Sung-Chyr Lin

ENHANCED BIOSURFACTANT PRODUCTION BY A BACILLUS LICHENIFORMIS MUTANT

Abstract A Bacillus licheniformis mutant derived by random mutagenesis with n -methyl- n ′-nitro- n -nitrosoguanidine treatment producing high levels of the lipopeptide biosurfactants was selected by an ion-pair plate assay. The mutant, designated B. licheniformis KGL11, is capable of producing lipopeptide biosurfactants at concentrations up to 391 mg l −1 which is twelve times more than the parent strain. HPLC analysis, infrared spectroscopy analysis, and surface tension measurement indicated that the biosurfactants produced by the mutant were identical to those produced by the wildtype strain. The biosurfactants exhibited a low surface tension of 26.5 dyne cm −1 and a low critical micelle concentration of 10 mg l −1 . Similar to the wildtype strain, the mutant produced biosurfactants at the mid-exponential phase and subsequently deactivated biosurfactants at the onset of the stationary phase; nevertheless, the deactivated biosurfactants was re-excreted into the culture upon further incubation.

General approach for the development of high-performance liquid chromatography methods for biosurfactant analysis and purification

Abstract A general approach for the development of HPLC methods for biosurfactant analysis and purification was proposed. By comparing the chromatograms of the cell-free fermentation broth, the ultrafiltration filtrate, and the ultrafiltration filtrate of a methanol–surfactant mixture, the peaks corresponding to biosurfactants can be identified without any prior structural information of the biosurfactants. It can be assumed that the peaks observed only on the chromatogram of the filtrate of methanol–surfactant mixture but not on the chromatogram of the filtrate are biosurfactant peaks. This approach can be applied for the development of a HPLC assay for any biosurfactants as long as the concentration of biosurfactants in the fermentation broth is higher than the critical micelle concentration. The HPLC methods thus developed can also be adapted for the preparation of homogeneous biosurfactant samples useful for chemical analysis for the elucidation of chemical structure of biosurfactants and for the determination of the physical properties of biosurfactants.

Enhanced protein renaturation by temperature-responsive polymers

The application of temperature-sensitive polymer (PNIPAAm) for the renaturation of beta-lactamase from inclusion bodies was investigated. It was observed that PNIPAAm was more effective than PEG in enhancing protein renaturation. At a concentration of 0.1%, PNIPAAm improved the yield of beta-lactamase activity by 41% from 46. 5 to 65.4 IU/mL, compared to 26% with PEG from 46.5 to 58.7 IU/mL. Kinetic study indicated that PNIPAAm did not significantly affect the initial rate of protein renaturation but did increase final activity yield. In the presence of PEG and PNIPAAm, the activity yields increased with temperature, indicating that hydrophobic interactions between denatured protein and polymer molecules contributed to the enhanced protein renaturation with polymers. The sequential addition approach, aiming at enhancing protein renaturation by reducing local protein concentration during renaturation, was also shown effective in enhancing protein renaturation, especially in the presence of polymers. With the sequential addition approach, the activity yield was increased by 60. 5% from 46.5 to 74.6 IU/mL with PNIPAAm. Similar behavior was also observed with PEG. PNIPAAm exhibited similar behavior as PEG on the renaturation of beta-lactamase in terms of temperature effect and concentration effect, indicating that the mechanism for enhanced protein renaturation for the two polymers might be similar. PNIPAAm exhibits a lower critical solution temperature (LCST) of 32 degrees C and can be effectively separated from aqueous solution and recycled. A protein renaturation process employing PNIPAAm, which offers the advantages of enhanced renaturation efficiency, minimum loss of protein aggregates, and ease of polymers recycling, was proposed.

Production of d-amino acid precursors with permeabilized recombinant Escherichia coli with d-hydantoinase activity

Abstract Recombinant Escherichia coli cells expressing d -hydantoinase were used as the biocatalysts for the production of N-carbamoyl- d -hydroxyphenylglycine from dl -p-hydroxyphenylhydantoin. Although high concentrations of DMSO could lead to enzyme denaturation, in the presence of 1.5% DMSO, the rate of product formation increased by more than 80% due to enhanced permeability of the cell membrane and increased substrate concentration. Reduced mass transfer resistance, achieved by the permeabilization of cell membrane with CTAB and glutaraldehyde, led to a 60% increase in the rate of production. However, in addition to causing a shift of optimal pH toward lower pH, permeabilization of the cell membrane resulted in reduced enzyme stability toward thermal and organic denaturation. Nevertheless, the stability of the d -hydantoinase of the recombinant cells toward pH, temperature and organic solvents can be significantly enhanced by immobilization.

PRODUCTION OF N-CARBAMOYL-D-HYDROXYPHENYLGLYCINE BY D-HYDANTOINASE ACTIVITY OF A RECOMBINANT ESCHERICHIA COLI

Abstract Recombinant Escherichia coli BL21 (DE3) harbouring plasmid pET36 encoding d -hydantoinase from Pseudomonas putida were used as biocatalyst for the production of N -carbamoyl- d -hydroxyphenylglycine from dl - p -hydroxyphenylhydantoin. The optimum d -hydantoinase activity was observed at 40°C and pH 8.5. At a substrate concentration of 300 mg/l, an initial reaction rate of 24.52 mM/h g cells was obtained and 91% of the substrate was converted into product after a 24-h reaction. Recombinant cells were immobilized within calcium alginate beads with diameters ranging from 2 to 3 mm. The specific activity of the immobilized cells increased with cell loads, probably due to reduced mass transfer resistance. The immobilized cells also exhibited an optimal pH of 8.5. However, under the conditions described above, the initial reaction rate with the immobilized cells as the biocatalysts was reduced by 87% to 3.19 mM/h g cells, probably due to the formation of cell aggregates inaccessible to substrate. Thermostability and reusability of d -hydantoinase were increased upon immobilization. The initial reaction rate with immobilized cells was increased with temperature at least up to 60°C. More than 95% of the d -hydantoinase activity was recovered after three cycles for the immobilized cells, compared to 22% for the free cell systems.

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers.

Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.