Journal of Molecular Catalysis B-enzymatic最新文献

In this study, Y. lipolytica lipase LIP2 (Lip2) was immobilized on the macroporous adsorptive resin DA201-C in n-heptane and then used to catalyze the continuous synthesis of geranyl propionate in a continuous stirred tank reactor (CSTR). With the lipase loading of 0.01 g/g resin, 25 °C and adsorption for 3 h, the immobilization efficiency can be up to 98.6% and the synthetic activity of the lipase was 690.8 U/g after immobilization, representing an increase of 28.5% compared with the free lipase (synthetic activity 537.2 U/g). The immobilized Lip2 was used to generate geranyl propionate in CSTR, the continuous production of geranyl propionate was performed with 3 g of immobilized Lip2 at 35 °C. At the flow rate of 6 mL/h the process can maintain its steady state at least for 12 h, which was about three times of the average residence time, and the esterification ratio of 72.8% could be attained. Finally, FT-IR and NMR were used to identify the ester.

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.

Presently the production and use of biodiesel is not cost-effective in comparison to traditional fossil fuels. Naturally occurring enzymes, such as lipases and esterases, can potentially be engineered to lower the cost of certain steps in the biodiesel synthesis process that would otherwise be more costly. However, these enzymes have evolved to perform biologically relevant functions, and not necessarily to manufacture biodiesel under commercially viable conditions. To this end, we have identified, cloned, expressed, purified, and characterized two proteins from the staphylococcal lipase family that are capable of catalyzing the formation of fatty acid alkyl esters. In an effort to explore strategies for improving these fatty acid modifying enzymes (FAMEs), we have engineered a chimeric fusion protein that significantly increases the esterification of free fatty acid with ethanol. The fusion protein, which consists of a staphylococcal FAME fused to a Drosophila ethanol binding protein, demonstrably improves the rate of catalysis by providing an additional substrate binding site and concomitant increase in the local concentration of substrate. This results in greater overall substrate (ethanol) residence in proximity to the catalytic domain, and a faster rate of catalysis, without the necessity of altering the amino acid sequence of the FAME protein.

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).

Toluene Dioxygenase (TDO) enzymatic complex has been widely used for preparation of enantiopure cis-cyclohexadienediols for synthetic applications. Along the last 30 years, a variety of mono- and di-substituted arenes have been studied as substrates for this enzyme, and some interesting observations have been reported regarding the yield and selectivity of the biotransformation. Nevertheless, none of them has been explained considering the active site’ structural and electronic features. In this work we present the first computational model of TDO’s active site, with a description of its architecture and interactions with the substrate to understand and predict substrate orientation. Our findings indicate that in the O₂-free TDO, the iron(II) is stabilized by the coordination of an oxygen atom from the neighboring Gln215 residue. Besides, the active site is comprised by four pockets with different relative affinities for the substrates’ substituents. Monosubstituted arenes adopt a pose in which the alkyl chains maximize the London dispersion interactions with minimal steric clashes, giving an explanation for the observed trend in the benzylic hydroxylation yields. Finally, the computational results allowed us to rationalize the enantiomeric excess of 1,4-disubstituted arenes and the regioselectivity of the dihydroxylation of fluorobenzene. These data were used to develop and validate an affinity index (A_I), as a quantitative indicator of the orientation preference for these substrates. This practical and easy-to-use tool can be applied to successfully predict the orientation of the para-disubstituted benzenes into the TDO active site.

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.

Amino acid composition, protein conformation, weak interactions are some of the factors determining protein stability. Two point mutants of Bacillus LipJ (Ala81Tyr and Asn166Tyr) were designed and generated for comparison. Residue Ala81 targeted for substitution was buried in protein core with 0% accessibility. On other hand, residue Asn166 at protein COOH terminal was predicted to be located within αF helix near surface. Point variants Ala81Tyr and Asn166Tyr were generated by site directed mutagenesis. In comparison to native protein LipJ, point variant Ala81Tyr was observed to be more thermostable and demonstrated 1.6 times relative activity after 1 h exposure at 50 °C. However, point variant Asn166Tyr showed dramatic shift in its thermostability, specific activity and substrate specificity pattern compared to wild type LipJ. Asn166Tyr variant was found to remain stable for hours at 50 °C. Specific activity of Asn166Tyr was 20% of parent enzyme. Substrate specificity of Asn166Tyr was significantly shifted to fatty acyl chains of shorter lengths. Homolog modelling predicted global vs local structure change to explain molecular basis of differential behavior of point variants Asn166Tyr and Ala81Tyr respectively.

A thermophilic lipase from Thermus thermophilus (lipaseTt) was heterologously overexpressed in Escherichia coli BL21(DE3). The tolerance of (lipaseTt) towards polar organic solvents and the associated activation phenomena were investigated. The catalytic efficiency (Kcat/K_M) of lipaseTt in 56% DMSO was up to 25.64 times higher than that in phosphate buffer. Molecular dynamic (MD) simulations were carried out to investigate the activation mechanism of the lipaseTt by polar organic solvents. After refining the homology modeling of lipaseTt by using MD-simulation, a new secondary structure in the lid region indicates a rigid structure, corresponding to its nature of thermostability. Subsequently, the loss of a secondary structure of lipaseTt in the presence of polar organic solvent was observed in both MD-simulation and Circular dichroism spectra. The results of MD-simulation demonstrate that the overall structure maintained stable with or without polar organic solvent; however, the lid region was found more flexible in the presence of polar organic solvent. The flexible lid facilitates the substrate to access the catalytic site inside the lipase and the lipase displays enhanced activity in the presence of a polar organic solvent.

The asymmetric synthesis of chiral 1,3-oxathiolan-5-ones has attracted a great deal of attention owing to their broad biological activities and importance as intermediates for a range of useful pharmaceuticals. However, the enantiomeric purity of desired enantiomer was moderate while maintaining high yield. Here, high enantiomerically pure chiral 1,3-oxathiolane intermediate was synthesized by cyclization-acetylation reaction in organic media using immobilized Trichosporon laibachii (T. laibachii) lipase with one-pot process. Response surface methodology (RSM) integrated a desirability function approach was used to study and optimize the reaction conditions. The effects of several reaction parameters (initial water content of reaction medium, lipase amount and reaction temperature) on enantiomeric excess (ee) and the conversion of benzoyl aldehyde (C) were evaluated. The enzyme activity as well as enantioselectivity was markedly influenced by the initial water content of reaction medium and reaction temperature. The maximum overall desirability (D) of 0.958 was achieved at initial water content of 0.46%, lipase amount of 4.58% and reaction temperature 43 °C. The corresponding maximum ee and C were 94.33% and 97.40%, respectively. A good correlation between the predicted and actual responses showed that the generated model could adequately predict ee and C.