Co-Immobilization of GOD & HRP on Y-Shaped DNA Scaffold and the Regulation of Inter-Enzyme Distance.

In multienzymes cascade reaction, the inter-enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y-shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP-DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter-enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance-dependent. Moreover, the inter-enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg-1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.

Improving the Encapsulation Efficiency of Lipase in Molecular Cages and Its Application.

Here, lipase encapsulation is constructed by locking enzyme molecules in nanomolecular cages on the surface of SH-PEI@PVAC magnetic microspheres. To improve the encapsulation efficiency in enzyme loading, the thiol group is efficiently modified on the grafted polyethyleneimine (PEI) using 3-mercaptopropionic acid. N2 adsorption-desorption isotherms reveal the existence of mesoporous molecular cages on the microsphere surface. The robust immobilizing strength of carriers to lipase demonstrates the successful encapsulation of enzymes in nanomolecular cages. The encapsulated lipase shows high enzyme loading (52.9 mg/g) and high activity (51.4 U/mg). Different sizes of molecular cages are established, and the cage size showed important effects on lipase encapsulation. It shows that enzyme loading is low at a small size of molecular cages, which is attributed to that the nanomolecular cage is too small to house lipase. The investigation in lipase conformation suggests that the encapsulated lipase retains its active conformation. Compared with the adsorbed lipase, the encapsulated lipase shows higher thermal stability (4.9 times) and higher resistance to denaturants (5.0 times). Encouragingly, the encapsulated lipase shows high activity and reusability in lipase-catalyzed synthesis of propyl laurate, suggesting the potential application value of encapsulated lipase.

Fabricating cholyglycine-glucose-6-phosphate dehydrogenase conjugates for cholyglycine detection.

To establish cholyglycine (CG) detection via enzyme multiplied immunoassay technique (EMIT), glucose-6-phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten-enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine and CG-G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG-G6PD conjugates were investigated. Consequently, CG amount, NADH, D-glucose-6-phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG-G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG-G6PD conjugate as test kit, the cholyglycine-EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten-G6PD conjugates. This article is protected by copyright. All rights reserved.

Fabricating cholyglycine-glucose-6-phosphate dehydrogenase conjugates for cholyglycine detection.

To establish cholyglycine (CG) detection via enzyme-multiplied immunoassay technique (EMIT), glucose-6-phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten-enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine, and CG-G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG-G6PD conjugates were investigated. Consequently, CG amount, nicotinamide adenine dinucleotide, d-glucose-6-phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG-G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG-G6PD conjugate as test kit, the cholyglycine-EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten-G6PD conjugates.

Kinetic biosynthesis of L-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM).

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of L-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of L-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing L-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of L-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K(m,a) and V(max) were 80.085 mM and 0.747 mM min(-1), respectively. As a result, Lipozyme TLIM-catalyzed esterification of L: -ascorbic acid gave a maximum conversion of 99%.

Biosynthesis of benzyl cinnamate using an efficient immobilized lipase entrapped in nano-molecular cages.

To improve the performance of lipase in biosynthesis of benzyl cinnamate, a new immobilized lipase by entrapping enzyme into nano-molecular cages was designed. Consequently, the entrapped lipase showed a robust immobilization, which diminished the leakage of lipase notably in use. Moreover, the entrapped lipase exhibited higher activity (57.1 U/mg) than free lipase (50.0 U/mg), demonstrating that the native conformation of lipase was not destroyed during immobilization. Compared with the adsorbed lipase (half-life 40.7 min) and free lipase (half-life 29.8 min), the entrapped lipase (half-life 85.3 min) increased the stability by about 2-3 times. Furthermore, the entrapped lipase was applied in biosynthesis of benzyl cinnamate, where it showed excellent activity and re-usability. After 7 cycles, the yield of benzyl cinnamate catalyzed by the entrapped lipase remained 70.2%, while the yield catalyzed by the adsorbed lipase was only about 10%. These results indicated that the nano-molecular cages could inhibit denaturation of lipase and maintain its activity well.

PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics.

Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1 mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.

High-yield synthesis of bioactive ethyl cinnamate by enzymatic esterification of cinnamic acid.

In this paper, Lipozyme TLIM-catalyzed synthesis of ethyl cinnamate through esterification of cinnamic acid with ethanol was studied. In order to increase the yield of ethyl cinnamate, several media, including acetone, isooctane, DMSO and solvent-free medium, were investigated in this reaction. The reaction showed a high yield by using isooctane as reaction medium, which was found to be much higher than the yields reported previously. Furthermore, several parameters such as shaking rate, water activity, reaction temperature, substrate molar ratio and enzyme loading had important influences on this reaction. For instance, when temperature increased from 10 to 50 °C, the initial reaction rate increased by 18 times and the yield of ethyl cinnamate increased by 6.2 times. Under the optimum conditions, lipase-catalyzed synthesis of ethyl cinnamate gave a maximum yield of 99%, which was of general interest for developing industrial processes for the preparation of ethyl cinnamate.

Synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid.

In this study, lipase catalysis was successfully applied in synthesis of benzyl cinnamate through esterification of cinnamic acid with benzyl alcohol. Lipozyme TLIM was found to be more efficient for catalyzing this reaction than Novozym 435. In order to increase the yield of benzyl cinnamate, several media, including acetone, trichloromethane, methylbenzene, and isooctane, were used in this reaction. The reaction showed a high yield using isooctane as medium. Furthermore, the effects of several parameters such as water activity, reaction temperature, etc, on this reaction were analyzed. It was pointed out that too much benzyl alcohol would inhibit lipase activity. Under the optimum conditions, lipase-catalyzed synthesis of benzyl cinnamate gave a maximum yield of 97.3%. Besides, reusable experiment of enzyme demonstrated that Lipozyme TLIM retained 63% of its initial activity after three cycles. These results were of general interest for developing industrial processes for the preparation of benzyl cinnamate.

Kinetic and thermodynamic investigation of enzymatic L-ascorbyl acetate synthesis.

Kinetics and thermodynamics of lipase-catalyzed esterification of l-ascorbic acid in acetone were investigated by using vinyl acetate as acyl donor. The results showed that l-ascorbic acid could generate inhibition effect on lipase activity. A suitable model, Ping-Pong Bi-Bi mechanism having substrate inhibition, was thus introduced to describe the enzymatic kinetics. Furthermore, the kinetic and thermodynamic parameters were calculated from a series of experimental data according to the kinetic model. The inhibition constant of L-ascorbic acid was also obtained, which seemed to imply that enhancing reaction temperature could depress the substrate inhibition. Besides, the activation energy values of the first-step and the second-step reaction were estimated to be 37.31 and 4.94 kJ/mol, respectively, demonstrating that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature.

Effect of hydrophobic/hydrophilic characteristics of magnetic microspheres on the immobilization of BSA.

Core-shell magnetic poly(styrene-acrylamide-acrylic acid) microspheres with carboxyl groups were successfully synthesized via dispersion copolymerization in the presence of nano-particle of Fe(3)O(4). The microspheres were characterized by FTIR spectra. They were used as carrier to immobilize bovine serum albumin (BSA). To investigate the effect of the microsphere surface properties on the immobilization of BSA, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. The results showed that microspheres with different hydrophobicities/hydrophilicities had different immobilized ratios of BSA. In comparison with microspheres having hydrophilic characteristics those with hydrophobic characteristics had a much higher immobilized ratio. The possible reasons for these observations are discussed. In addition, ester activation and coupling times were optimized with respect to immobilized ratio.

Structure monomorphism of RNA G-quadruplex that is independent of surrounding condition.

G-quadruplex structures in nucleic acids have become attractive targets of study because their distinct structural forms may have diverse biological functions. Here, we preliminary investigated the structure and thermodynamics of G-quadruplexes of RNAs and corresponding DNAs of the same sequence by CD, UV and PAGE. Structural analysis demonstrated that the RNA sequences folded into a parallel-stranded G-quadruplex independently of surrounding conditions with different cations (K(+) and Na(+)) under both dilute and molecular crowding conditions. In contrast, G-quadruplexes of their corresponding DNA sequences showed structural polymorphism depending on the surrounding conditions. Moreover, it was demonstrated that parallel G-quadruplexes of the RNA sequences were more stable than either parallel or antiparallel G-quadruplexes of the same DNA sequences. These results allow us to propose a single, robust RNA G-quadruplex structure that exists under various conditions.