Journal of Molecular Catalysis B-enzymatic最新文献

Dextransucrase from Leuconostoc mesenteroides B-512F was covalently immobilized on glutaraldehyde-actived chitosan particles. The best initial protein loading (400 mg/g of dried support) showed 197 U/g of catalytic activity. The optimal reaction pH and temperature of this new biocatalyst were determined to be 4.5 and 20 °C, respectively. Regarding the thermal stability, the immobilization enhanced enzyme protection against high temperatures, whereas glucose and maltose acted as stabilizers. The biocatalyst was stable under storage at 5 °C for a month. The biocatalyst presented good operational stability, retaining up to 40% of its initial activity after ten batch cycles of reaction to obtain oligosaccharides. These results suggest the use of the immobilized dextransucrase on chitosan particles as a promising novel biocatalyst to produce dextran and oligosaccharides.

A blackberry-shaped Fe₃O₄@SiO₂ nanoparticles were prepared, characterized and applied in covalently binding catalase. The enzyme loading decreased with the increase of pH, however, the activity recovery increased simultaneously. To elucidate the influence factor of the activity recovery, the enzyme loading was further regulated by changing the initial free enzyme content. The relationship between enzyme loading and activity recovery showed the consistent trend, whether the variation of enzyme loading was incurred by pH or by initial enzyme content. The simulated parameters showed the similar values according to the experiment at different conditions. It was concluded that activity recovery was dominated by protein density on surface, not by the orientation of the enzyme on surface, due to the negligible diffusion limit for H₂O₂ as the substrate of catalase. The immobilized catalase at pH 7.0 has a high activity recovery of 100% at 14.4 enzyme μg/mg nanoparticles. The K_m and V_max of the immobilized enzyme above are 0.215 mol and 0.797 mol/min, similar to 0.167 mol and 0.727 mol/min for the free enzyme, respectively.

Biodiesel is considered to be a good alternative in renewable energy generation; therefore it is well studied throughout for its efficient, economic and greener production. Present study illustrated the use of waste cooking oil and dimethyl carbonate (DMC) as a reactants, enzyme as catalyst that facilitated the biodiesel production by providing low cost reactant, ecofriendly methodology and glycerol carbonate as marketable by-product. It also includes resolution to the problems conjured using above combination like prolonged reaction time by applying microwave technology. Additionally the rate of reaction, activation energy and advantages of microwave technology over conventional method in terms of reduced requirement of DMC is also summarized in this manuscript. It is found that, about 94% conversion was obtained in just four hours using microwave irradiation when operated at optimised parameters which include temperature, enzyme loading, water content, molar ratio reactants and addition of surfactant. Lipase 435 used as a catalyst was found to recover 88% of its activity after catalysing six successive reaction cycles. Biodiesel obtained was observed to fit ASTM D 6751 standards after least downstream steps.

A recombinant Candida antarctica lipase B expressed in Pichia pastoris (LIPB) was immobilized on pore-expanded SBA-15 previously modified 3-amino-propyltriethoxysilane (APTES) and activated with two bifunctional reagents, glutaraldehyde (GA) or divinylsulfone (DVS), producing the biocatalysts: SBA-15-APTES-GA-LIPB and SBA-15-APTES-DVS-LIPB, respectively. After LIPB immobilization, both preparations were then modified with glutaraldehyde, producing the biocatalysts: SBA-15-APTES-GA-LIPB-GA, SBA-15-APTES-DVS-LIPB-DVS. Alternatively, LIPB was immobilized on SBA-15-APTES-DVS at pH 10.2 and the biocatalyst was named SBA-15-APTES-DVS-LIPB-pH10. The different biocatalysts were assayed to check the effect of the immobilization strategies on the stability and in the substrate specificity during the kinetic resolution of (R,S)-Phenylethyl acetate. The thermal stability of some new preparations were higher than LIPB adsorbed on SBA-15 (SBA-15-LIPB) and LIPB immobilized on Glyoxyl-agarose. High conversions in the enzymatic kinetic resolution were obtained (43–50%) for all biocatalysts studied. Regarding activity and stability, the SBA-15-APTES-DVS-LIPB-pH10 was the most successful strategy, since, in first cycle, the maximum conversion was obtained (50%), and the biocatalyst remained active and enantioselective even after five successive cycles.

A sarcosine oxidase (SOX) gene from Bacillus sp. (AY626822.2) was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies. A 3D model of SOX was then built and refined, and molecular docking was used to investigate the interactions between SOX and natural or coenzyme-like ligands, including flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); riboflavin; isoalloxazine; 7-methyl-8-chloro-10-(1′-d-ribityl) isoalloxazine (7-M-8-C); 7-bromo-8-methyl-10-(1′-d-ribityl) isoalloxazine (7-B-8-M); 7-methyl-8-bromo-10-(1′-d-ribityl) isoalloxazine (7-M-8-B); 7-chloro-8-ethyl-10-(1′-d-ribityl) isoalloxazine (7-C-8-E); 7,8-diethyl-10-(1′-d-ribityl) isoalloxazine (7,8-D); and 3-methyl-7,8-dimethyl-10-(1′-d-ribityl) isoalloxazine (3-M-7,8-D). Unfolded SOX was extracted from inclusion bodies, and reconstructed with these ligands via a refolding process. The reconstructed enzymes were then subjected to structural and catalytic analysis. After structural simulation, refinement, and molecular docking, all ligands were able to recognize the coenzyme site of SOX. In addition, when the position 7- or 8-site of the compounds was modified, new pi-cation/sigma interactions were formed in the SOX-ligand complex. Fluorescent detection revealed that all the ligands could be successfully reconstructed with unfolded SOX. Circular dichroism (CD) spectra and nano differential scanning calorimetry (DSC) analysis indicated that the loss of phosphoric acid and adeninein natural coenzymes could significantly reduce the α-helix content, transition temperature (T_m), and calorimetric enthalpy (ΔH). In addition, although reconstruction with the position 7- or 8-site modified compounds led to variations in secondary structure, no significant shifts in T_m and ΔH were observed. Furthermore, in the evaluation of catalytic kinetic parameters, when SOX was reconstructed with ligands containing halogen atoms at the 7- or 8-sites, much higher relative specificities in the presence of organic solvents were noted.

It is usually time-consuming to determine intrinsic kinetic parameters of bisubstrate enzymes, especially when experimental kinetic data deviate from a linear Lineweaver-Burk plot due to complex inhibition patterns. A typical example is ω-transaminase (ω-TA) which is an industrially important enzyme for asymmetric synthesis of chiral amines. ω-TA catalyzes transfer of an amino group between a donor (D) and an acceptor (A) via a ping-pong bi-bi mechanism and often displays substrate inhibitions by reactive amino acceptors, which leads one to prefer to determine apparent kinetic parameters rather than intrinsic ones despite limited applicability for precise understanding of enzyme properties. Here, we developed a new method to determine intrinsic kinetic parameters of ω-TA by double-reciprocal analysis using only two sets of kinetic data. First, linear regression of 1/initial rate (v_i) against 1/[A] was carried out with one set of kinetic data measured at a fixed [D] while [A] lay far below the concentration range under the influence of substrate inhibition. Second, another linear regression of 1/[D] vs 1/v_i was conducted with one set of kinetic data obtained at a fixed [A] within a substantial substrate inhibition range. The resulting four equations obtained from the y-intercepts and slopes of the two regression lines were used for determination of four intrinsic kinetic parameters, i.e. turnover number (k_cat), substrate inhibition constant (K_SI) for A and Michaelis constants (K_M) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including K_SI.

Three new metabolites were obtained on incubation of androgenic steroid mesterolone (1) with Cunninghamella blakesleeana. These metabolites were identified as 1α-methyl-11β,14α,17β-trihydroxy-5α-androstan-3-one (2), 1α-methyl-7β,17β-dihydroxy-5α-androstan-3-one (3), and 1α-methyl,17β-hydroxy-5α-androstan-3,7-dione (4). During this study, hydroxylation at C-11, C-14, and C-15, and oxidation at C-7 of substrate 1 were observed. β-Hydroxylation at C-11 is a rather unique transformation by C. blakesleeana, as α-hydroxylation is reported to be catalyzed by most of the other microorganisms.

Lipases from Candida antarctica (A and B) (CALA and CALB), Candida rugosa (CRL), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML), as well as the chimeric phospholipase Lecitase Ultra (LU) were immobilized on octyl agarose or on heterofunctional octyl supports. RML, CRL and TLL were covalently immobilized on octyl agarose beads activated with divinyl sulfone (OCDVS), while the other lipases were immobilized on octyl-glyoxyl beads (OCGLX). The 12 biocatalysts were utilized in the production of esters using tributyrin and 20% (v/v) methanol, ethanol or isopropanol via a kinetically controlled strategy. All preparations produced the desired ester, except RML, TLL and LU for isopropyl butyrate. CALA showed the best performance in these reactions, with maximum yields over 40%. The immobilization on heterofunctional supports usually reduced the activity and even the maximum yields, although some exceptions were relevant (e.g., CALA or CALB in the production of ethyl butyrate). The effect of the nucleophile was also very different using the just physically adsorbed or the covalently immobilized preparations, some instances one preparation has as best substrate an alcohol while the best substrate was other alcohol using the other lipase preparation.

Using CALB as model enzyme, we have shown the advantages of the use of the covalent preparation. The increase of the alcohol permitted the increase in maximum ester yields. However, the combined presence of dibutyrin and alcohol prevented the reuse of OC-CALB due to the enzyme desorption, while the covalent preparation could be reused by 6 cycles.

Inulin is a type of fructose polymer that is commonly present in plants as a storage carbohydrate. Enzymatic hydrolysis of inulin via exo-inulinase to produce fructose is an efficient, green and state-of-the-art technique. To achieve the high-level secretory expression of inulinase and to realize enzymatic preparation of fructose syrup from inulin, an Aspergillus exo-inulinase gene inu was codon-optimized and co-expressed with the endoplasmic reticulum secretion protein in Pichia cells. After inducible expression in a 500-L pilot scale bioreactor, the inulinase activity of the recombinant strains reached 10,480 U/mL of cultivation broth. Next, according to the determined enzymatic characteristics of inulinase INU, we optimized the parameters for inulinase to hydrolyse inulin. Under the optimal condition of the enzyme/inulin ratio of 5000 U/g, 15% substrate and an incubation temperature of 50 °C for 4 h, the hydrolysis ratio of inulin reached 100%. The hydrolysis products of inulin contain two components, 95% fructose, and 5% glucose. This study has fulfilled the scaled-up production of inulinase and facilitated its industrial application for enzymatic preparation of fructose from inulin.