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Expression, purification and characterisation of recombinant cyclodextrin glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli


Citation

Nik Pa, Nik Ida Mardiana (2020) Expression, purification and characterisation of recombinant cyclodextrin glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli. Doctoral thesis, Universiti Putra Malaysia.

Abstract

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) represents one of the most important groups of microbial amylolytic enzymes that degrade starch for the production of cyclodextrin through cyclisation reactions. The cyclodextrins are able to form complexes with variety of organic and inorganic guest molecules, thus bring to wide range of applications in food, pharmaceutical, chemical industries, agriculture and environmental engineering. The expression of recombinant β-CGTase from Escherichia coli is widely adopted as an efficient approach to produce higher yield of enzyme. However, a major concern in the recombinant β-CGTase production is inadequate enzyme secretion across the two membranes of E. coli cells. Hence, the extracellular expression of recombinant β-CGTase from E. coli and its secretion efficiency need to be improved. Therefore, this study aimed to express the N-terminal His-tagged β- CGTase in E. coli JM109 for the production of functionally expressed enzyme at higher yield. Then, the optimisation of medium supplementation was carried out to enhance the secretion of β-CGTase from E. coli into the culture medium. Further enhancement was also done by optimising the systematic codon usage, followed by optimisation of glycine supplementation using response surface methodology (RSM). The expressed enzyme from the recombinant E. coli was then purified and characterised. In this study, the previously isolated cgt-BS gene from Bacillus sp. NR5 UPM was used to enhance the recombinant β-CGTase production. The cgt-BS gene was cloned into pQE30xa (pQECGT-BS) and expressed in E. coli JM109. The maximum secretion of recombinant β-CGTase was obtained at 12 hours of fermentation with the activity of 30.371 U/mL. The amino acid sequence of CGTBS protein was then characterised by using in-silico analyses to confirm the presence of active, substrate binding and cyclisation sites of this protein. This was done to ensure the CGT-BS protein was functionally expressed in E. coli. The extracellular secretion of β-CGTase from E. coli was optimised by supplementing the medium supplements using one factor at a time approach. The parameters optimised were the types of medium supplements including glycine, xylose and Triton X-100. Then, the medium supplement concentration, time of induction and post-induction temperature were optimised after selecting the suitable medium supplement. With the addition of 1.2 mM glycine as medium supplement, induction at 2 hours of cultivation and post induction temperature at 37°C, the extracellular secretion of β-CGTase was enhanced and increased up to 1.3-fold. Through nucleic acid double staining assay based on flow cytometry, the supplementation of glycine at optimal conditions was shown to not give any apparent effect on the viability of E. coli cells, hence the release of β-CGTase into the culture medium was due to the increased membrane permeability of E. coli cells and not due to cell lysis. Further study on the enhancement of extracellular β-CGTase from E. coli was conducted by optimising the systematic codon usage. The cgt-BS gene from Bacillus sp. NR5 UPM was redesigned by optimising the systematic codon usage to favourably match codon frequencies of E. coli, without changing the amino acid sequence. Following synthesis, the codon-optimised cgt-BS gene was cloned into pQE30xa, namely pQEcoCGT-BS, then expressed in E. coli JM109. The expression of codon optimised recombinant β-CGTase was achieved at 38.782 U/mL at 12 hours of fermentation without supplementation of any medium supplement. The recombinant β-CGTase expression increased up to 1.29-fold, suggesting that the optimised codon usage is crucial for translational efficiency to increase protein expression. Then, the subsequent enhancement by glycine supplementation was done by selecting three factors to be optimised using RSM which were the glycine concentration, time of induction and post-induction temperature. The optimisation process consisted of 20 experiments with six replicates at the centre point. The highest secretion of recombinant β-CGTase (65.524 U/mL) from E. coli harbouring pQEcoCGT-BS was obtained at 12 hours of fermentation with addition of 1.2 mM of glycine, induced at 2 hour of fermentation at 37°C. The validation study was done to confirm the optimal factors for β-CGTase production. In this study, the optimum recombinant β-CGTase activity was obtained at 12 hours of fermentation, which was 1.7-fold increment as compared to a single approach of codon optimisation. Further to that, the recombinant β- CGTase expressed in E. coli was successfully purified by diafiltration method, followed by Ni-NTA chromatography and the pooled elution fraction was concentrated by ultrafiltration method. These purification strategies resulted in 21.55-fold and 23.82-fold of purified β-CGTase expressed in E. coli harbouring pQECGT-BS and pQEcoCGT-BS, respectively. The purified enzyme was characterised using SDS-PAGE which showed the estimated size of 78.6 kDa. As an overall conclusion, a combined approach of codon usage modification and glycine supplementation was able to enhance the extracellular expression of the recombinant β-CGTase up to 65.524 U/mL, which was 2.2-fold than without any optimisation process and 5.6-fold as compared to the wild type strain when using soluble starch as a substrate. This combined strategy could be potentially used to enhance the secretion of recombinant proteins into the culture medium, particularly the recombinant β-CGTase from E. coli. Meanwhile, with the presence of CGTase active sites, sites for cyclisation reaction and starch binding site suggests CGT-BS to be a functional protein.


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Additional Metadata

Item Type: Thesis (Doctoral)
Call Number: FBSB 2020 18
Chairman Supervisor: Assoc. Prof. Norhayati Ramli, PhD
Divisions: Faculty of Biotechnology and Biomolecular Sciences
Depositing User: Mas Norain Hashim
Date Deposited: 16 Aug 2021 05:43
Last Modified: 16 Aug 2021 05:43
URI: http://psasir.upm.edu.my/id/eprint/90518
Statistic Details: View Download Statistic

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