Enzyme immobilization has been known to be one of the methods to improve the stability and reusability of enzyme. In this study, a strategy to optimize laccase immobilization on polyethylene terephthalate grafted with maleic anhydride electrospun nanofiber mat (PET-g-MAH ENM) was developed. The development involves the screening and optimization processes of the crucial factors that influence the immobilization yield such as enzyme concentration, pH values, covalent bonding (CV) time, CV temperature, crosslinking (CL) time, CL temperature and glutaraldehyde concentration using two-level factorial design and Box-Behnken design (BBD), respectively. It was found that laccase concentration, pH values and glutaraldehyde concentration play important role in enhancing the immobilization yield of laccase on PET-g-MAH ENM in the screening process. Subsequently, the optimization result showed at 0.28 mg/ml laccase concentration, pH 3 and 0.45% (v/v) glutaraldehyde concentrations gave the highest immobilization yield at 87.64% which was 81.2% increment from the immobilization yield before optimization. Under the optimum condition, the immobilized laccase was able to oxidize 2, 2-azino-bis 3-ethylbenzothiazoline-6- sulfonic acid (ABTS) in a broad range of pH (pH 3-6) and temperature (20- 70 °C). Meanwhile, the kinetic parameters for Km and Vmax were 1.331 mM and 0.041 mM/min, respectively. It was concluded that the optimization of immobilized laccase on PET-g-MAH ENM enhance the performance of this biocatalyst.
E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L-1) and a productivity of 0.22 g L-1 h-1. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L-1 and 0.26 g L-1 h-1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L-1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.
A suitable nanofiber sheet was formulated and developed based on its efficacy in the immobilization of recombinant Escherichia coli (E. coli) to enhance xylitol production. The effects of different types of nanofibers and solvents on cell immobilization and xylitol production were studied. The most applicable nanofiber membrane was selected via preliminary screening of four types of nanofiber membrane, followed by the selection of six different solvents. Polyvinylidene fluoride (PVDF) nanofiber sheet synthesized using dimethylformamide (DMF) solvent was found to be the most suitable carrier for immobilization and xylitol production. The thin, beaded PVDF (DMF) nanofibers were more favourable for microbial adhesion, with the number of immobilized cells as high as 96 × 106 ± 3.0 cfu/ml. The attraction force between positively charged PVDF nanofibers and the negatively charged E. coli indicates that the electrostatic interaction plays a significant role in cell adsorption. The use of DMF has also produced PVDF nanofibers biocatalyst capable of synthesizing the highest xylitol concentration (2.168 g/l) and productivity (0.090 g/l/h) and 55-69% reduction in cell lysis compared with DMSO solvent and free cells. This finding suggests that recombinant E. coli immobilized on nanofibers shows great potential as a whole-cell biocatalyst for xylitol production.
Cross-linked enzyme aggregates (CLEAs) are influenced by mass diffusion limitations such as the degree of molecular cross-linking attained, which affects substrate accessibility. Thus, this study seeks to improve substrate accessibility using macromolecular cross-linkers in cross-linked levanase aggregates (CLLAs) formation for levan-type fructooligosaccharides (L-FOS) production. Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-D and with bovine serum albumin (BSA) to form CLLAs-DB which showed activity recoveries of 65.6% and 81.6%, respectively. After cross-linking, the pH (6-10) and thermal stability (30-40 °C) increased, and organic solvent tolerance resulted in the activation of CLLAs. Likewise, CLLAs-DB had higher substrate affinity and accessibility and a higher effectiveness factors than CLLAs-D. The total L-FOS yield of CLLAs-DB (78.9% (w/v)) was higher than that of CLLAs-D (62.4% (w/v)). Therefore, as a cross-linker, DAST may have application prospects as a promising and green biocatalyst for product formation.