Construction and characterization of a xylanase cell surface display system using ice nucleation protein in escherichia coli for degradation of pineapple hemicellulose

• Main Author: Wee, Mei Yuin
• Format: Thesis
• Language: English
• Published: 2020
• Subjects: TP Chemical technology
• Online Access: http://eprints.utm.my/id/eprint/92407/1/WeeMeiYuinJoannePSChE2020.pdf.pdf; http://eprints.utm.my/id/eprint/92407/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:138776

Cell surface display is a method of anchoring enzymes on the surface of cells. It can be used as a whole-cell biocatalyst to catalyse the breakdown of substrates extracellularly. An anchoring motif is an essential tool for the construction of a surface display system in cells. By far, ice nucleation protein (INP) has been among the most successful anchoring motifs studied. However, some problems still arise in relation to its limited expression on the cell surface and also its stability during display. Therefore, exploring a wider selection of INP anchors could be useful in providing one with better surface display efficiency. An INP from Erwinia ananas, InaA, is the anchor employed in this study for the functional display of xylanase enzyme (EC 3.2.1.8). The surface display using InaA fused to xylanase (InaAxyl) was compared with two other established INPs and gave the highest enzyme activity of 92.2 U/g dry cell weight which was up to three times higher than the other two INPs used. The proper expression of InaAxyl on the surface of the cell was confirmed by SDS-PAGE, Western blot, immunofluorescence microscopy and flow cytometry analysis. Quantitative data from flow cytometry showed that surface anchoring using InaA was up to four times more effective than the other two constructs used. Conditions for expression of InaAxyl were optimized using one-factor-at-a-time (OFAT) method. The conditions are post-induction harvest time of 8 h using LB medium with 0.3 mM inducer concentration and agitation rate of 200 rpm at 25 °C. The cell surface display system was then used for the hydrolysis of hemicellulose from pineapple waste. The degradation of lignocellulosic biomass has not been done using a bacterial surface display system before. After subjecting the raw pineapple waste to pretreatment for the breakdown of lignin, hemicellulose extraction was carried out. The hemicellulose extracted pineapple was analysed using Fourier-transform infrared spectroscopy which confirmed the successful extraction of hemicellulose. The morphology of the pineapple waste before and after hemicellulose extraction was also studied using field emission scanning electron microscope. The rough surfaces of the recalcitrant lignin structure before the pretreatment changed to smooth after the extraction of hemicellulose. Then, screening of reaction conditions for InaAxyl with pineapple waste was studied using OFAT. Optimization for pH, cell loading and temperature of reaction was investigated using response surface methodology Box-Behnken design in the DESIGN EXPERT software. A total of 2.129 mg/ml of reducing sugar was produced under the optimized conditions of pH 7.5 using 100 g/L wet cell weight of cells at 30 °C. High performance liquid chromatography (HPLC) was used for qualitative and quantitative analysis to determine the type and amount of xylooligosaccharides (XOS) produced from the reaction. The XOS detected were xylobiose and xylotriose with a total yield of 5.4 mg/g of pineapple substrate. Based on the results of this study, it can be concluded that InaAxyl was well expressed on the cell surface in its active and stable form. The cell surface display system successfully degraded pineapple waste into XOS.