Abstract
Lignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Immobilization might be an effective approach for recovering the hydrolysis products and improving the stability and reusability of the enzymes. In this study, we used a recombinant genetic engineering approach to construct a scaffold protein gene (CipA) and a xylanase gene (XynC) fused to a dockerin gene (DocT). After expressing CipA and XynC-DocT (XynCt) genes using E. coli hosts, the crude extracts were collected. An immobilized metal ion affinity membrane/Co2+ ion (IMAM-Co2+) system was prepared to adsorb CipA in its crude extract, thereby allowing simultaneous purification and immobilization of CipA protein. A similar approach was applied for the adsorption of XynCt protein, exploiting the interaction between the cohesin units in IMAM-Co2+-CipA and the dockerin unit in XynCt. The activity of the xylanase unit was enhanced in the presence of Co2+ for both the free XynCt enzymes and the immobilized CipA-XynCt. The heat resistance and stability over a wide range of values of pH of the immobilized CipA-XynCt were superior to those of the free XynCt. Furthermore, the immobilized CipA-XynCt retained approximately 80% of its initial activity after seven reaction cycles. The values of Km and νmax of IMAM-Co2+-CipA-XynCt (1.513 mg/mL and 3.831 U/mg, respectively) were the best among those of the other tested forms of XynCt.
Highlights
Hemicellulose is the second largest chemical component of woody and grassy biomass, and is linked with cellulose in the cell walls of almost all terrestrial plants [1,2]
Constructs of CipA and XynCt pET21d-CipA was constructed following the procedure in Figure 2a; it was transformed into E. coli
(pET21b-XynCt), which was transformed into E. coli DH5α for preservation, and transformed into
Summary
Hemicellulose is the second largest chemical component of woody and grassy biomass, and is linked with cellulose in the cell walls of almost all terrestrial plants [1,2]. As an alternative chemical treatment, the use of enzymes for the degradation of celluloses has the advantages of environmental friendliness, good specificity, and lower by-product formation [4,5]. The structure of xylan is as complex as that of cellulose. The side chains of xylan must be cleaved before the xylan backbone can be completely hydrolyzed, necessitating several enzymes with different specificities to complete its hydrolysis. Because xylan does not form a densely packed crystal structure as cellulose does, it is more readily hydrolyzed enzymatically [6].
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