Enzymatic Process Research Articles

Screening, identification, and application of anaerobic ammonia oxidizing bacteria in activated sludge systems: A comprehensive review.

Anaerobic ammonium oxidation (Anammox) has garnered significant attention due to its ability to eliminate the need for aeration and supplementary carbon sources in biological nitrogen removal process, relying on the capacity of anaerobic ammonium oxidizing bacteria (AnAOB) to directly convert ammonium and nitrite nitrogen into nitrogen gas. This review consolidates the latest advancements in AnAOB research, outlining the mechanisms and enzymatic processes of Anammox, and summarizing the molecular biological techniques used for studying AnAOB, such as 16s rRNA sequencing, qPCR, and metagenomic sequencing. Additionally, it also overviews the currently identified AnAOB species and their distinct metabolic traits, while consolidating strategies to improve their performance. It further delineates coupled processes that utilize Anammox technology, offering practical insights for process selection. Eventually, the review concludes by suggesting future research directions and highlighting critical areas for further investigation. This review serves as a theoretical reference for the enrichment and cultivation of AnAOB, environmental impact management, and the selection of effective treatment processes.

Identification of Fungal Metabolite Gliotoxin as a Potent Inhibitor Against Bacterial O-Acetylserine Sulfhydrylase CysK and CysM

Cysteine is an essential amino acid for sustaining life, including protein synthesis, and serves as a precursor for antioxidant glutathione. Pathogenic bacteria synthesize cysteine via a two-step enzymatic process using serine as the starting material. The first step is catalyzed by serine acetyltransferase, also known as CysE, and the second by O-acetylserine sulfhydrylase (OASS), referred to as CysK or CysM. This cysteine biosynthetic pathway in bacteria differs significantly from that in mammals, making it an attractive target for the development of novel antibacterial agents. In this study, we aimed to identify OASS inhibitors. To achieve this, a high-throughput screening system was developed to analyze compounds capable of inhibiting CysK/CysM activity. Screening 168,640 compounds from a chemical library revealed that gliotoxin, a fungal metabolite, strongly inhibits both CysK and CysM. Furthermore, gliotoxin significantly suppressed the growth of Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, under cystine-deficient conditions. Gliotoxin possesses a unique disulfide structure classified as epipolythiodioxopiperazine. To date, no studies have reported OASS inhibition by compounds with this structural motif, highlighting its potential for future structural optimization. The screening system developed in this study is expected to accelerate the discovery of functional CysK/CysM inhibitors, providing a foundation for novel antibacterial strategies.

Clinical evidence on nutrological management and gut microbiota in inflammatory bowel diseases: a systematic review

Introduction: The main risk factor for inflammatory bowel disease (IBD) is a positive family history. Crohn's disease (CD) can affect individuals aged 15 to 40 and 50 to 80 years, with a higher percentage in women. Ulcerative colitis (UC) can start at any age. Metabolism encompasses the interactions between diet, the microbiome, and cellular enzymatic processes that generate the chemical pathways necessary to sustain life. Endogenous metabolites and dietary nutrients can directly influence epigenetic enzymes. Epigenetic modifications to DNA and histone proteins alter cell fate by controlling chromatin accessibility and downstream gene expression patterns. Objective: It was to highlight the main interactions between nutrition and gut microbiota in the treatment of inflammatory bowel diseases. Methods: The present study followed the international systematic review model (PRISMA). This study was carried out from July to September 2024. It included randomized controlled, prospective, and retrospective studies published from 2013 to 2023. The Cohen test was performed to calculate the Effect Size and the inverse error standard (precision or sample size) for the risk of bias (Funnel Plot). Results and Conclusion: A total of 30 articles were found, and 17 clinical studies on the modulation of diet to control IBD were included in this study. These studies have shown reductions in persistent gut symptoms, improved gut microbiota, reduced markers of inflammation, and improved quality of life. Diet has an important role in controlling and even remitting IBD. The studies were homogeneous in results, with X2 = 82.5%, which increases the reliability of clinical results on the importance of diet in modulating IBD.

Pathway Elucidation and Key Enzymatic Processes in the Biodegradation of Difenoconazole by Pseudomonas putida A-3.

The extensive agricultural use of the fungicide difenoconazole (DIF) and its associated toxicity increasingly damage ecosystems and human health. Thus, an urgent need is to develop environmentally friendly technological approaches capable of effectively removing DIF residues. In this study, strain Pseudomonas putida A-3 was isolated for the first time which can degrade DIF efficiently. After optimization of the degradation conditions, the degradation rate reached 75.98%. Moreover, a new DIF degradation pathway, including hydroxylation, hydrolysis, dechlorination, and ether bond breaking. The acute and chronic toxicity of DIF degradation products assessed using ECOSAR software showed lower toxicity than the parent compound. Furthermore, strain A-3 remarkably accelerated the degradation of DIF in contaminated water-sediment systems. We successfully predicted six potential key enzymes for DIF degradation based on the results of whole genome sequencing, RT-qPCR, and molecular docking. Overall, the results revealed novel pathways for DIF biodegradation and provide a strong candidate for bioremediation of DIF residue-polluted environments.

Taming Highly Enolizable Aldehydes via Enzyme Catalysis for Enantiocomplementary Construction of β-Hydroxyphosphonates.

Taming highly enolizable aldehydes for catalytic asymmetric C-C coupling with nucleophiles remains an elusive challenge compared to widely explored simple alkyl or aryl aldehydes. Herein, we use ThDP-dependent enzymes to realize the direct C-C coupling of highly enolizable 2-phosphonate aldehydes with in situ-generated dynamically reversible nucleophiles (acyl anions). Unlike NHC-mediated reactions that yield complex mixtures of multiple adducts, our enzymatic process selectively produces biologically active β-hydroxy phosphonates with high yields (up to 95%) and excellent enantioselectivities (up to 99% ee). The products can be obtained on gram scales and exhibit rich reactivity for downstream transformations to afford diverse molecules. PfBAL (or its mutant A28G) and PaBAL enzymes serve as enantiocomplementary pairs, enabling the synthesis of both product configurations. Mechanistic studies proved that the entrance directions of the active cavities of these two enzyme pairs were distinct, leading to acyl anions formed from these two enzyme pairs attacking 2-phosphonate aldehydes from different orientations.

Mercury toxicity resulting from enzyme alterations- minireview.

Mercury is widely known for its detrimental effects on living organisms, whether in its elemental or bonded states. Recent comparative studies have shed light on the biochemical implications of mercury ingestion, both in low, persistent concentrations and in elevated acute dosages. Studies have presented models that elucidate how mercury disrupts healthy cells. Mercury's unique ability to interfere with crucial enzymatic processes at deposition sites is a vital feature of these models. The strong affinity for the sulfhydryl moieties of enzyme catalytic sites leads to enzyme inactivation through permanent covalent modifications. This inactivation can have catastrophic effects on an organism's metabolic functions. Moreover, it has been found that mercury's binding to sulfhydryl moieties is highly nonspecific and can occur in various ways. This review aimed to explore the effects of mercury on a broad spectrum of enzymes with a specific focus on how these alterations can detrimentally affect several metabolic pathways.

Micelle-enabled bromination of α-oxo ketene dithioacetals: mild and scalable approach via enzymatic catalysis.

The bromination of α-oxo ketene dithioacetals using KBr/H2O2, catalyzed by Curvularia inaequalis vanadium chloroperoxidase (CiVCPO), has been successfully demonstrated. A comparative study of enzymatic processes "on water" versus "in water", using 2 wt% of the surfactant TPGS-750-M revealed that the in-water protocol not only provides higher yields but also accommodates a broader substrate scope. This bromination method in an aqueous micellar medium enabled the preparation of brominated α-oxo ketene dithioacetals in fair to excellent yields (23 examples). In the micellar system, the substrate concentration was increased up to 50 mM, and the amounts of the brominating agents KBr and H2O2 were reduced to approximately 2.0 equivalents without compromising efficiency. Notably, this process allows for the gram-scale preparation of brominated α-oxo ketene dithioacetals in high yields. Key advantages of this method include its benign and eco-friendly nature, the use of water as a green solvent, and its potential for large-scale production of brominated α-oxo ketene dithioacetals.

Exo- and Endo-1,5-α-L-Arabinanases and Prebiotic Arabino-Oligosaccharides Production.

There is growing interest in pentose-based prebiotic oligosaccharides as alternatives to traditional hexose-based prebiotics. Among these, arabino-oligosaccharides (AOS), derived from the enzymatic hydrolysis of arabinan polymers, have gained significant attention. AOS can selectively stimulate the growth of beneficial gut bacteria, including Bifidobacterium and Bacteroides species, and contribute to health-benefit functions such as blood sugar control, positioning AOS as a promising synbiotic candidate. For the industrial production of AOS, the development of efficient enzymatic processes is essential, with exo- and endo-1,5-α-L-arabinanases (exo- and endo-ABNs) playing a crucial catalytic role. Most ABNs belong to the glycoside hydrolase (GH) family 43, characterized by a five-bladed β-propeller fold structure. These enzymes hydrolyze internal α-1,5-L-arabinofuranosidic linkages, producing AOS with varying degrees of polymerization. Some ABNs GH43 were known to exhibit exo-type hydrolytic modes of action, producing specific AOS products such as arabinotriose. Additionally, exo-ABNs from GH93, which feature a six-bladed β-propeller fold, exclusively release arabinobiose through their exo-type catalytic mechanism. This review represents the first comprehensive analysis of exo- and endo-ABNs, offering scientific insights into their biotechnological potential for AOS production. It systematically compares enzyme classification, structural differences, catalytic mechanisms, paving the way for innovative applications in health, food, and pharmaceutical industries.

Identification and Characterization of a New Thermophilic κ-Carrageenan Sulfatase.

Carrageenans are sulfated polysaccharides found in the cell wall of certain red seaweeds. They are widely used in the food industry for their gelling and stabilizing properties. In nature, carrageenans undergo enzymatic modification and degradation by marine organisms. Characterizing these enzymes is crucial for understanding carrageenan utilization and may eventually enable the development of targeted processes to modify carrageenans for industrial applications. In our study, we characterized a κ-carrageenan sulfatase, AMOR_S1_16A, belonging to the sulfatase S1_16 subfamily, which selectively desulfates the nonreducing end galactoses of κ-carrageenan oligomers in an exomode. Notably, AMOR_S1_16A represents the first κ-carrageenan sulfatase within the S1_16 subfamily and exhibits a novel enzymatic activity. This study provides further understanding of the substrate specificity and characteristics of the S1_16 subfamily. Moreover, this research highlights that many processes and enzymes remain to be discovered to fully understand carrageenan utilization pathways and to develop enzymatic processes for carrageenan modification and processing.

Coexpression of D-Allulose 3-Epimerase and L-Rhamnose Isomerase in Bacillus subtilis through a Dual Promoter Enables High-Level Biosynthesis of D-Allose from D-Fructose in One Pot.

D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of Blautia produca D-allulose 3-epimerase (Bp-DAE) and Bacillus subtilis L-rhamnose isomerase (BsL-RI) within a single cell using B. subtilis WB800N as the host. Using this system, D-allose was synthesized via a simple, cost-effective, one-pot enzymatic process, employing whole cells as catalysts and D-fructose as the substrate. The system exhibited optimal activity at 65 °C, pH 8.5, with 1 mM Mn2+ and 20 g/L of whole-cell dry weight. Initial production reached 12.5 g/L D-allose with a 12.5% yield from 100 g/L D-fructose. Optimization of dual promoter combinations enhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields of 15.00, 14.55, and 14.40%, respectively. This D-allose production biocatalyst offers a scalable and economically viable platform for the industrial production of rare sugar.

Protein hydrolysates derived from superworm (Zophobas morio): Composition, bioactivity, and techno-functional properties.

This study aimed to produce protein hydrolysates from superworm (Zophobas morio) flour using the enzymes alcalase (HA), protamex (HP), or flavourzyme (HF), and to characterize their nutritional composition, techno-functional properties, bioactive capacity, and bioaccessibility index. The enzymatic process increased the total amino acid and crude protein contents of the hydrolysates by approximately 36% and 46%, respectively, generating better foaming capacity, oil retention, and emulsification capacity, when compared to raw flour. Although 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical capture was similar between the hydrolysates, HA (1479,66μM FeSO4/g) and HP (1514,66μM FeSO4/g) showed greater antimicrobial and iron reducing power (FRAP) activity, while HF has a higher scavenging efficiency for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (27.53%). The best antimicrobial activity was observed for HA against Vibrio corallilyticus (400mg/mL), and HP showed a better antioxidant response scavenging for DPPH radical. The antioxidant capacity against ABTS radical after in vitro simulation of gastrointestinal digestion (GID) was as follows: HA (79.07±1.53%), HP (74.65±5.85%), and HF (57.95±8.31%). Therefore, insect flour is a promising ingredient for the production of protein hydrolysates and their application in animal and human feeds.

ADAR1 Gene Polymorphism and Hematological Analysis in Responders and Relapse Patients with Chronic Myeloid Leukemia

Abstract Background: Chronic myeloid leukemia (CML) is defined by the Break point cluster region and Abelson Genes (BCR-ABL) fusion gene, which produces a continuously active tyrosine kinase known as the primary driver responsible for initiating and sustaining the disease. The RNA editing enzyme adenosine deaminase acting on double-stranded RNA 1 (ADAR1) converts adenosine into inosine in double-stranded RNA substrates. This enzymatic process is crucial in cellular RNA editing and can impact disease progression and therapeutic responses in CML. Objective: Analysis of ADAR1 gene polymorphisms, ADAR1 levels, and hematological parameters in Iraqi patients with CML. Materials and Methods: From January 2021 to February 2023, one hundred and twenty samples of whole blood were collected from patients with CML at the National Center of Hematology/Mustansiriyah University. The control group in this study comprised thirty samples of fresh whole blood. Total DNA genomic extraction was done to detect the ADAR1 gene polymorphism by sequencing. Furthermore, serum was used to detect ADAR1 enzyme level. Results: According to the age and male/female ratio, the patients’ groups were matching with control group. A group of CML patients on treatment, 65 out of 100 patients were on imatinib, while 35 were treated with other tyrosine kinase inhibitors. The patient groups in terms of age and gender ratio were matched with the control group. Among CML patients receiving treatment, 65 out of 100 were using imatinib, while 35 were treated with other tyrosine kinase inhibitors. In the newly diagnosed CML group, ADAR mutations were present in 35% of cases, and among treated CML patients, this proportion was 36%, whereas the control group showed no mutations (P < 0.001). The distribution of DNA polymorphisms—A/G, G/T, and G/G—was 42%, 30%, and 28%, respectively, among patients with CML, compared to 34%, 20%, and 46%, respectively, in the control group. There are significant differences between different groups according to genotyping of ADAR1 (P < 0.05). Based on ADAR1 mutation, there is a significant difference observed between the newly diagnosed CML group and the CML patients in the treatment group compared to the control group (P < 0.001). Specifically, the differences between the new diagnosis CML group and the treatment group were not statistically significant (P = 0.326). Furthermore, both the new diagnosis CML group and the relapse group showed significant differences compared to the control group (P < 0.001). The P value associated with the correlation between ADAR mutation and these groups is <0.001, indicating a highly significant correlation. Conclusion: The current findings suggest that ADAR1 polymorphisms and ADAR1 enzyme levels in Iraqi patients with CML could potentially influence disease progression and deepen our understanding of its mechanisms. These factors may also contribute to disease development and affect how patients respond to treatment.

Proteogenomics for Non-model Ocean-Derived Fungi.

Most biological and biomedical experiments are designed and studied using the most common model organisms (MOs) like humans, mice, Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, worms, fruit flies, zebrafish, and Arabidopsis thaliana. These model organisms have been extensively studied and have a well-established set of genetic, physiological, and other tools available for research. In contrast, non-model organisms (NMOs) are those that are not traditionally used in scientific research and do not have a well-established set of genetic or other biological tools available for their study. The majority of MOs are associated with land habitats but rarely with ocean environments. The ocean forms the largest portion of our planet, yet ocean-derived organisms are the least explored, and these organisms are primarily NMOs. However, these are thrilling living entities, such as ocean-derived fungi (ODF). These ODFs are a diverse group of fungi that live in different ocean sectors, including the ocean, estuaries, and coastal ecosystems. These fungi are found to colonize and adapt to different substrates. They are important decomposers in marine ecosystems, breaking down dead organic matter and recycling nutrients. ODFs have adapted to survive in the unique and challenging conditions of the ocean environment, including high salt concentrations, low nutrient availability, and exposure to waves and currents. ODFs are potent producers of natural compounds with pharmaceutical and industrial applications, such as antibiotics, anticancer agents, antivirals, and enzymes for industrial processes. ODFs are an exciting group of fungi; however, these are the least studied because of the nonavailability of MOs from this group. Hence, there is a massive scope of expanding our current knowledge about ODFs, their genetic traits, potential future drug-producing capabilities, and lifestyle traits.With the advent of next-generation DNA sequencing, there is huge potential for the characterization of the genetic material of ODF as NMOs. Parallel proteomic methods also pose huge potential. A marriage of NGS and proteomic methods generates a new avenue called proteogenomics, which focuses on better annotation of existing genomic data. Both methods are getting cheaper and accessible to the research community for studying the proteogenomics of NMOs. Herein, the proteogenomic protocol development and data analyses are illustrated for the ocean-derived fungus Scopulariopsis brevicaulis.

High-rate nitrogen loading accelerates organic phosphorus loss through enzymatic and non-enzymatic processes in a semi-arid grassland

Semi-arid grasslands suffer from increased nitrogen (N) loading, which affects phosphorus (P) cycling. Phosphorus is essential but limited for all living organisms. Organic P (Po) is crucial for P supply in grasslands response to N loading, but its transformation process remains unclear. To elucidate P cycling, we evaluated P fractions, phosphatase activities, and their coding genes (pho genes phoD, phoX, and phoC) at both DNA and RNA levels in response to a nitrogen loading gradient. The results showed that the total P and Po contents significantly decreased with increasing N loading rates. In contrast, plant and litter biomass P exhibited an opposite trend but were insufficient to offset soil P loss. Unexpectedly, neither the phosphatase activities nor microbial biomass increased with increasing N loading rates, although the DNA abundance of pho genes responded positively to N enrichment. A partial correlation network indicated that N loading rates decreased Po levels through phosphatase activities, which were significantly associated with soil pH and the RNA abundance of pho genes. Moreover, non-enzymatic mineralization may be enhanced in Po cycling in semi-arid grasslands undergoing high N loading, especially following the initial stage of enzymatic mineralization. Although the activity of phosphatases did not continue to rise with increased N loading, the strategy for addressing N-induced P limitation through overconsumption of Po via non-enzymatic processes would accelerate the degradation of the ecosystem in the future.

Cassava starch extraction with a polygalacturonase from a wild yeast strain with disintegration activity on plant tissues.

The objective of the present study was to optimize an enzymatic starch extraction process from cassava roots using a polyglacturonase (PGase) from a wild yeast strain (Wickerhanomyces anomalus). The supernatant of W. anomalus culture, with PGase activity, was used as source of enzyme (enzymatic extract, EE). The cassava starch extractions with the EE were done at lab scale and the effects of several factors were studied by using statistical designs. Chemical and functional properties of the starch obtained (enzymatic starch, ES) and those of a commercial starch (CS), were determined. The highest extraction yield was obtained using processed cassava tissues, a solid-liquid ratio of 3:6, EE with an enzyme dosage of 15 EU mL-1, at 40°C for 5h. Proximal analysis of the ES did not show significant differences with the CS. Swelling power and water solubility of ES increased with the temperature up to 70°C (1.97 gg-1) and 90°C (4.27% w/w), respectively, similar to those of CS. Viscoamylographic profiles of ES showed a pasting temperature of 63.5°C, a viscosity peak of 422 BU, an stability of the pasta during cooking of 202 BU and a retrograde trend of 98 BU. These values were slightly different compared to those of CS. Clarity assays determined that ES paste is considered a clear or transparent paste. Microscopic evaluation of the ES grains revealed that they were intact without any mechanical damage. The production of cassava starch by enzyme technology seems to be an interesting alternative to the traditional method of extraction by mechanical means, resulting in a novel biotechnological process.

Coupling metabolomics and exome sequencing reveals graded effects of rare damaging heterozygous variants on gene function and human traits

Genetic studies of the metabolome can uncover enzymatic and transport processes shaping human metabolism. Using rare variant aggregation testing based on whole-exome sequencing data to detect genes associated with levels of 1,294 plasma and 1,396 urine metabolites, we discovered 235 gene–metabolite associations, many previously unreported. Complementary approaches (genetic, computational (in silico gene knockouts in whole-body models of human metabolism) and one experimental proof of principle) provided orthogonal evidence that studies of rare, damaging variants in the heterozygous state permit inferences concordant with those from inborn errors of metabolism. Allelic series of functional variants in transporters responsible for transcellular sulfate reabsorption (SLC13A1, SLC26A1) exhibited graded effects on plasma sulfate and human height and pinpointed alleles associated with increased odds of diverse musculoskeletal traits and diseases in the population. This integrative approach can identify new players in incompletely characterized human metabolic reactions and reveal metabolic readouts informative of human traits and diseases.

Studying the Function of tRNA Modifications: Experimental Challenges and Opportunities.

tRNAs are essential molecules in protein synthesis, responsible for translating the four-nucleotide genetic code into the corresponding amino acid sequence. RNA modifications play a crucial role in influencing tRNA folding, structure, and function. These modifications, ranging from simple methylations to complex hypermodified species, are distributed throughout the tRNA molecule. Depending on their type and position, they contribute to the accuracy and efficiency of decoding by participating in a complex network of interactions. The enzymatic processes introducing these modifications are equally intricate and diverse, adding further complexity. As a result, studying tRNA modifications faces limitations at multiple levels. This review addresses the challenges involved in manipulating and studying the function of tRNA modifications and discusses experimental strategies and possibilities to overcome these obstacles.

A novel chitosan microsphere as food processing enzyme immobilization carrier and its application in nucleotide production.

Developing a robust and safe carrier for enzyme immobilization is crucial for their application in the food processing industry. In this study, a novel crosslinked chitosan microspheres (CSMs) were prepared using glutaraldehyde (GA) as the crosslinking agent, using a newly developed emulsification-neutralization combined method. Nuclease P1 (NP1) was immobilized onto these microspheres, the maximum activity of NP1@CSMs-GA reach to 53,859.4U/g. The activity recovery yield reach to 75%. Compared to the free NP1, the stability of NP1@CSMs-GA was significantly enhanced. Its vmax and Km is 895.71mg/(g·min) and 77.27mg/mL respectively. This NP1@CSMs-GA was utilized in production of nucleotides through hydrolysis of RNA. In BSTR, NP1@CSMs-GA retained more than 75.1% initial activity after 10cycles of reuse. Moreover, in PBR, the RNA hydrolysis conversion rate maintained 81% after 24h of continuous operation. These results demonstrate that NP1@CSMs-GA exhibits excellent reusability and production stability in practical processes.

Solubilization Inclusion Bodies from Synthetic Recombinant PGA Gene Expressed in E. coli BL21(DE3) by Denaturing and Non-denaturing Agents

Background: With the rise in green chemistry, the synthesis of antibiotic compounds through enzymatic processes is a preferred option. Penicillin-G acylase (PGA) is an important enzyme for producing important antibiotics, such as penicillin and its derivatives. Therefore, studies on PGA have been conducted worldwide. In the penicillin biosynthetic pathway, PGA catalyzes the conversion of penicillin G into 6-amino penicillanic acid (6-APA), a precursor for the enzymatic synthesis of penicillin derivatives. Unfortunately, bacteria naturally produce PGA in small quantities. Objective: One strategy for producing this enzyme in large quantities is DNA recombination, which is expressed in Escherichia coli. The formation of inclusion bodies (IBs) is a common obstacle to protein overexpression in Escherichia coli. In this study, we discuss IBs solubilization methods for recombinant PGA derived from E. coli (rPGAEc) expressed in E. coli BL21 (DE3). Recombinant E. coli BL21 (DE3) cells harboring rPGAEc were induced with IPTG for enzyme expression. Induction was performed at 16 °C for 4 h and 24 h. The PGA enzyme expressed in the IBs form was then incubated in two solutions containing 8 M urea and 0.2% sarcosine to obtain a soluble enzyme. Results: Based on protein analysis by SDS-PAGE, a solution containing 8 M urea solubilized PGA more abundantly than 0,2% sarcosine. Conclusion: The solubilization technique of PGA expressed by E. coli proposed in this study is an alternative solution that can be considered for this purpose.

Efficacy of Citromazinc for the biological control of the Aphis spiraecola Patch (Hemiptera: Aphididae) on apple trees in Bulgaria under laboratory and field conditions

The invasive Aphis spiraecola Patch, which originated in Eastern Asia, recently became a common pest on apple in Bulgaria. The aim of the study was to determine the efficacy of the biological insecticide Citromazinc in concentrations of 0.15% and 0.3% under laboratory and field conditions, against the green citrus aphid (Aphis spiraecola) on apple. Citromazinc is a corrector of manganese and zinc deficiency, which are essential for the formation of chlorophyll. Manganese also acts as a catalyst in many enzymatic processes. Zinc acts as an activator of some important functions and participates in the formation of auxins and growth hormones. Citromazinc showed excellent biological efficacy against the green citrus aphid under laboratory conditions. In both applied concentrations (0.15% and 0.3%), the efficacy reached 100% on the 7th day after treatment. The effect of the product was also very good under field conditions. On the 7th day after treatment, the efficacy reached almost 100% in the higher concentration (0.3%) and 90% in the lower concentration (0.15%). The tested biological insecticide Citromazinc is suitable for the efficient control of the green citrus aphid in apple orchards, even at its lower applied concentration. Keywords: Green citrus aphid, biological efficacy, insecticide, apple