Journal of Molecular Catalysis B-enzymatic最新文献

The four isomeric forms of the flavour linalool oxide (pyranoid) were synthesized starting from the inexpensive monoterpene terpinolene. The key steps of the process include mCPBA epoxidation of the starting diene, alumina-catalysed rearrangement of the obtained diepoxide and the diastereoselective reduction of the ketone 2,2,6-trimethyl-6-vinyldihydro-2H-pyran-3(4H)-one. The resulting racemic cis- and trans-linalool oxides were resolved through an enzyme-mediated acetylation procedure. More specifically, we found that Candida rugosa lipase and lipase PS are the catalysts of choice for the resolution of cis- and trans-linalool oxide, respectively.

Monolignol oxidoreductases from the berberine bridge enzyme-like (BBE-like) protein family (pfam 08031) catalyze the oxidation of monolignols to the corresponding aldehydes. In this report, we explore the potential of a monolignol oxidoreductase from Arabidopsis thaliana (AtBBE-like protein 15) as biocatalyst for oxidative reactions. For this study we employed a variant with enhanced reactivity towards oxygen, which was obtained by a single amino acid exchange (L182V). The pH and temperature optima of the purified AtBBE-like protein 15 L182V were determined as well as the tolerance toward organic co-solvents; furthermore the substrate scope was characterized. The enzyme has a temperature optimum of 50 °C and retains more than 50% activity between pH 5 and pH 10 within 5 min. The enzyme shows increased activity in the presence of various co-solvents (10–50% v/v), including acetonitrile, 2-propanol, 1,4-dioxane, and dimethyl sulfoxide. Primary benzylic and primary or secondary allylic alcohols were accepted as substrates. The enantioselectivity E in the oxidation of secondary alcohols was good to excellent (E>34 to >200).

Lysine decarboxylases (LDCs) catalyze the conversion of l-lysine to cadaverine, a highly attractive building block for bio-based polyamides. Due to economic and environmental concerns, LDCs active at elevated pH are highly desirable. In this study, a new LDC from Aliivibrio salmonicida (AsLdc) was discovered, expressed, and characterized. Compared to the LDCs from Escherichia coli, LdcC and CadA, the latter was frequently used for cadaverine production, the purified AsLdc showed much higher activities at alkaline pH 7.0–8.5, for instance, 205.1 U/mg at pH 7.5 with 10 μg/mL enzyme, in comparison to 68.3 and 51.5 U/mg for CadA and LdcC, respectively. The activities of AsLdc and CadA correlated well with the proportions of decamers at the pH range of 5.0–8.5. AsLdc with a melting temperature of 79 °C was more thermostable than CadA (73.6 °C). When used for whole-cell biotransformation of l-lysine to cadaverine at pH 7.5, AsLdc completed the transformation within 7 h while the CadA did only 82.8%. These results indicate the high potential of the new AsLdc for the industrial production of cadaverine.

6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone), the major gingerol present in ginger rhizomes, has been found to prevent fat accumulation and improve blood flow. However, its low solubility in water and strong pungent odor often limit its potential applications such as in functional foods. In order to overcome these disadvantages, we investigated microbial glucosylation of 6-gingerol by the soil bacterium Ensifer sp. M-26. 5-O-α-Glucosylgingerol was found to be produced from a mixture of maltose and 6-gingerol by the action of washed cells of Ensifer sp. M-26. The addition of organic solvents, such as hexane, to the reaction mixture enhanced 5-O-α-glucosylgingerol production. When the microbial reaction was carried out with 1.3 g/L of 6-gingerol and 100 g/L of maltose under optimized conditions, 0.40 g/L of 5-O-α-glucosylgingerol was accumulated in 25 h. The molar yield of 5-O-α-glucosylgingerol was 19.9 mol%.

An efficient, cost effective and environmentally friendly protocol has been developed for the Michael addition of malononitrile on 1,3-diaryl-2-propen-1-ones (Chalcones) using very cheaper, easily available natural catalyst, baker’s yeast. The whole cells of yeast excellently worked in nonaqueous medium, ethanol without decrease in catalytic activity.

Biotransformation of steroid compounds: androst-4-ene-3, 17-dione (1), 19-nor-4-androstene-3, 17-dione (2), 13β-ethyl-4-gonene-3, 17-dione (3) using Penicillium decumbens as biocatalyst was investigated, and selective hydroxylation of 1–3 was observed. Biotransformation of 1 afforded 1α-hydroxy-androst-4-ene-3, 17-dione (4) and 1α, 6β-dihydroxy-androst-4-ene-3, 17-dione (5). Incubation of 19-nor-4-androstene-3, 17-dione (2) with P. decumbens culture resulted in the isolation of compounds 1α-hydroxy-19-nor-4-androstene-3, 17-dione (6) and 1α, 15β-dihydroxy-19-nor-4-androstene-3, 17-dione (7). Biotransformation of 3 gave one product 1α-hydroxy-13β-ethyl-4-gonene-3, 17-dione (8) at the same condition. The four metabolic products 5–8 were reported for the first time and the identified C1-α hydroxylation reaction of steroid by P. decumbens provides an another possibility to perform chemical modifications on C-1.

The xylanase gene (xynTF16) from a thermophilic bacterium Geobacillus sp. TF16 was cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant enzyme (GsXynTF16) was purified 52-fold by nickel affinity chromatography, and determined as a single band 39.8 kDa on SDS-PAGE with a specific activity of 246 U/mg protein. The recombinant enzyme was immobilized on chitosan with a yield of 85.6%. The enzyme showed the highest activity towards xylan. The immobilized enzyme displayed an increase in optimum temperature from 55 to 65 °C in comparison with free enzyme. While the free enzyme was optimally active at pH 8.5, immobilized enzyme showed higher activity in the pH range 6.0–8.5. Thermal and pH stability of immobilized enzyme was determined to be higher than that of the free enzyme. Immobilized xylanase could be reused for 6 consecutive cycles retaining 80% of its initial activity. It was also found to be effective in releasing the reducing sugar from juice and poultry feed and oven spring in bakery. These results suggest that this study provides an alternative xylanase enzyme with enhanced properties.

Thermophilic enzymes have attracted widespread attention due to their unique characteristics such as temperature, solvent, and pH stability. In this study, two novel esterases were identified in hyperthermophilic bacterium Aquifex aeolicus VF5 based on the typical characteristics of lipolytic enzymes. Two genes, Aaeo1 and Aaeo2 encoded 207 and 226 amino acids respectively, were synthesized after optimization according to the P. pastoris codon preferences. Using sequence alignment and phylogenetic analysis, Aaeo1 and Aaeo2, were classified into the bacterial lipolytic enzyme family VIII and V, respectively. Two genes were cloned and expressed in P. pastoris cells, and the recombinant enzymes were purified from culture supernatants by Ni²⁺-NTA affinity chromatography and characterized. The optimum temperatures of Aaeo1 and Aaeo2 were 80 °C and 85 °C, respectively, and both of the optimal pH was 8.0. Aaeo1 had the activity preference both to short and long chain p-nitrophenyl esters and triacylglycerides, and the K_m and k_cat of the best substrate, p-nitrophenyl butyrate (p-NPC₄) were 5.11 ± 0.31 mM and 16.12 ± 2.32 s⁻¹, respectively. While Aaeo2 only displayed high activity towards short chain p-nitrophenyl esters, p-nitrophenyl butyrate (p-NPC₄), and exhibited K_m and k_cat of 0.79 ± 0.03 mM and 3.59 ± 1.15 s⁻¹, respectively. The activities of both esterases were inhibited by PMSF, DEPC, and EDTA, but were activated by DTT. The two esterases exhibited certain tolerance to various organic solvents and some detergents. These characteristics make them very promising for the industrial application.

Alkaline proteases produced from protein-rich waste (hair waste and soya residues) by solid state fermentation (SSF) were immobilised onto functionalized magnetic iron oxide nanoparticles (MNPs) using glutaraldehyde as a crosslinking agent. The covalent binding method had a better immobilisation yield compared to simple adsorption, retaining 93%–96% (459 ± 106 U/mg nanoparticles, 319 ± 34 U/mg nanoparticles) of hair waste and soya residues proteases, respectively after crosslinking with 5% glutaraldehyde for 6 h. However, the adsorption immobilisation yield was 47%–54% after 8 h for both proteases. MNPs and immobilised proteases were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and electron diffraction. Our results indicated successful crosslinking between the proteases and amino-functionalized MNPs. The operational stability (pH and temperature) and storage stability of free and immobilised enzyme were also analysed. Despite the fact that the optimum pH of free and immobilised proteases was identical in the alkaline region, the immobilised proteases reached their optimum condition at higher temperatures (40 °C–60 °C). After 2 months of storage at 4 °C, the immobilised proteases showed good stability, retaining more than 85% of their initial activity. The high magnetic response of MNPs render an ease of separation and reusability, which contributes to the residual activity of both immobilised proteases on MNPs retaining more than 60% of their initial values after seven hydrolytic cycles. These results showed the enhancement of the stability of the crosslinking interactions between the proteases and nanoparticles. The immobilised proteases were capable of hydrolysing selected proteins (casein, oat bran protein isolate, and egg white albumin). However, differences in the degree of hydrolysis were observed, depending on the combination of the protease and type of substrate used.

l-Ornithine, a non-protein amino acid, is widely used as a food supplement and nutrition product that is usually produced by the enzymatic hydrolysis of l-arginine. However, urea, a by-product, complicates purification of l-ornithine. Here, we describe a highly efficient coupled system, which involves Rummeliibacillus pycnus arginase and Jack bean urease, for the production of l-ornithine without urea residue. Although R. pycnus arginase exhibited the highest enzyme activity at pH 9.5 with Mn²⁺, it could be activated by Ni²⁺ at 70 °C and pH 6.5. Kinetic study showed that, compared with Mn²⁺-containing arginase, Ni²⁺-containing arginase had a higher substrate affinity and catalysis efficiency. Based on the properties of commercial Jack bean urease and recombinant arginase, both enzymes showed robust activity at pH 6.5 and 40–50 °C. Using a coupled system involving arginase and urease for bioconversion, l-ornithine production of 37.8 g l⁻¹ was achieved with a 99.7% molar yield within 5 h. Thus, this coupled system represents an applicable strategy for the biosynthesis of l-ornithine.