Selective extraction and quantitative analysis of pyrroloquinoline quinone from food.

Pyrroloquinoline quinone (PQQ) is a bioactive compound that has attracted significant attention due to its potential health benefits. In this study, we developed a new magnetic molecularly imprinted nanoparticle (MMIN) for the selective extraction and determination of PQQ from food samples. The MMIN was synthesized using a surface molecular imprinting technique with PQQ as the template molecule, Fe3O4 nanoparticles as the magnetic core, and methacrylic acid as the functional monomer. The MMIN exhibited high selectivity and affinity towards PQQ, allowing for efficient extraction and preconcentration of PQQ from complex food matrices. The extracted PQQ was then quantified using HPLC-DAD. The developed method showed good linearity (R2 = 0.9985) and low limits of detection (0.03 µg L-1). The accuracy and precision of the method were evaluated by analyzing spiked food samples, with average recoveries close to 89.8%. The MMIN also demonstrated good reusability, with negligible decrease in extraction efficiency after five cycles of use. Overall, the developed MMIN-based method provides a reliable and efficient approach for the analysis of PQQ in food samples.

A new α-amylase inhibitory peptide from Gynura medica extract.

In this study, we discovered a novel peptide, Gymepeptide A, with α-amylase inhibitory activity in the water extract of Gynura medica. The structure of Gymepeptide A was determined as CGDREETR using HR-MS, 1H NMR, 13C NMR, and 2D-NMR techniques. Notably, Gymepeptide A possesses a rare double arginine residue structure and exhibits strong α-amylase inhibitory activity. Enzyme dynamic assays, molecular docking experiments, and isothermal titration calorimetry indicated that the double arginine residue structure of Gymepeptide A interacts with amino acid residues in the nearby active site region of α-amylase through hydrogen bonds and van der Waals forces. This interaction effectively inhibits the hydrolysis activity of α-amylase. Furthermore, in vitro starch digestion tests revealed that Gymepeptide A significantly reduced the digestion rate of starch and the concentration of glucose produced after starch digestion. These findings highlight the great potential of Gymepeptide A in decreasing postprandial blood glucose levels.

Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction.

In this paper, new supramolecular extractants, which contained surfactant, alkane and alkanol, were designed and used to separate PQQ. After a series of tests, the optimal extractant composition was determined as benzalkalonium (C8-C16) chloride (BC): n-hexane:n-pentanol, and the highest extraction rate could reach 98%. The extraction equilibrium could be reached in five minutes. The mechanism of the extraction selectivity was inferred as an ion-pair and π-π complexation interaction between PQQ and BC, which was indicated by UV and fluorescence quenching experiments. To recycle the organic extractant, the extract was back-extracted with sodium chloride solution. After extraction, back extraction and crystallization, an isolated product with a purity of 97.5% was obtained from G. oxydans fermentation broth. The product was identified as PQQ by HPLC analysis and MS. Above all, the present research developed a simple and efficient method for the separation of PQQ from fermentation broth.

Antifungal Macrocyclic Trichothecenes from the Insect-Associated Fungus Myrothecium roridum.

Three new macrocyclic trichothecenes possessing rare 6'-ketal moieties, roridoxins A-C (1-3), and five known compounds (4-8) were isolated from the insect-associated fungus Myrothecium roridum. Their structures were confirmed by a combination of NMR and HRESIMS data, while their absolute configurations were unambiguously determined by single-crystal X-ray analysis and electronic circular dichroism experiments. Trichothecenes 1 and 3 showed potent antifungal activities against four strains of phytopathogenic fungi. In addition, 1, 3, 5, and 6 were found to significantly inhibit the cell growth of Candida albicans with minimal inhibitory concentration values from 8.8 to 18.5 µg/mL. Moreover, they were able to inhibit the biofilm formation of C. albicans better than the positive control.

Pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth enhances superoxide anion-scavenging capacity of Cu/Zn-SOD.

A bioassay-guided fractionation of extract from Gluconobacter oxydans fermentation broth afforded Compound 1, which was identified as pyrroloquinoline quinone (PQQ) by spectroscopic methods. PQQ has been shown to enhance the superoxide anion-scavenging capacity significantly for Cu/Zn-SOD. To illustrate the mechanism, the interaction between PQQ and Cu/Zn-SOD was investigated. The multiple binding sites involving hydrogen bonds and van der Waals force between PQQ and Cu/Zn-SOD were revealed by isothermal titration calorimetry. The α-helix content was increased in the Cu/Zn-SOD structure with the addition of PQQ into the solution through ultraviolet (UV) spectroscopy. These results indicated that PQQ could change the conformation of Cu/Zn-SOD through interaction, which could enhance its superoxide anion-scavenging capacity. Therefore, PQQ is a potential natural antioxidant.

A new family-3 glycoside hydrolase from Penicillium oxalicum BL 3005 catalyzing tyrosol glucosylation to form salidroside.

A glycoside hydrolase from Penicillium oxalicum BL 3005 was purified to apparent homogeneity. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The enzyme was identified to be a new member of family-3 by peptide sequence. High transglycosylation activity was found in the hydrolytic reaction of cellobiose. In the reaction, salidroside (4-hydroxyphenethyl O-ß-d-glucopyranoside) was formed by adding tyrosol as the glycosyl acceptor. The optimum reaction pH and temperature were pH 6.5 and 55 °C, respectively. The maximum yield of salidroside was almost 20 g/L. These results indicated that the ß-glucosidase of P. oxalicum can be considered as a very promising catalyst for the synthesis of salidroside.

The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.

Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

Bioconversion of lutein to form aroma compounds by Pantoea dispersa.

OBJECTIVES: To investigate the conversion of lutein, a carotenoid, to aroma compounds by Pantoea dispersa Y08, a lutein-degrading bacterium isolated from marigold flower residue. Bioconversion conditions, including substrate concentration, applied co-solvent and reaction time, were optimized. RESULTS: A maximum biodegradation yield of 80 % for lutein at 10 g/l was achieved. The intermediate, 3-hydroxy-ß-ionone, and final ß-ionone products were revealed by GC-MS. A bioconversion pathway of lutein is proposed to involve cleavage at the 9-10 double bond position, followed by de-hydroxylation at the 3-hydroxy position. CONCLUSIONS: This is the first report of the ability of a bacterium, P. dispersa, to sequentially convert lutein to 3-hydroxy-ß-ionone and then ß-ionone.

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation--up to 6 mM--in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

Membrane-bound pyrroloquinoline quinone-dependent dehydrogenase in Gluconobacter oxydans M5, responsible for production of 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose.

A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent D-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.

A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism.

A membrane-bound pyrroloquinoline quinine (PQQ)-dependent D-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of D-sorbitol to L-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by D-sorbitol), while products are released in sequence (first L-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for D-sorbitol, in addition to PQQ-binding and Mg(2+)-binding sites in the catalytic center.

Penicillin acylase catalysis in the presence of ionic liquids.

Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA.

[The structure-function relationship of thermostable beta-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102].

Beta-glycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostable monomeric protein and adopts the (beta/alpha)8 barrel fold, is an excellent model system to be investigated for the thermostable mechanism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly (Wang et al, 2003) and comparison of other proteins structure of homofamily, Glu164 and Glu338 may act as proton donor and nucleophile in the hydrolysis reaction respectively; proline located at N1 of alpha-helix and arginine which can form ion link may contribute to the thermostability. We aim to further identify the critical sites and the amino acid residue(s) responsible for the activity, the thermal stability and the substrate specificity. Mutations had been constructed by site-directed mutagenesis. They are Glu164Gln, Glu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified to SDS-PAGE purity. Changes in the conformations were examined by means of CD. The Glu338Ala mutant showed no detectable hydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. The Glu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-type were determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at N1 positions of alpha-helix, and Arg325 that form salt bridge between alpha-helices 5 and alpha-helices 6, are the critical sites to protein thermostabilization.