Shau-Wei Tsai

Dynamic synergistic effect on Trichoderma reesei cellulases by novel β-glucosidases from Taiwanese fungi.

Dynamic synergistic effects in cellulosic bioconversion have been revealed between Trichoderma reesei cellulases and β-glucosidases (BGLs) from six Taiwanese fungi. A high level of synergy (8.9-fold) was observed with the addition of Chaetomella raphigera BGL to T. reesei cellulases. In addition, the C. raphigera BGL possessed the highest activity (V(max)/K(m)=46.6 U/mg mM) and lowest glucose inhibition (Ki=4.6mM) with the substrate 4-nitrophenyl β-d-glucopyranoside. For the natural cellobiose substrate, however, the previously isolated Aspergillus niger BGL Novo-188 had the highest V(max)/K(m) (0.72 U/mg mM) and lowest Ki (59.5mM). The demonstrated dynamic synergistic effects between some BGLs and the T. reesei cellulase system suggest that BGLs not only prevent the inhibition by cellobiose, but also enhance activities of endo- and exo-cellulases in cellulosic bioconversion. Comparisons of kinetic parameters and synergism analyses between BGLs and T. reesei cellulases can be used for further optimization of the cellulosic bioconversion process.

Lipase-catalyzed dynamic resolution of naproxen 2,2,2-trifluoroethyl thioester by hydrolysis in isooctane

A lipase-catalyzed enantioselective hydrolysis process under continuous in situ racemization of substrate by using trioctylamine as an organic base was developed for the production of (S)-naproxen from racemic naproxen thioesters in isooctane. Naproxen 2,2, 2-trifluoroethyl thioester and 45 degrees C were selected as the best substrate and temperature, respectively, by comparing the time-course variations for the racemization of (S)-naproxen thioesters containing an electron-withdrawing group. A detailed investigation of the effect of trioctylamine concentration on the kinetic behaviors of the thioester in racemization and enzymatic reaction was conducted, in which more than 70% conversion of the racemate (or 67.2% yield of (S)-naproxen) with eep value higher than 92% was obtained. Copyright 1999 John Wiley & Sons, Inc.

Enhancement of (S)-naproxen ester productivity from racemic naproxen by lipase in organic solvents

The kinetics of enantioselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase (triacylglycerol ester hydrolases, EC 3.1.1.3) were examined in various organic mixtures. The effects of solvent hydrophobicity on the activity, selectivity and stability of the enzyme and Naproxen solubility were investigated. Parabolic correlation for the dependence of the kinetic constants and Naproxen solubility on solvent hydrophobicity was found. A mixture of 60% isooctane and 40% toluene (v/v) was selected as the best reaction medium in which improvement of (S)-Naproxen ester productivity was obtained.

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)‐mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55°C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from VS/(Et) = 1.62 mmol/h g and VS/VR = 43.6 of (R,S)‐ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM‐87 from the producer) to 16.7 mmol/h g and 867 of (R,S)‐2‐methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)‐2‐hydroxycarboxylic acid esters, giving improvements of the enzyme performance from VS/(Et) = 1.56 mmol/h g and VS/VR = 41.9 of (R,S)‐ethyl 3‐chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)‐2‐methoxyethyl 3‐chloromandelate (16) for the immobilized enzyme, VS/(Et) = 5.46 mmol/h g and VS/VR = 8.27 of (R,S)‐ethyl 4‐chloromandelate (10) for free SNSM‐87 to 33.5 mmol/h g and 123 of (R,S)‐methyl 4‐chloromandelate (14) for the immobilized enzyme, as well as VS/(Et) = 3.0 mmol/h g and VS/VR = 7.94 of (R,S)‐ethyl 3‐phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)‐2‐methoxyethyl 3‐phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach. Biotechnol. Bioeng. 2008;101: 460–469.

Surfactant enhancement of (S)‐naproxen ester productivity from racemic naproxen by lipase in isooctane

In the enantioselective esterification of racemic Naproxen with trimethylsilyl methanol in isooctane by Candida cylindracea lipase, improvements in (S)‐naproxen ester productivity and enzyme selectivity were demonstrated by adding bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) as the best surfactant. The effect of water content on the enhancement of enzyme activity was elucidated from the reduced adsorption of surfactant molecules on the lipase. A competitive inhibition by the alcohol and a noncompetitive inhibition by the surfactant to the enzyme were found from the kinetic analysis. By using a two‐phase extraction, a complete separation of the surfactant from the organic solution was obtained.

Formulation of two-layer dissolving polymeric microneedle patches for insulin transdermal delivery in diabetic mice

Dissolving microneedles (MNs) display high efficiency in delivering poorly permeable drugs and vaccines. Here, two-layer dissolving polymeric MN patches composed of gelatin and sodium carboxymethyl cellulose (CMC) were fabricated with a two-step casting and centrifuging process to localize the insulin in the needle and achieve efficient transdermal delivery of insulin. In vitro skin insertion capability was determined by staining with tissue-marking dye after insertion, and the real-time penetration depth was monitored using optical coherence tomography. Confocal microscopy images revealed that the rhodamine 6G and fluorescein isothiocyanate-labeled insulin (insulin-FITC) can gradually diffuse from the puncture sites to deeper tissue. Ex vivo drug-release profiles showed that 50% of the insulin was released and penetrated across the skin after 1 h, and the cumulative permeation reached 80% after 5 h. In vivo and pharmacodynamic studies were then conducted to estimate the feasibility of the administration of insulin-loaded dissolving MN patches on diabetic mice for glucose regulation. The total area above the glucose level versus time curve as an index of hypoglycemic effect was 128.4 ± 28.3 (% h) at 0.25 IU/kg. The relative pharmacologic availability and relative bioavailability (RBA) of insulin from MN patches were 95.6 and 85.7%, respectively. This study verified that the use of gelatin/CMC MN patches for insulin delivery achieved a satisfactory RBA compared to traditional hypodermic injection and presented a promising device to deliver poorly permeable protein drugs for diabetic therapy.

Kinetics of Enantioselective Esterification of Naproxen by Lipase in Organic Solvents

The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed.

Improvements in lipase production and recovery from Acinetobacter radioresistens in presence of polypropylene powders filled with carbon sources

Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports. A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth, less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step.

Lipase‐catalyzed enantioselective esterification of S(+)‐naproxen ester prodrugs in cyclohexane

A lipase-catalyzed enantioselective esterification process in cyclohexane was developed for the synthesis of S(+)-naproxen ester prodrugs containing the moiety of N,N-dialkylamino, ethylene glycol or alkyl ether of ethylene glycol. A high enantiomeric ratio of 44 was obtained when di(ethylene glycol) was selected as the best acyl acceptor. A reversible ping-pong Bi Bi mechanism has been employed to elucidate the enzymatic behavior of the initial conversion rate for S(+)-naproxen and the time-course conversions for both enantiomers. Improvement of the enzyme activity was demonstrated when alcohol in excess of its cyclohexane solubility limit was used. The application of excess racemic naproxen in the presence of solid substrate suspensions showed enhanced productivity and enantioselectivity for the desired S(+)-ester. Studies of the recovery and racemization of the remaining R(-)-naproxen are also reported.

Effects of hydrolysis and esterification side-reactions on the kinetic resolution of enzyme-catalyzed irreversible transesterification in organic solvents

A simplified kinetic model for the enzyme-catalyzed irreversible transesterification in organic solvents was proposed by considering the hydrolysis and esterification side reactions. The analysis was extended to the kinetic resolution of activated racemic esters containing a chiral center in the acid moiety, and was confirmed from the lipase-catalyzed enantioselective transesterification between (R,S)-suprofen 2,2,2-trifluoroethyl ester and 2-N-morpholinoethanol (or di(ethylene glycol)) in cyclohexane. Theoretical analysis indicates that the time-course yield and/or the enantiomeric excess for the desired (S)-ester product may be improved if the employed enzyme has a higher activity and/or enantioselectivity for the hydrolysis and esterification than the transesterification.