Expression of xylanase on Escherichia coli using a truncated ice nucleation protein of Erwinia ananas (InaA)

Cell surface display involves the anchoring of enzymes on the surface of cells to be used as whole-cell biocatalysts. In this study, an ice nucleation protein (INP) anchor from Erwinia ananas IN-10, InaA, was used to display xylanase. The ability of InaA to be targeted to the outer membrane was compared to cells displaying xylanase using two other INPs, InaK and InaZ. SDS-PAGE and western blot of the outer membrane fraction proved that surface targeting was successful. Whole-cell xylanase activity showed that InaA anchored xylanase gave an activity of 92.2 U/g dry cell weight which was up to three times higher than the other two display constructs used. Surface anchoring of InaA was up to four times better compared to the other two INP anchors as was confirmed by flow cytometry. Expression of InaA on the surface was optimized by one-factor-at-a-time (OFAT) to obtain the optimum parameter conditions for highest surface expression. The results presented showed that InaA is an excellent INP for surface display for xylanase and has great potential in the degradation of xylan

Bioconversion of pineapple pomace for xylooligosaccharide synthesis using surface display of xylanase on Escherichia coli

Cell surface display of xylanase on Escherichia coli was used for the hydrolysis of hemicellulose from pineapple pomace. The feasibility of bioconversion of lignocellulosic biomass into xylooligosaccharides (XOS) was investigated. In this study, pineapple pomace was pretreated, and the hemicellulose fraction was obtained for reaction with the whole-cell biocatalyst. FESEM and FTIR analyses were used to observe morphological and compositional changes of pineapple pomace respectively after pretreatment. Factors affecting hydrolysis reaction were investigated and optimized using the Box-Behnken Design. The highest amount of reducing sugar was produced at pH 7.5, cell loading of 100 g/L wet cell weight, and temperature of 30 °C. The amount of reducing sugar produced was 2.129 mg/ml. HPLC analysis indicated that the XOS produced were xylobiose and xylotriose with a total yield of 5.4 mg/g of pineapple hemicellulose. FESEM analysis on the surface structure of pineapple pomace after the hydrolysis reaction showed clear signs of degradation by xylanase. Based on the results presented, it can be deduced that the application of cell surface display on E. coli for degradation of lignocellulosic biomass is possible and should be explored as it offers great potential for the production of XOS in industry.