UDC 577.154
MOLECULAR MUTAGENESIS AT Tyr-101 OF THE AMYLOMALTASE TRANSCRIBED FROM A GENE ISOLATED FROM SOIL DNA
© 2014 S. Watanasatitarpa*, P. Rudeekulthamrong**, K. Krusong***, W. Srisimarat***, W. Zimmermann****, P. Pongsawasdi***, J. Kaulpiboon*****
*Biochemistry and Molecular Biology Program, Faculty of Medicine, Thammasat University, Pathumthani 12120 Thailand; **Department of Biochemistry, Phramongkutklao College of Medicine,
Bangkok 10400 Thailand ***Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand ****Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig 04130 Germany *****Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University,
Pathumthani 12120 Thailand e-mail: jkaulpiboon@yahoo.com Received September 6, 2013
The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportion-ation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for dis-proportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.
DOI: 10.7868/S0555109914030325
Amylomaltase (EC 2.4.1.25) is a member of the 4-a-glucanotransferase group within the a-amylase family, with a high transferase but low hydrolytic activity [1—3]. The enzyme is categorized in the glycoside hydrolase (GH) family 77 due to the lack of domain C succeeding the catalytic (P/a)8 barrel of the main GH 13 in the a-amylase family [1]. This enzyme is unique in the ability to convert starch or related saccharides to CAs or large-ring cyclodextrins with a degree of polymerization of 16 glucose units upwards through the intramolecular transglycosylation (cy-clization) reaction. The enzyme can also catalyze 3 types of intermolecular transglycosylation reaction (disproportionation, coupling, and hydrolysis reactions). Most amylomaltases have relatively high disproportionation activity whereby the glycosyl group is transferred from one a-1,4 glucan molecule (donor) to another molecule (acceptor). The other two activities are of minor to amylomaltase, the coupling and hydrolysis reactions by which CAs are cleaved and the obtained linear glucan is transferred to an acceptor or
water molecule, respectively [3]. Presently, this enzyme has received interest due to beneficial use in producing prebiotic oligosaccharides [4], a thermorevers-ible starch gel for food use [5], and CAs. CAs have high potential in the pharmaceutical, food, agricultural and cosmetic industries [6—8].
Amylomaltase was first found in Escherichia coli as a maltose-inducible enzyme, which is essential for the metabolism of maltose [9]. The amylomaltase gene has been cloned from several bacteria and archaea, e.g. Clostridium butyricum NClMB 7423 [10], Thermus aquaticus ATCC 33923 [11], Aquifex aeolicus [12], Py-robaculum aerophilum IM2 [5], Borrelia burgdorferi [1] and Corynebacterium glutamicum [13]. Based on the study of gits 3D structure, Przylas et al. [14] have analyzed the crystal structure of amylomaltase from T. aquaticus in complex formation with the acarbose inhibitor. They found that acarbose molecule bound at 2 binding sites, the active site and the second glucan binding site (GBS). The active site includes the catalytic and the primary binding sites with 7 conserved
residues (Asp-293, Glu-340, Asp-395, Tyr-59, Asp-213, Arg-291 and His-394) localized in the central (ß/a)8-barrel of domain A. In addition to domain A, this enzyme contains several insertions between the strands of the barrel. All insertions are presented at the C-terminal side of the 3D-barrel, where the second GBS (Tyr-54 and Tyr-101, T. aquaticus numbering [14]) is also located. These insertions are divided into 3 subdomains: subdomain B1, B2 and B3, which might be responsible for the reaction specificity and determination of the product ring size [15, 16]. Previous studies indicated that the binding of glucan substrate to the second GBS through an interaction with the aromatic side chains of Tyr-54 and Tyr-101 is a trigger for amylomaltase to take a completely active conformation for all 4 types of activity, however it obscures the cyclization reaction since the flexibility of the glucan is restricted by such binding [16, 17]. Therefore, to engineer amylomaltase for efficient production of CAs, the amino acid substitution at Tyr-54/101 (in T. aquaticus) [16, 17] or the corresponding Tyr-172 in C. glutamicum [18] were studied. The aim of this study was to clone the recombinant thermostable amyloma-ltase using the gene isolated from the soil DNA and to investigate enzyme properties. Since mutation at Tyr-54 has been extensively studied in previous reports, we here focused on Tyr-101 mutation in the attempt to understand more on the role of the second GBS.
MATERIALS AND METHODS
Bacterial strains, plasmids and chemicals. Escherichia coli BL21 (DE3) was purchased from BioLab (UK). The pET-19b expression vector was obtained from Novagen (Germany). Restriction enzymes, DNA ligase and DNA polymerase, were the products of New England Biolabs Inc. (USA). HisTrap FF™ column was obtained from GE Healthcare (UK). Soluble potato starch, glucose, malto-oligosaccharides and BSA were purchased from Sigma (USA). Yeast extract and tryptone were obtained from Difco (USA). Rhizopus sp. glucoamylase was purchased from Fluka (Germany). CA standard (CA22 to CA50) was kindly provided by Prof. T. Endo, Hoshi University, Tokyo (Japan). Pea starch was purchased from EmslandStärke GmbH (Germany). The commercial glucose oxidase kit was obtained from Human GmbH (Germany). All other chemicals used were of analytical grade.
Construction of WT (p 19bAMY) and mutant (p19bAMY-Y101S) plasmids. Soil samples were collected from potential sites at Ban Nong Khrok hot spring in Thailand [19]. The bacterial DNA from soil samples was directly extracted by ISOIL kit (Nippon gene, Japan). A 1.5 kb amylomaltase gene was amplified using the AMYp13 forward and reverse primers. The amylomaltase gene was first constructed in pET17b and named as p17bAMY recombinant plas-mid [19]. To purify enzyme easily, the amylomaltase
gene from p17bAMY plasmid was sub-cloned into the NdeI and EcoRI sites of pET-19b vector containing an NH2-terminal His-tag sequence. The obtained plasmid was renamed as p19bAMY and transformed into E. coli BL21 (DE3) host cells by heat shock technique as described by the manufacturer (New England Biolabs, USA). Transformant cells were selected by blue-white colony screening. The recombinant plasmid was extracted, purified and checked for plasmid size before DNA sequencing.
The mutant plasmid (p19bAMY-Y101S) was constructed by PCR-mediated site-directed mutagenesis using the p19bAMY recombinant plasmid as the template with the mutagenic primers changing the codon for Y-101 by S-101, as underlined (forward mutagenic primer: GGCCTCCT CTCCGCCTGGAAGTGGC and reverse mutagenic primer: GCCACTTCCA GGCGGAGAGGAGGCC). The PCR conditions used were as follows: an initial denaturation at 95°C for 2 min, followed by 16 cycles of amplification at 95°C for 1 min, 60°C for 1 min, 72°C for 7 min and final elongation at 72° C for 5 min. The reaction mixture was treated with DpnI at 37°C for 24 h, following which only Y101S mutated plasmid was transformed into E. coli BL21 (DE3). Transformants were selected on LB plates containing 100 ^g/mL ampicillin, and the existence of mutated plasmid was proved by DNA sequencing.
Expression and purification of amylomaltase. E. coli cells harboring the p19bAMY or the p19bAMY-Y101S recombinant plasmids were grown in an LB medium containing 100 ^g/mL ampicillin at 37°C for 24 h. The expression of both recombinant enzymes was induced by 0.8 mM IPTG when OD600 of the culture reached 0.5. After 4 h, cells were harvested and sonicated using a Vibra Cell™ VCX130 (tip diameter of 5 mm, Sonics, USA) in an ice bath with 50% amplitude for 3 cycles of 5 min pulse and 5 min pause. Bacterial cell debris was removed by centrifugation at 12,000 x g, 4°C for 2 h. The obtained supernatant was used as a crude enzyme. It was put into dialysis tubing (molecular weight cut-off at 10 kDa, Sigma-Aldrich, USA) and then concentrated by AQUASORBTM before loading to HisTrap FFTM column. The column (0.7 x 2.5 cm) was equilibrated with at least 5 column volumes of 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and 20 mM imidazole at a flow rate of 1 mL/min. Then, the crude enzyme from the WT or mutant cells was applied to the column and washed with the same buffer until OD280 of eluent decreased to a steady baseline. The bound proteins were eluted by 500 mM imidazole in the same buffer. The active fractions were pooled and assayed for enzyme activity by starch transglycosylation assay [20], and the protein concentration was determined by Bradford method [21], using BSA as the standard.
Assay of amylomaltase a
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