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CASE: Intrauterine device (IUD) is used worldwide as an effective contraceptive method, but the migration of IUD is a serious complication. We report the case of IUD migration leading to bladder calculus formation and a minimally invasive transurethral surgical approach was performed for treatment. Holmium laser was used to break up the bladder calculus and cut through the bladder mucosa where the IUD was attached, finally the IUD was removed through the urethra. This minimally invasive procedure is a safe and effective treatment for IUD migration, and similar cases have not been reported in the literature. CONCLUSION: That the secondary bladder calculus were smashed by intense pulse mode of holmium laser, and the bladder tissue around the attached IUD was opened by cutting mode of holmium laser, and finally the IUD was completely removed from urethra, this surgical method is safe and effective, and there is no case report on IUD removal of transurethral cystoscope in the literature.
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Many drugs and prebiotics derive their activities from sugar substituents. Due to the prevalence and complexity of these biologically active compounds, enzymatic glycodiversification that facilitates easier access to these compounds can make profound contributions to the pharmaceutical, food, and feed industries. Amylosucrases (ASases) are attractive tools for glycodiversification because of their broad acceptor substrate specificity, but the lack of structural information and their poor thermostability limit their industrial applications. Herein, we reported the crystal structure of ASase from Calidithermus timidus, which displays a homotetrameric quaternary organization not previously observed for other ASases. We employed a workflow composed of five common strategies, including interface engineering, folding energy calculations, consensus sequence, hydrophobic effects enhancement, and B-factor analysis, to enhance the thermostability of C. timidus ASase. As a result, we obtained a quadruple-point mutant M31 ASase with a half-life at 65 °C increased from 22.91 h to 52.93 h, which could facilitate biosynthesis of glucans with a degree of polymerization of more than 20 using sucrose as a substrate at 50 °C. In conclusion, this study provides a structural basis for understanding the multifunctional biocatalyst ASase and presents a powerful methodology to effectively and systematically enhance protein thermostability.
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Amilose , Glucosiltransferases , Estabilidade Enzimática , Glucanos , Glucosiltransferases/metabolismo , Engenharia de Proteínas , Especificidade por Substrato , Sacarose/metabolismoRESUMO
Human milk oligosaccharides (HMOs) are receiving wide interest and high attention due to their health benefits, especially for newborns. The HMOs-fortified products are expected to mimic human milk not only in the kinds of added oligosaccharides components but also the appropriate proportion between these components, and further provide the nutrition and physiological effects of human milk to newborns as closely as possible. In comparison to intensively studied 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) has less attention in almost all respects. Nerveless, 3-FL naturally occurs in breast milk and increases roughly over the course of lactation with a nonnegligible content, and plays an irreplaceable role in human milk and delivers functional properties to newborns. According to the safety evaluation, 3-FL shows no acute oral toxicity, genetic toxicity, and subchronic toxicity. It has been approved as generally recognized as safe (GRAS). Biological production of 3-FL can be realized by enzymatic and cell factory approaches. The α1,3- or α1,3/4-fucosyltransferase is the key enzyme for 3-FL biosynthesis. Various metabolic engineering strategies have been applied to enhance 3-FL yield using cell factory approach. In conclusion, this review gives an overview of the recent scientific literatures regarding occurrence, bioactive properties, safety evaluation, and biotechnological preparation of 3-FL.
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Leite Humano , Oligossacarídeos , Feminino , Humanos , Recém-Nascido , Oligossacarídeos/metabolismo , Trissacarídeos/genética , Trissacarídeos/metabolismo , Aleitamento Materno , Lactação , BiotecnologiaRESUMO
Glucobiose is a range of disaccharides consisting of two glucose molecules, generally including trehalose, kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, and gentiobiose. The difference glycosidic bonds of two glucose molecules result in the diverse molecular structures, physiochemical properties and physiological functions of these glucobioses. Some glucobioses are abundant in nature but have unconspicuous roles on health like maltose, whereas some rare glucobioses display remarkable biological effects. It is unpractical process to extract these rare glucobioses from natural resources, while biological synthesis is a feasible approach. Recently, the production and application of glucobiose have attracted considerable attention. This review provides a comprehensive overview of glucobioses, including their natural sources and physicochemical properties like structure, sweetness, digestive performance, toxicology, and cariogenicity. Specific enzymes used for the production of various glucobioses and fermentation production processes are summarized. Additionally, their versatile functions and broad applications are also introduced.
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Enzymes can produce high-quality food with low pollution, high function, high acceptability, and medical aid. However, most enzymes, in their native form, do not meet the industrial requirements. Sequence-based and structure-based methods are the two main strategies used for enzyme modification. Molecular Dynamics (MD) simulation is a sufficiently comprehensive technology, from a molecular perspective, which has been widely used for structure information analysis and enzyme modification. In this review, we summarize the progress and development of MD simulation, particularly for software, force fields, and a standard procedure. Subsequently, we review the application of MD simulation in various food enzymes for thermostability and catalytic improvement was reviewed in depth. Finally, the limitations and prospects of MD simulation in food enzyme modification research are discussed. This review highlights the significance of MD simulation and its prospects in food enzyme modification.
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AIMS: l-Fuculose is a valuable rare sugar that is used to treat a variety of ailments, including HIV, cancer, Hepatitis B, human lysosomal disease (fucosidosis), and cardio-protective medications. The enzymatic approach for the production of l-fuculose using l-fucose as a substrate would be an advantageous method with a wide range of industrial applications. The objective of this study is the characterization of recombinant l-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI) for the production of l-fuculose from an inexpensive and natural source (fucoidan) as well as its comparison with commercial l-fucose (Sigma-Aldrich). METHODS AND RESULTS: Fucoidan, a fucose-containing polysaccharide (FPs), was isolated from Undaria pinnatifida, subsequently hydrolyzed, and characterized before the enzymatic production of l-fuculose. The results elaborate that FPs contain 35.9% of fucose along with other kinds of monosaccharides. The purified Pa-LFI exhibited a single band at 65 kDa and showed it as a hexamer with a native molecular mass of 396 kDa. The highest activity of 104.5 U mg-1 of Pa-LFI was perceived at a temperature of 50°C and pH 6.5 in the presence of 1 mM of Mn2+. The Pa-LFI revealed a melting temperature (Tm) of 75°C and a half-life of 12.6 h at 50°C. It exhibited that Pa-LFI with aldose substrate (l-fucose), has a stronger isomerizing activity, disclosing Km,kcat, and kcat/Km 86.2 mM, 32 831 min-1, and 335 min-1 mM-1, respectively. After reaching equilibrium, Pa-LFI efficiently catalyzed the reaction to convert l-fucose into l-fuculose and the conversion ratios of l-fuculose from 100 g L-1 of FPs and commercial fucose were around 6% (5.6 g L-1) and 30% (30.2 g L-1), respectively. CONCLUSIONS: According to the findings of the current study, the Pa-LFI will be useful in the manufacturing of l-fuculose using an effective and easy approach that produces no by-products.
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Fucose , Polissacarídeos , Humanos , Fucose/química , Polissacarídeos/químicaRESUMO
The trisaccharide, 2'-fucosyllactose (Fucα1-2Galß1-4Glc; 2'-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2'-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2'-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2'-FL as well as its applications. In addition, several approaches for 2'-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2'-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2'-FL bioprocess also should be taken into consideration.
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Leite Humano , Trissacarídeos , Humanos , Lactente , Engenharia Metabólica , Leite Humano/química , Oligossacarídeos , Trissacarídeos/análise , Trissacarídeos/farmacologiaRESUMO
Kestoses, the smallest fructooligosaccharides, are trisaccharides composed of a fructose molecule and a sucrose molecule linked by either ß-(2,1) or ß-(2,6) linkage. 1-kestose, 6-kestose and neokestose are the three types of kestoses occurring in nature. As the main kind of fructooligosaccharide, kestoses share similar physiological effects with other fructooligosaccharides, and they have recently been determined to show more notable effects in promoting the growth of probiotics including Faecalibacterium prausnitzii and Bifidobacterium than those of other fructooligosaccharides. Kestoses exist in many plants, but the relatively low content and the isolation and purification are the main barriers limiting their industrial application. The production of kestoses by enzymatic biosynthesis and microbial fermentation has the potential to facilitate its production and industrial use. In this article, the recent advances in the research of kestoses were overviewed, including those studying their functions and production. Kestose-producing enzymes were introduced in detail, and microbial production and fermentation optimization techniques for enhancing the yield of kestoses were addressed. ß-Fructofuranosidase is the main one used to produce kestoses because of the extensive range of microbial sources. Therefore, the production of kestoses by microorganisms containing ß-fructofuranosidase has also been reviewed. However, few molecular modification studies have attempted to change the production profile of some enzymes and improve the yield of kestoses, which is a topic that should garner more attention. Additionally, the production of kestoses using food-grade microorganisms may be beneficial to their application in the food industry.
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Biotecnologia , Oligossacarídeos , Biotecnologia/tendências , Enzimas/metabolismo , Fermentação , Microbiologia de Alimentos , Oligossacarídeos/metabolismoRESUMO
Targeted molecular therapy, for example, with sorafenib (SF) is considered as a new and potent strategy for glioblastoma (GBM) that remains hard to treat today. Several clinical trials with SF, as monotherapy or combination therapy with current treatments, have not met the clinical endpoints, likely as a result of the blood-brain barrier (BBB) and inferior GBM delivery. Here, we designed and explored small, smart, and LDLR-specific micelles to load SF (LDLR-mSF) and to improve SF therapy of GBM by enhancing BBB penetration, GBM accumulation, and cell uptake. LDLR-mSF with 2.5% ApoE peptide functionality based on poly(ethylene glycol)-poly(ε-caprolactone-co-dithiolane trimethylene carbonate)-mefenamate exhibited nearly quantitative SF loading, small size (24 nm), high colloidal stability, and glutathione-activated SF release. The in vitro and in vivo studies certified that LDLR-mSF greatly enhanced BBB permeability and U-87 MG cell uptake and caused 10.6- and 12.9-fold stronger anti-GBM activity and 6.0- and 2.5-fold higher GBM accumulation compared with free SF and non-LDLR mSF controls, respectively. The treatment of an orthotopic human GBM tumor model revealed that LDLR-mSF at a safe dosage of 15 mg of SF/kg significantly retarded tumor progression and improved the survival rate by inducing tumor cell apoptosis and inhibiting tumor angiogenesis. These small, smart, and LDLR-specific micelles provide a potential solution to enhance targeted molecular therapy of GBM.
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Neoplasias Encefálicas , Glioblastoma , Barreira Hematoencefálica , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Micelas , Sorafenibe/farmacologiaRESUMO
OBJECTIVE: To evaluate the effect of real-time transrectal ultrasound-guided seminal vesiculoscopy (TRUS-SVS) in the treatment of azoospermia secondary to ejaculatory duct obstruction. METHODS: This retrospective study included 40 cases of azoospermia secondary to bilateral ejaculatory ducts obstruction treated by TRUS-SVS from June 2016 to June 2018 after failure to enter the vesiculoscope through the ejaculatory duct or prostatic utricle. We analyzed the success rate of surgery, operation time, postoperative complications, treatment results, and application value of TRUS-SVS. RESULTS: Real-time TRUS-SVS was successfully performed in 36 (90.0%) of the cases, 33 through bilateral and the other 3 through unilateral seminal vesicle, with a mean operation time of (32.8 ± 16.6) min. Thirty-seven of the cases were followed up for 6ï¼15 (mean 9.3) months, of which sperm were found in 31 at 1ï¼3 months and in 25 at 3ï¼12 months, and pregnancies achieved in 9 cases within 12 months after surgery. No serious complications as retrograde ejaculation, urinary incontinence and rectal injury were observed postoperatively, except 2 cases of epididymitis and 2 cases of hematuria, which were all cured. CONCLUSIONS: For the patients who failed in seminal vesiculoscopy through the ejaculatory duct or prostatic utricle, real-time TRUS-SVS is a recommended procedure with the advantages of a high success rate, less damage to the prostate and rectum, and benefit to the improvement of semen quality.
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Azoospermia , Ductos Ejaculatórios , Azoospermia/diagnóstico por imagem , Azoospermia/etiologia , Azoospermia/cirurgia , Ductos Ejaculatórios/diagnóstico por imagem , Ductos Ejaculatórios/cirurgia , Humanos , Masculino , Estudos Retrospectivos , Análise do Sêmen , Ultrassonografia de IntervençãoRESUMO
Levan and inulin are two types of fructan. Levan is composed of ß-(2, 6) fructosyl linkage and inulin is composed of ß-(2, 1) linkage. Both levan and inulin have been accepted and applied in the food, medicinal and chemical industries for their outstanding physicochemical properties in recent years. Microbial levansucrase and inulosucrase are key enzymes responsible for the synthesis of fructan from sucrose. In this review, levansucrase and inulosucrase are discussed together for the first time regarding the evolutionary relationships, bacteria origin, crystal structure, product-forming mechanism and commercial applications. Particularly, some insights into the product specificity about levansucrase and inulosucrase as well as the mechanism for product elongation for fructan are also discussed in the article.
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Hexosiltransferases , Bactérias/enzimologia , Evolução Molecular , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Estrutura Terciária de ProteínaRESUMO
Functional carbohydrates are ideal substitutes for table sugar and make up a large share of the worldwide functional food market because of their numerous physiological benefits. Growing attention has been focused on levan, a ß-(2,6) fructan that possesses more favorable physicochemical properties, such as lower intrinsic viscosity and greater colloidal stability, than ß-(2,1) inulin. Levan can be used not only as a functional carbohydrate but also as feedstock for the production of levan-type fructooligosaccharides (L-FOSs). Three types of levan-degrading enzymes (LDEs), including levanase (EC 3.2.1.65), ß-(2,6)-fructan 6-levanbiohydrolase (LF2ase, EC 3.2.1.64), and levan fructotransferase (LFTase, EC 4.2.2.16), play significant roles in the biological production of L-FOSs. These three enzymes convert levan into different L-FOSs, levanbiose, and difructose anhydride IV (DFA IV), respectively. The prebiotic properties of both L-FOSs and DFA IV have been confirmed in recent years. Although levanase, LF2ase, and LFTase belong to the same O-glycoside hydrolase 32 family (GH32), their catalytic properties and product spectra differ significantly. In this paper, recent studies on these LDEs are reviewed, including those investigating microbial source and catalytic properties. Additionally, comparisons of LDEs, including those of their differing cleavage behavior and applications for different L-FOSs, are presented in detail.
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Bactérias/enzimologia , Frutanos/metabolismo , Fungos/enzimologia , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/metabolismo , BiotransformaçãoRESUMO
Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and ß-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed ß-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and ß-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS.
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Hexosiltransferases , Transferases , Transferases/metabolismo , beta-Frutofuranosidase/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Frutanos/metabolismo , Catálise , Sacarose/metabolismo , Especificidade por SubstratoRESUMO
Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.
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Bacillus subtilis , Lactose , Lactulose , Lactulose/metabolismo , Lactulose/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Lactose/metabolismo , Fermentação , Engenharia Metabólica/métodos , Engenharia GenéticaRESUMO
d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.
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Bacillus subtilis , Racemases e Epimerases , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Frutose/metabolismo , CetosesRESUMO
Enzymatic production of D-mannose attracts increasing attention because of the health effects and commercial values of D-mannose. Several kinds of epimerases or isomerases have been used for enzymatic production of D-mannose from D-glucose or D-fructose. D-Mannose epimerase (MEase), belonging to N-acyl-D-glucosamine 2-epimerase superfamily enzymes, catalyzes the C-2 epimerization between D-glucose and D-mannose. In this study, a novel MEase was identified from Cytophagaceae bacterium SJW1-29. Sequence and structure alignments indicate that it is highly conserved with the reported R. slithyformis MEase with the known crystal structure. It was a metal-independent enzyme, with an optimal pH of 8.0 and an optimal temperature of 40⯰C. The specific activities on D-glucose and D-mannose were 2.90 and 2.96â¯U/mg, respectively. The Km, kcat, and kcat/Km on D-glucose were measured to be 194.9â¯mM, 2.72â¯s-1, and 0.014â¯mM-1 s-1, respectively. The purified enzyme produced 23.15â¯g/L of D-mannose from 100â¯g/L of D-glucose at pH 8.0 and 40 °C for 8â¯h, with a conversion rate of 23.15â¯%.
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Carboidratos Epimerases , Glucose , Manose , Manose/metabolismo , Glucose/metabolismo , Especificidade por Substrato , Cinética , Carboidratos Epimerases/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Temperatura , Modelos Moleculares , Alinhamento de SequênciaRESUMO
Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.
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Proteínas de Bactérias , Estabilidade Enzimática , Hexosiltransferases , Inulina , Lactobacillus , Mutagênese Sítio-Dirigida , Inulina/metabolismo , Inulina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hexosiltransferases/química , Lactobacillus/enzimologia , Lactobacillus/genética , Lactobacillus/metabolismo , Cinética , Temperatura Alta , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Carbohydrate degradation is crucial for living organisms due to their essential functions in providing energy and composing various metabolic pathways. Nevertheless, in the catalytic cycle of polysaccharide degradation, the details of how the substrates bind and how the products release need more case studies. Here, we choose an inulin fructotransferase (SpIFTase) as a model system, which can degrade inulin into functionally difructose anhydride I. At first, the crystal structures of SpIFTase in the absence of carbohydrates and complex with fructosyl-nystose (GF4), difructose anhydride I, and fructose are obtained, giving the substrate trajectory and product path of SpIFTase, which are further supported by steered molecular dynamics simulations (MDSs) along with mutagenesis. Furthermore, structural topology variations at the active centers of inulin fructotransferases are suggested as the structural base for product release, subsequently proven by substitution mutagenesis and MDSs. Therefore, this study provides a case in point for a deep understanding of the catalytic cycle with substrate trajectory and product path.
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Hexosiltransferases , Inulina , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , Inulina/metabolismo , Inulina/química , Especificidade por Substrato , Simulação de Dinâmica Molecular , Domínio Catalítico , Biocatálise , Catálise , Frutose/metabolismo , Frutose/químicaRESUMO
d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni2+. Its kcat and kcat/Km values toward d-fructose were measured to be 248.5 min-1 and 2.117 mM-1·min-1, respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.
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BACKGROUND: Diabetes damages the seminal vesicle tissues leading to a decrease in seminal fluid secretion, so investigations are ongoing to identify specific therapeutic approaches to address diabetes-induced damage to seminal vesicles. OBJECTIVE: This study investigated the secretory dysfunction of seminal vesicles and how curcumin can ameliorate this dysfunction. MATERIALS AND METHODS: First, 40 diabetic males (DM group) and 40 nondiabetic males (control group) underwent seminal vesicle ultrasound evaluation and ejaculate volume measurements. Then, the effects of curcumin on seminal vesicle function were investigated in a diabetic rat model. Fifty 8-week-old SPF-grade SD rats were categorized into five groups: control, DM (diabetes mellitus), low-dose CUR (curcumin 50 mg/kg/d), medium-dose CUR (curcumin 100 mg/kg/d), and high-dose CUR (curcumin 150 mg/kg/d). After a month-long diet with varying curcumin doses, key parameters such as body weight, blood glucose levels, seminal vesicle volume, and seminal fluid secretion were measured. Transcriptome sequencing was performed to assess differences in gene expression and structural changes in rat seminal vesicle tissues were examined by HE staining. Finally, human seminal vesicle cell lines were cultured and divided into five groups (HG-CON, HG-CUR-5 µM, HG-CUR-10 µM, HG-CUR-20 µM, and HG-CUR-50 µM) to measure the fructose levels in the seminal vesicle cell culture fluids and evaluate the expression of CASP1, GSDMD, and TRPV6. Post TRPV6 interference, variations in the gene expression of CASP1, GSDMD, and TRPV6 were monitored. RESULTS: Diabetic patients exhibited a notable reduction in seminal vesicle volume and ejaculate volume compared with the control group, with a direct correlation between the decrease in ejaculate and seminal vesicle volume. Animal studies demonstrated that curcumin supplementation significantly augmented seminal vesicle volume in diabetic rats and notably improved their seminal vesicle secretory dysfunction, particularly in the high-dose curcumin group. Transcriptome sequencing and experimental verification pinpointed the differential expression of TPRV6 and pyroptosis-associated genes (CASP1, GSDMD), with reduced TRPV6 expression but increased markers of pyroptosis (CASP1 and GSDMD) in diabetic rats. Curcumin treatment reversed these effects with an increase in TRPV6 and a decrease in GSDMD and CASP1. Cell transfection experiments indicated that TRPV6 downregulation increased GSDMD and CASP1 gene expression. CONCLUSION: Curcumin effectively activates TRPV6, thereby diminishing pyroptosis in the seminal vesicle tissues of diabetic rats. This activation not only leads to an increase in the seminal vesicle volume but also significantly ameliorates the seminal vesicle secretory dysfunction in diabetic rats.