Journal of Molecular Catalysis B-enzymatic最新文献

Product inhibition of β-glucosidase is considered as one of the central rate limiting steps as it starts accumulation of intermediates responsible for the slowdown of the cellulose hydrolysis. Feedback inhibitions exhibited by glucose and other oligosaccharides on the cellulose hydrolyzing enzyme reduces the rate of hydrolysis bringing the entire process to standstill. However, the exact mechanism of this catalytic slowdown is still elusive. In present study, β-glucosidases were investigated for their activities under high glucose and cellobiose concentrations. β-glucosidases recognizes cellobiose a true substrate and hydrolyzes it resulting in glucose or transglycosylates it to give cellotriose. Our observation highlight that rates of reaction for cellotriose synthesis and glucose formation are mainly concentration driven and are dynamically adjusted based on cellobiose concentration in the reaction system. We therefore conclude that critical concentration of DP2: DP3 influences hydrolysis or transglycosylation and any modulation to this ratio influences the dynamics of β-glucosidases hydrolysis.

In order to remove the residual lactose in crude galacto-oligosaccharides (GOS), different commercial soluble β-galactosidases from Kluyveromyces lactis (Lactozym Pure 6500L, Maxilact L2000, Lactase NL and Biolactasa-NL) and reaction conditions (temperature, total carbohydrate concentration and enzyme:substrate mass ratio) were evaluated. To select the best biocatalyst, the hydrolytic activity on o-NPG and thermal stability of all enzymes were evaluated in the absence and presence of three cations (Co²⁺, Mg²⁺, Mn²⁺) at different concentrations. The enzyme source, cation and cation concentration were selected to obtain the highest hydrolytic activity and thermal stability. Then lactose hydrolysis of raw GOS was assessed varying the temperature (30 °C–45 °C), total carbohydrate concentration (10%-50%) and enzyme:substrate mass ratio (50 IU g⁻¹–400 IU g⁻¹) and considering the lactose percentage decrease as response parameter (D_L). Lactase NL was selected as the best enzyme, with a hydrolytic activity of 286 IU mg⁻¹ and a half-life of 9 h at 35 °C in the presence of 1 mM Mn²⁺. The best reaction conditions for lactose hydrolysis employing the selected enzyme were 35 °C, 50% initial carbohydrate concentration and 135 IU g⁻¹. At such conditions of lactose hydrolysis, 70% reduction of lactose in raw GOS was obtained, with an increase of 48% in monosaccharides and of 30% in GOS. This pre-hydrolytic step is a key aspect for the subsequent purification of GOS by nanofiltration or selected bioconversion, in which monosaccharides can be removed efficiently producing GOS of high purity.

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.

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.

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.

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.

Here, we report regioselectively functionalized synthesis, as well as photo physical, electrochemical, and thermal, of a novel water-soluble conjugated polymer. For this purpose, horseradish peroxidase (HRP)-catalyzed polymerization of a multifunctional monomer, 7-amino-4-hydroxy-2-naphthalene sulfonic acid (AHNAPSA) was carried out by using hydrogen peroxide as the oxidant at room temperature for 24 h under air. The structure of poly(7-amino-4-hydroxy-2-naphthalene sulfonic acid), (PAHNAPSA) was identified by using nuclear magnetic resonance, infrared and ultraviolet-visible. Further characterization was performed by means of gel permeation chromatography (GPC), thermogravimetry (TG), differential scanning calorimetry (DSC), cyclic voltammetry (CV), photoluminescence (PL), dynamic light scattering (DLS) and solid state conductivity measurements. The spectral analysis results exhibited functional group selective polymerization of the monomer containing a multi-active center. Solvent effects on the optical, electrochemical and photo physical properties of PAHNAPSA were investigated by using five different solvents. PAHNAPSA presented an irreversible redox characterization at different scan rates. Optical band gap of PAHNAPSA is also found in the range of 3.18 eV to 3.55 eV. The fluorescence measurements were utilized to investigate the photochemical behaviors of PAHNAPSA in selected polar solvents. Accordingly, PAHNAPSA surprisingly presented multicolor emission behavior with relatively high quantum yield in all selected solvents. In addition, PAHNAPSA presented a reversible pH-responsive behavior and also had high selectivity and sensitivity towards chromium ions. Finally, the kinetic parameters associated with the solid state thermal degradation of PAHNAPSA were calculated from isoconversional methods. The TG/DTG analysis showed that PAHNAPSA followed a diffusion controlled degradation mechanism in N₂.

A major drawback to the industrial production of β-cyclodextrin is the limited β-cyclization activity of cyclodextrin glycosyltransferase (CGTase). Here, we construct mutants of the β-CGTase from Bacillus circulans strain STB01 that contain single substitutions at Tyr89 and double substitutions at Tyr89 and Asp577. The results show that the double mutants Y89G/D577R, Y89D/D577R, and Y89N/D577R display enhanced β-cyclization activity, and have higher β-cyclization activity than that of the three single Tyr89 mutants. The double mutant Y89D/D577R exhibited the highest β-cyclization activity and β-cyclodextrin production, increasing 35.1% and 12.4% compared with those of the wild-type CGTase, respectively. The β-cyclization activity of double mutant Y89D/D577R is also higher than that of the single mutant D577R, which had the highest β-cyclization activity among the mutants prepared in our previous studies. The enhanced β-cyclization activity of these mutants may be a result of intermolecular interactions that stabilize intermediates in the β-cyclization reaction. Thus, double mutant Y89D/D577R is much more suitable for industrial β-cyclodextrin production than the wild-type enzyme.

The immobilization of Candida rugosa lipase by adsorption was performed onto commercial titania powder (Degussa P25). The change of titania particles surface was diagnosed by means of FTIR and FESEM analysis, as well as by shift of zeta potential value towards that of lipase. A detailed study of the effect of immobilization on enzyme kinetic, temperature stability, as well as on potential for its reuse in aqueous organic media was undertaken. Immobilization of lipase altered enzyme affinity toward substrates with different length of carbon chain in hydrolytic reaction. The Vmax value decreased 2–8-fold, where major constraint was registered for the ester containing the longest carbon chain. Thermostability of lipase improved more than 7-fold at 60 °C. Significant potential for reuse in water solutions was also found after immobilization. In cyclohexane immobilized lipase catalyzed synthesis of amyl octanoate by ping-pong bi–bi mechanism with inhibition by amyl alcohol. Obtained kinetic constants were Vmax = 26.4 μmol min⁻¹, K_Ac = 0.52 mol/L, K_Al = 0.2 mol/L and K_i,Al = 0.644 mol/L. Esterification activity remained 60% after 5 reuse cycles in cyclohexane indicating moderate reuse stability.