Enhancing the immobilization of cyclodextrin glucanotransferase producing escherichia coli for direct conversion of starch to ß-cyclodextrin

Abstract

Enzyme can be considered as one of the most used and effective biocatalyst both in research and industry. However, some of the major drawbacks of using enzyme are their cost and the unavoidable one-time-usage. This problem has been temporary solved by enzyme immobilization. Nevertheless, there are still disadvantages especially in terms of the enzyme stability and reduction of catalytic activity. Therefore, whole-cell immobilization, also known as whole-cell biocatalyst, can provide a better solution as the immobilized whole-cell can provide fresh enzyme for each reaction and has high reusability. In this study, Escherichia coli (E. coli) harbouring the enzyme cyclodextrin glucanotransferase (CGTase) was immobilized to a commercial activated charcoal (ACh) for the direct conversion of starch to ß-cyclodextrin (ß-CD). The immobilization of the E. coli was enhanced by employing two methods; first, treatment to the ACh, and second, manipulating the appendages of the E. coli called curli. This is done because cell immobilization can become problematic in terms of the amount of cell successfully immobilized. The treatment of the ACh was done through chemical treatment, using four different chemicals, sodium hydroxide (NaOH), hydrochloric acid (HCl), ammonium hydroxide (NH4OH), and acetic acid (CH3COOH). The treatment was done relatively at mild conditions. Treatment with NaOH showed the highest increment in cell immobilized with more than 120 % increase. This is mainly attributed by the higher surface area and pore volume resulted from the NaOH treatment. The manipulation of the curli was done by the addition of nickel (II) chloride during the immobilization process. The added nickel triggered the natural response of the cell, forcing the cell to produce its curli and making the cell sticky and easily attached to surfaces. This in turn further increased the cell immobilized by at least 50 %. The resulting enhanced-immobilized cell increases CGTase activity by 10 % and can be reused up to 10 cycles for CGTase expression and showed lower cell lysis compare to both the free cell and to the cell immobilized without any treatment. The optimization of starch direct conversion using the immobilized cell was done using the Box-Behnken design in the design expert software. Under the optimized condition, the highest yield of 15.45 mg/mL ß-CD was obtained, which was analysed using high performance liquid chromatography, and the immobilized cell was managed to be reused up to 6 cycles. Based on the results of this study, it can be concluded that the immobilization of E. coli was greatly enhanced by the combination of the two methods, ACh treatment and curli manipulation. Also, the enhancement resulted in less cell lysis and stronger cell attachment to the support material. The immobilized cell was successful in directly converting starch to ß-CD.