposed pathway involving direct Mg2+ coordination to the substrate coupled with leaving group protonation through a metal-activated water is not feasible due to an inconducive I-PpoI active site alignment. Despite QM cluster models of varying size uncovering a pathway involving leaving group protonation by a metal-activated water, indirect (water-mediated) metal coordination to the substrate is required to afford this pathway, which renders this mechanism energetically infeasible. Instead, QM cluster models reveal that the preferred pathway involves direct Mg2+-O3' coordination to stabilize the charged substrate and assist leaving group departure, while H98 activates the water nucleophile. These calculations also underscore that both catalytic residues that directly interact with the substrate and secondary amino acids that position or stabilize these residues are required for efficient catalysis. QM/MM calculations on the solvated enzyme-DNA complex verify the preferred mechanism, which is fully consistent with experimental kinetic, structural, and mutational data. The fundamental understanding of the I-PpoI mechanism of action, gained from the present work can be used to further explore potential uses of this enzyme in biotechnology and medicine, and direct future computational investigations of other members of the understudied HE family.

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation.

Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in the amy13E gene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis of amy13E found that this gene was essential for kojibiose and isomaltose metabolism in C. japonicus. Interestingly, a Δamy13E mutant was not deficient for cyclodextrin or pullulan utilization in C. japonicus; however, heterologous expression of the gene in E. coli was sufficient for cyclodextrin-dependent growth. Biochemical analyses found that CjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of the CjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications.IMPORTANCEUnderstanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Production of Mono-Hydroxylated Derivatives of Terpinen-4-ol by Bacterial CYP102A1 Enzymes.

CYP102A1 from Bacillus megaterium is an important enzyme in biotechnology, because engineered CYP102A1 enzymes can react with diverse substrates and produce human cytochrome P450-like metabolites. Therefore, CYP102A1 can be applied to drug metabolite production. Terpinen-4-ol is a cyclic monoterpene and the primary component of essential tea tree oil. Terpinen-4-ol was known for therapeutic effects, including antibacterial, antifungal, antiviral, and anti-inflammatory. Because terpenes are natural compounds, examining novel terpenes and investigating the therapeutic effects of terpenes represent responses to social demands for eco-friendly compounds. In this study, we investigated the catalytic activity of engineered CYP102A1 on terpinen-4-ol. Among CYP102A1 mutants tested here, the R47L/F81I/F87V/E143G/L188Q/N213S/E267V mutant showed the highest activity to terpinen-4-ol. Two major metabolites of terpinen-4-ol were generated by engineered CYP102A1. Characterization of major metabolites was confirmed by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy (NMR). Based on the LC-MS results, the difference in mass-to-charge ratio of an ion (m/z) between terpinen-4-ol and its major metabolites was 16. One major metabolite was defined as 1,4-dihydroxy-p-menth-2-ene by NMR. Given these results, we speculate that another major metabolite is also a mono-hydroxylated product. Taken together, we suggest that CYP102A1 can be applied to make novel terpene derivatives.

Multifaceted personality and roles of heme enzymes in industrial biotechnology.

Heme enzymes are the most prominent category of iron-containing metalloenzymes with the capability of catalyzing an astonishingly wide range of reactions like epoxidation, hydroxylation, demethylation, desaturation, reduction, sulfoxidation, and decarboxylation. Various enzymes in this category are P450s, heme peroxidases, catalases, myoglobin, cytochrome C, and others. Besides this, the natural promiscuity and amenability of these enzymes to protein engineering and evolution have also added several non-native reactions such as C-H, N-H, S-H insertions, cyclopropanation, and other industrially important reactions to their capabilities. Surprisingly, all of these reactions and their wide substrate scopes are attributed to changes in the active site scaffold of different heme enzymes as the center of all enzymes is constituted by a porphyrin ring containing iron. Multiple prominent research groups across the world, including 2018, Nobel Laureate Frances Arnold's group, have shown keen interest in engineering and evolving these enzymes for utilizing their industrial potential. Besides engineering the active site, researchers have also explored the possibility of these enzymes catalyzing non-native reactions by replacing the center porphyrin ring with other cofactors or by changing the iron in the porphyrin ring with other metal ions along with engineering the active site and thereby creating novel artificial metalloenzymes. Thus, in this mini-review from our group, for the first time, we are trying to catalog various activities catalyzed by heme enzymes and their engineered variants and their active usage in various industries along with shedding light on their potential for use in various applications in the future.

FACTORS ON THE BIOSYNTHESIS OF AMYLOLITIC ENZYMES OF STREPTOMYCETE ORIGIN

Modern enzyme biotechnology is a promising and rapidly developing field that requires the latest research on the conditions of enzyme biosynthesis. Optimizing the composition of the nutrient medium depending on the needs of microorganisms and physicochemical factors directly affect the increase in the efficiency of the biosynthesis of amylolytic enzymes, namely the biosynthetic capacity of the strain Streptomyces recifensis var. lyticus 2P-15. Modulation of the biosynthetic activity of strains producing amylolytic enzymes will allow to significantly increase their economic yield. Aim. The purpose of this work is to optimize the biosynthetic capacity of the strain Streptomyces recifensis var. lyticus 2P-15 in terms of the synthesis of amylolytic enzymes and the study of the dynamics of the influence of physical and chemical factors on optimization. Methods. The object of the study is the strain Streptomyces recifensis var. lyticus 2P-15, obtained by three-stage selection of the producer. The simplex method of selecting the composition of the environment was used for the research. The ratio of amylolytic activity to the level of biomass accumulation was taken as the biosynthetic capacity of the strain. A photocolometric method was used to determine amylolytic activity. The level of biomass accumulation was determined by the weight method. Results. It was established that as a result of optimizing the composition of the simplex nutrient medium by the method of mathematical modeling, the biosynthetic capacity increased by 3.63 compared to the control variant. It was also investigated that the optimal concentration of such a component of the nutrient medium as monosodium glutamate С5Н8NO4Na・H2O is 1.5%, which increases the amylolytic activity by 2.63 and increases the accumulation of biomass. Separately, it should be noted the obtained results of the study of the optimal concentrations of heavy metal ions added to the optimized version of the nutrient medium, which allow further research in this aspect to be continued and the use of Co, Mo, Cd ions in the composition of the nutrient medium. With the obtained results, there is an increase in amylolytic activity in the best response by 3.54. The obtained results have theoretical and practical significance for further research in the biotechnology of enzymes. Conclusions. The prospect of further research into the optimization of the biosynthesis of actinomycetes by the simplex method of other aspects of its regulation will be to increase the biosynthetic capacity of the studied strain, which will have a positive effect on the economic output of the production of amylolytic enzyme preparations by obtaining microbial synthesis.

A (S)-3-Hydroxybutyrate Dehydrogenase Belonging to the 3-Hydroxyacyl-CoA Dehydrogenase Family Facilitates Hydroxyacid Degradation in Anaerobic Bacteria.

Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for β-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.

Food Security in Emerging Global Situations-Functional Foods and Food Biotechnology Approaches: A Review

People having access to physical, social and economic food that is sufficient, safe and nutritious to meet their dietary needs and food preferences, for an active and healthy life, could be a mirage without the involvement of local communities, in global effort to mitigate malnutrition. World leaders have identified the lacuna in the drive for food security and therefore, seek a sustainable approach through which biotechnology and functional food technology could be tailored to forestall food insecurity, especially in emerging global situations. Consequently, researchers in food microbiology, enzyme biotechnology, among other disciplines are making concerted effort to provide solutions to some emerging world problems such as banditry, wars, climate change, internal displacement, migration, among others. Emphasis on the role of functional foods and biotechnology for sustainable food security should be paramount on the global scale as championed by United Nations Organization (UNO), Food and Agricultural Organization (FAO), World Health Organization/World Bank (WHO/WB) and World Food Programme on Malnutrition. Functional food components such as biotin, carotenoids, lycopene, monosaturated and polyunsaturated fatty acids, among others could be tailored to reduce the incidences coronary heart diseases, diabetes and free radicals in the body for improved nutrition, while nutritional education, alongside global support and empowerment initiatives for sustainability in agricultural biotechnological approaches would be highly imperative. It is therefore imperative for the global support agencies to reduce malnutrition to emphasize food functionality such as the role of carotenoids, fatty acids, lycopene, vitamins, minerals, biotin, fibre, etc in combating food insecurity through nutritional education, and in farming operations. It is in doing so that the role of food biotechnology would impact positively on the affected population, whereby foods are not just in abundance but also have the ability and capacity to provide the needed food ingredients to functionally prevent cardiovascular diseases, diabetes, neutralize free radicals and prevent antioxidant activity in the body.

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives.

The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes.

Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I.

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius's analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants.

Обоснование местного применения протеолитических ферментов в ветеринарии

The research is devoted to the search for possible use of proteolytic enzymes in Biomedicine, veterinary and biotechnology. In recent decades, thanks to new data in the field of biochemistry, physiology, immunology and practical medicine enzyme preparations have been widely used in the treatment of a number of diseases. These drugs have anti-inflammatory, decongestant, fibrinolytic, immunomodulatory and secondary analgesic effect, due to the influence on key pathophysiological processes in the body. The article describes the results of local application "Chymopsin" traumatic purulent and ulcerative skin lesions in horses. In the course of the study, good results were obtained and the effectiveness of the drug was proved both in the form of monotherapy and as part of complex therapy. Studies have shown that the inclusion of the drug "Chymopsin" in the standard treatment regimen leads to a significant reduction in the time of epithelialization and regeneration of the skin.

Purification and characterization of thermostable alpha‐amylase from Geobacillus sp. DS3 from Sikidang Crater, Central Java, Indonesia

Amylases are considered the most essential enzymes in biotechnology since they are widely utilized in the textile, food processing, and detergent industries. It is necessary to explore extracellular enzymatic activity in several microorganisms to discover a new potential application from amylases. In a previous study, thermophilic bacteria Geobacillus sp. DS3 isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia showed amylase activity in starch medium at 70 °C. This study aimed to purify and characterize the thermostable alpha‐amylase from Geobacillus sp. DS3. The alpha‐amylase was produced and purified using ammonium sulfate and DEAE Sephadex A‐25 column. The enzyme activity was determined using the 3,5‐dinitrosalicylic acid (DNS) method. Geobacillus sp. DS3 optimally produced the alpha‐amylase at 60 °C for 15 h. The alpha‐amylase exhibited high enzymatic activity in 40–60% saturated ammonium sulfate extract. The molecular weight of the enzyme was estimated to be 58 kDa. The thermostable alpha‐amylase showed activity at the optimum temperature of 50 °C in 200 mM sodium phosphate buffer pH 7.0. The enzyme was inhibited by EDTA, PMSF, 2‐ME, and mostly by HgCl2. The Km and Vmax of the pure enzyme were 235.43 mM and 1428.57 U/mL, respectively. The result suggested that the purified thermostable alpha‐amylase from Geobacillus sp. DS3 offers potential application in areas of the food industry, such as the bakery industry.

Identification and Potential Biotechnological Characterization of Lactic Acid Bacteria Isolated from White Cheese Samples

In this study, the isolation of lactic acid bacteria was carried out from one hundred white cheese samples collected from different regions of Turkey. Subsequently, phenotypic and genotypic characterization of the isolates was performed. Biochemical characteristics of the isolates were determined by API 50CHL. Furthermore, the biotechnological enzyme production potential of the isolates was screened. Genomic fingerprint profiles of the test isolates were detected by using rep-PCR (BOX-PCR), which has been used successfully in the differentiation of microorganisms at the species, subspecies, and even strain levels. The results showed that a total of forty-one bacteria were isolated and seventeen of which are found to be different species. The isolates generally grew at 4-6 pH values, 0-8% NaCl and 30-40°C. Later, isolates thought to be different species were identified by 16S rRNA gene sequence analyses. According to 16S rRNA sequence results, MA56 showed a 96.41% similarity match to Lentilactobacillus buchneri, it is thought to be a new species. In addition, MA19, MA25, MA43, and MA47 were determined to have multi-enzyme production potential. MA43 has a plantaricin gene and it showed a high antagonistic effect on Escherichia coli O157:H7 ATCC 43888 and Pseudomonas aeruginosa ATCC 9027. Inhibition zones were measured at 19 mm and 16 mm respectively.