Journal of Molecular Catalysis B-enzymatic最新文献

This work extends the present knowledge about the ability of filamentous fungi and Baker’s Yeast to selectively transform oxygen-containing compounds. Previously, it has been demonstrated that several species of the Aspergillus genus are able to perform selective oxidation of benzopyrans. Isochroman or 3,4-dihydro-1H-benzopyran (1) was chosen as model substrate for the biotransformation since related motifs are often found in the structure of natural products with important biological and pharmacological activities. All the tested strains showed the ability to oxidize 1. Chemodivergent reaction pathways between the employed microorganisms were observed. The use of cytochrome P450 enzyme inhibitors, and different oxygenation conditions allowed to inquire about the type of enzymes involved in the process. The results obtained were compared with chemical one-electron oxidation of compound 1 and thus, a metabolic pathway was proposed.

Toluene dioxygenase-catalysed (TDO) oxidation converts substituted phenol substrates into catechols, hydroquinones, and chiral cyclohexenone cis-diol products. The ratio between the isolated products varied widely even between similar substrates, e.g. o-cresol, m-cresol and p-cresol. These differences are caused by different binding interactions within the active site of TDO. This study provides insight into the binding interactions by molecular docking using AutoDock tools. The nature of binding of phenolic substrates was of major interest, in order to explain the observed regio- and stereo-selectiviy of product formation. The ellipse-shaped binding pocket of TDO consists of a polar and a hydrophobic region, limiting the possible substrate orientations. The phenolic hydroxyl group was preferentially hydrogen bonded with Gln-215 and His-311 in the active site. In some cases, a hydrogen bond was formed with other amino acids, e.g. Asp-219 and Met-220, instead. The position and type of the substituent on the phenol ring influences the formation of transient intermediates, and thus the nature and stability of the major isolated product.

In this study, a recombinant carotenoid cleavage dioxygenase 4 was produced from Osmanthus fragrans by E. coli under different bacterial growth conditions and used to develop a biotechnological method for preparation of natural β-ionone from β-carotene. β-ionone was analyzed by HPLC and OfCCD4 activity was measured based on concentration change of β-ionone. At pH 8.0 and 35 °C, the greatest activity of the purified recombinant protein was 14.3 U/mg and the maximum concentration of β-ionone was 71.186 mg/L within 1 h. Both the enzyme activity and the concentration of β-ionone could increase by nearly 6 times with addition of 9% Triton X-100 and 2% liquid paraffin.

The catalase activity of three unspecific peroxygenases (UPOs) from the agaric basidiomycetes Agrocybe aegerita, Coprinopsis cinerea and Marasmius rotula was investigated. The study included analysis of pH dependency of the catalase reaction and H₂O₂ mediated enzyme inactivation as well as experiments on the influence of a second substrate on the course of catalase reaction. Apparent kinetic parameters (K_m, k_cat) for the catalase activity of UPOs were determined. Inactivation of UPOs by H₂O₂ is discussed with regard to O₂ production and remaining UPO activity. Furthermore formation of biliverdin as heme destruction product was demonstrated along with the formation of UPO compound III as a possible intermediate that forces the destruction process. Radical trapping experiments with methyl benzoate gave indication for the formation of hydroxyl radicals in the presence of excess H₂O₂. Eventually, a plausible pathway of heme destruction has been proposed, proceeding via UPO compound III and subsequent hydroxyl radical formation, which in turn may cause heme bleaching and verdoheme and biliverdin formation.

Chondroitin lyases are therapeutically important enzymes. The functional aspects of a chondroitin AC lyase (PsPL8A) from Pedobacter saltans DSM12145 were investigated. PsPL8A was cloned in to pET28a(+) vector, expressed in E. coli BL-21(DE3) cells exhibited a molecular size of approximately, 77 kDa. PsPL8A displayed maximum activity with chondroitin 4-sulphate, C4S (489 U mg⁻¹) followed by chondroitin 6-sulphate, C6S (214 U mg⁻¹) and hyaluronic acid (43.2 U mg⁻¹). PsPL8A was maximally active at 39 °C and pH 7.2. 100 mM Na⁺ and 20 mM Ca²⁺ ions enhanced the activity of PsPL8A by 2-fold. The time dependent TLC analysis of PsPL8A degraded products of C4S revealed the presence of higher degree of polymerization (DP) chondroitin sulphate (CS) oligosaccharides at initial stage, but after 1 h, only ΔC4S disaccharide was produced as the major product. This result displayed that PsPL8A follows initially a concomitant endo- and exo-lytic mode which finally shifted to exolytic mode of catalysis. The oligosaccharides released were identified as di-, hexa-, octa- and dodeca-saccharide by ESI–MS analysis. The ΔC4S disaccharide showed a peak at m/z 458 (ESI–MS) while in MS/MS mode it gave the peak at m/z 300. ESI–MS/MS, ¹H- and ¹³C- NMR analyses confirmed the structure of ΔC4S disaccharide product obtained after 24 h reaction of C4S with PsPL8A. The enzyme reported in present study can be used for cancer mitigation, spinal cord injury treatment and CS oligosaccharides production which act as anti-inflammatory agents. This is the first study reporting the cloning and expression of chondroitin AC lyase from Pedobacter saltans DSM 12145.

Here we studied non-edible fat waste as a starting material to prepare eutectic mixtures of biomaterials. Initially, the fat was hydrolyzed using water and R. oryzae resting cells. The hydrolysis was performed in organic solvent-free media and the degree of hydrolysis at 1 h was 42% while hydrolytic values of 86% and 98% were achieved at 12 and 48 h, respectively. To recover the resting cells, they were extracted in consecutive cycles with solvents or supercritical CO₂. Compared with solvents, supercritical CO₂ allowed the highest reuse. Hence, R. oryzae was used for 336 h (7 reaction cycles), yielding 56.5 g of free fatty acid/g biocatalyst. Crude glycerol was recovered, showing a purity of 66.0% and an ash and water content of 2.3% and 1.8%, respectively. The hydrolyzed fat was crystallized with several solvents to yield palmitic and stearic acid mixtures with melting point characteristics of eutectic mixtures. We recovered 76% to 90% of the palmitic and stearic acids present in the initial hydrolyzed animal fat, depending on the solvent. The palmitic:stearic acid ratios determined by GC-FID were similar to those reported for eutectic mixtures whit phase change materials properties, as were the melting points, which ranged from 51.5 °C to 54.8 °C.

Palm kernel cake (PKC), mainly composed of mannan, lignin and protein, is abundant renewable resource with commercial value. To develop clean and efficient way for PKC refinery, the method based on the synergism of hot water pretreatment (HWP), steam pretreatment (SP) and enzymatic hydrolysis were developed. HWP of 180 °C, 20 min and SP of 121 °C, 20 min showed similar performance for sugar release from PKC. The main saccharides produced from PKC by HWP and SP were mannose and manno-oligosaccharides, while no furfural formed. The surface structure analyzed by SEM showed that HWP enhanced the microporosity of PKC, and the accessibility of which was increased thereafter. When HWP pretreated PKC was further hydrolyzed with enzyme cocktail (cellulase, xylanase, endo-mannanase), 45% of PKC was solubilized compared with the control. The manno-oligosaccharides produced by HWP and SP were converted to mannose and mannobiose by endo-mannanase. The results suggested that both HWP and SP promote enzymatic hydrolysis of PKC by releasing oligosaccharides and enhancing microporosity, and the synergism of which was effective for PKC decomposition.

Newly isolated strains of Wickerhamomyces subpelliculosus were recognized as excellent whole-cell biocatalyst for bioreduction of various ketones. The biocatalytic properties of the new strains were demonstrated in this study by stereoselective bioreduction of acetophenone 1a, 2-heptanone 1b, phenylacetone 1c, 3,4-dimethoxyphenylacetone 1d and 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e. Our study is the first report on application of W. subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of prochiral ketones. In these processes, both the freshly harvested cell paste and the lyophilized cell powder were tested as biocatalyst using glucose or 2-propanol at various concentrations as cosubstrates for cofactor regeneration. The newly isolated strains of W. subpelliculosus showed diverse characteristics, including optimal pH, temperature and organic solvent tolerance. Bioreductions of phenylacetone 1c applying glucose as cosubstrate under various mild conditions resulted (S)-1-phenylpropanol [(S)-2c] in good to excellent conversion (c = 63.4%–99.9%) with excellent enantiomeric excess [ee_(S)-2c = 98.7%–99.8%].

In this study, we developed a whole-cell biocatalysis process for high-level conversion of l-lysine into 5-aminovalerate. To obtain the highly efficient whole-cell biocatalyst, five expression plasmids were constructed to optimize the expression of 5-aminovaleramide amidohydrolase and l-lysine 2-monooxygenase in Escherichia coli. The engineered strain BL-22A-RB-YB harboring plasmid pET22b-davA, pRSFDuet-davB and pACYCDuet-davB was correspondingly obtained. Subsequently, the effects of induction conditions, reaction temperature, metal ion additives, and cell permeability on the whole-cell biocatalyst system were evaluated to improve biocatalytic efficiency. Under optimized reaction conditions, 95.3 g/L 5-aminovalerate was synthesized from 120 g/L l-lysine with a yield of 99.1%, and 103.1 g/L 5-aminovalerate was produced from 150 g/L l-lysine with a molar yield of 85.7%. The 5-aminovalerate production was then further improved using a l-lysine fed-batch strategy, and a hyper 5-aminovalerate production of 240.7 g/L was achieved within 28 h with a yield of 86.8%. The whole-cell biocatalytic system described here demonstrated an environmentally friendly strategy for industrial production of 5-aminovalerate.