Journal of Molecular Catalysis B-enzymatic最新文献

Cyclodextrin glycosyltransferase (CGTase) can be used for the industrial production of cyclodextrins. However, product inhibition by cyclodextrins largely restrains the cyclization activities of CGTase and severely limits the application of cyclodextrins. In this paper, the kinetic mechanisms of the three kinds of cyclization reaction were studied, and the product inhibition modes of two CGTases from different sources were compared. The results confirm that the synthesis of each cyclodextrin is substantially inhibited by the corresponding cyclodextrin. Meanwhile, product inhibition studies indicate competitive inhibition for α-CGTase and a mixed pattern for β-CGTase. This demonstrates that the inhibition type is not decided by the kinds of cyclodextrins or the varieties of cyclization reactions, but by the structure of the CGTase.

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 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.

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.

Candida antarctica lipase B (CAL-B) is one of the most useful enzymes for preparation of optically active alcohols and amines. However, CAL-B substrate’s scope for enzymatic kinetic resolution (EKR) of carboxylic acids and their derivatives is limited, especially by low stereoselectivity. In an attempt to overcome this drawback, we decided to employ substrate engineering of enzymatic transesterification of 2-bromobutyric esters by changing the alcohol moiety of the structure. The modifications in the substrate resulted mainly in alterations of the conversion rate, but the inclusion of a chiral alcohol moiety such as (R)-1-phenylethanol resulted in inversion of CAL-B enantiopreference. When esters containing ordinary achiral aliphatic alcohol moiety were used, CAL-B presented S-selectivity. This selectivity was unexpectedly changed to R when an ester containing (R)-1-phenylethyl alcohol moiety was introduced. The use of (R)-1-phenylethanol and its derivatives as nucleophiles in the EKR of the 2-bromobutyric esters also resulted in the inversion of enzymatic enantiopreference. This is the first time that CAL-B acyl enantiopreference has been switched by the chiral nature of the alcohol moiety.

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.

Lipozyme TL IM exhibits high enantioselectivity for the resolution of dl-menthol by the esterification of l-menthol. However, in this study, some factors such as protein loss, enzyme inactivation, and acetaldehyde damage greatly reduced the reaction conversion. For relieving the effects of these factors, macroporous resin that absorbs more protein was selected to immobilize Lipozyme TL 100L lipase with trehalose as the modifying agent. The immobilized lipases retained 37.2% of their initial activity after 8 times of repeated use. A packed-bed reaction system was designed to prevent the leaching of adsorbed lipase molecules out of the macroporous resin pore and to outflow acetaldehyde with the product. The immobilized lipase was continuously used with ee_p > 99.0%. Over 83.9% of the initial conversion remained after the reaction solution of 100 column volumes was pumped into the lipase column. The average volumetric productivity of l-menthyl acetate was 0.76 g/L/h. This process is readily applicable to large-scale preparation for optically active menthol.

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.

Aspergillus flavipes l-methionine γ-lyase (AfMGL) has been recognized as a powerful broad range anticancer agent. However, catalytic instability and antigenicity are the main challenges of its applications in vivo. Thus, the objective of this study was to investigate the influence of conjugation with dextran on AfMGL biochemical properties and functionality. The activity of dextran AfMGL conjugates was 50% of the free MGL that consistent with the extent of occupied surface reactive amino groups. The accessibility of reactive ε-amino groups on dextran AfMGL surface was reduced by 70% normalizing to free enzyme. The thermal stability of dextran AfMGL was increased by two folds over the free enzyme. The dextran AfMGL had a higher resistance to proteinase K, retaining about 70% of its initial activity comparing to 20% to the native enzyme after 30 min of proteolysis at 37 °C. The in vivo half-life time of dextran modified AfMGL in New Zealand rabbits was increased by 2.3 folds comparing to free enzyme. The in vitro anticancer activity of the free and modified AfMGL was evaluated against five tumor cell lines (MCF-7, HEPG-2, HCT, PC3, HEP-2). The activity of AfMGL towards the tested tumor cells was significantly increased upon dextran conjugation suggesting the dramatic increasing of MGL hydrophilicity and catalytic efficiency. Dextran solution was used as negative control.