Molecular and Biochemical Characterization of α-glucosidase

Molecular and Biochemical Characterization of an α-glucosidase (MalA) from the Halophilic Archaeon Haloquadratum walsbyi

Mara Cuebas-Irizarry¹*, Ricardo Irizarry-Caro¹*, Carol López-Morales, Keyla Badillo-Flores², and Rafael Montalvo-Rodríguez^1§

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Abstract

Alpha-glucosidases are enzymes capable of hydrolyzing nonreducing terminal 1,4-linked D glucose residues. α-Glucosidases (EC. 3.2.1.20) constitute a widespread group of exo-acting glucoside hydrolases that catalyze the hydrolysis of α-D-glucose from the non reducing ends of various α-linked substrates (Alarico et al., 2008; Naested et al., 2006). Alpha glucosidases also catalyze transglucosylation reactions that are used in the biotechnological industry to conjugate sugars with biologically useful materials or to produce food oligosaccharides (Zhoug et al., 2009). These enzymes are important in carbohydrate metabolism, in glycoprotein processing and quality control (Ernst et al., 2006).

All of the alpha glucosidases characterized from archaea correspond to hypertermophilic archaea. There are no reports in the literature describing an extremely halophilic alpha glucosidase (maltase). In this work we cloned an alpha glucosidase gene from the extremely halophilic archaea Haloquadratum walsbyi. The recombinant protein was purified and partially characterized.

There have been many efforts to study different strategies and procedures in order to determine the best manner to produce desired halophilic enzymes in sufficient amounts for large scale use (Connaris et al., 1998). Some studies that deal with the use of halophilic archaeal enzymes are the following: the purification and biochemical characterization of an extracellular serine protease produced by ­atrialba madagii (Jiménez et al., 2000); the characterization of a high stable α- amylase from the halophilic archaeon Haloarcula hispanica (Hutcheon et al., 2005); α-Amylase activity from the archaeon Haloferax mediterrani (Pérez et al., 2003), α- amylase gene cloning and molecular characterization from the moderate halophile Halomonas meridiana (Coronado et al., 2000); glucose dehydrogenase characterization from the halophilic archaeon Haloferax mediterranei (Bonete et al., 1996); characterization of an alcohol dehydrogenase from the haloalkaliphilic archaeon ­atronomonas pharaonis (Cao et al., 2008) and the purification, sequencing and expression of the extremely halophilic β-galactosidase from Haloferax alicantei (Holmes et al., 1996; Holmes et al., 2000).

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II. Glycoside Hydrolases

Polysaccharides are the most abundant carbon source in biosphere and support heterotrophic growth in the three domains of life. Their utilization involves hydrolysis of them to generate monosaccharides (Verhees et al., 2003). Glycoside hydrolases and glycosyl transferases are important enzymes which are responsible for the hydrolysis and formation of Haloquadratum walsbyi was described for first time in 1980 by Anthony E Walsby. It was detected by conventional microscopy in samples collected from a salt crust in the surface of a hypersaline pool on the Sinai Peninsula (Bolhuis et al., 2004).

Such cells were commonly found as the dominant cell type in these and other waters including natural salt lakes and saltern crystallizer ponds, and were able to be characterized by 16S rRNA gene PCR and fluorescence in situ hybridization (FISH). But it was not until 2004 that the first isolates were obtained in pure culture by two independent groups: Bolhuis and coworkers from (Australia solar saltern) and Burns and coworkers from (Spanish solar slattern) (Burns et al., 2007). The isolates were preliminary named as “C23 and HBSQ001” (Bolhuis et al., 2004; Burns et al., 2004).

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Finally the genome of the strain HBSQ001 was published in the 2006 (Bolhuis et al., 2006).

This organism belongs to the group of halophilic archaea that is dominant in NaCl saturated thalassohaline waters around the world. Phylogenetically, the genus belongs to the family Halobacteriaceae. Haloquadratum’s cells are thin, square or rectangular sheets with sharp corners, many measuring 2-5 μm wide but no more Various type of α-glucosidases with different substrate specificity have been found in microorganisms, where they present a very low affinity for polysaccharides and therefore attack starch at a very slow rate (Galichet et al., 1999). These enzymes were widely distributed among aerobic and anaerobic microorganisms, and could be found intracellular, extracellular or as a cell bound enzyme. These proteins have been extensively studied at the biochemical and molecular levels in a number of bacteria, but very little is know in archaea. α-Glucosidases have been described and purified from members of the archaea like Sulfolobus shibatae, Pyrococcus furious, Pyrococcus woesi, and Thermococcus litoralis but only one gene of archaea has been characterized in S. solfataricus. Several members of the halophilic archaea re capable of using alpha linked sugars as carbon sources. However there no reports describing alpha-glucosidases in the halophilic archaea. This study therefore constitutes the first report about the cloning and biochemical characterization of an alpha-glucosidase with high salinity requirement. have been described so far for the extremely

Materials and methods

Isolation of the α-glucosidase gene in H. walsbyi 

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We used bioinformatics tools to analyze the genome of Haloquadratum walsbyi (http://www.genome.jp/kegg/pathway.html) for the presence of α-glucosidase gen sequences. Phylogenetically, this halophilic archaeon is closely related to H. borinquense (Burns et al., 2007) and it is possible that these organisms might share several genes with high homology. Previous studies made in our laboratoty demonstrated that H.

borinquense has the ability to utilize maltose as a sole carbon source. The finding of a gene annotated as a malA in the genome of H. waslbyi was the starting point for the current study. A set of degenerate primers were designed for this ORF and a PCR reaction was performed using H. walsbyi genomic DNA. The putative maltase gene obtained from H. walsbyi using this approach was cloned and expressed in E. coli Rosetta as described below (Yi Cao et al., 2008). The recombinant protein was isolated and partially characterized as described below also (Yi Cao et al., 2008).

PCR procedure for amplification

Microbial strains and growth conditions

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Haloquadratum walsbyi JCM 12705T (=DSM No.16854) was kindly provided by Dr. Mike Dyall-Smith (Martinsried, Germany.)

Construction of expression plasmids

The α- glucosidase gene (malA) was amplified by PCR using genomic DNA from H. walsbyi. It was amplified by polymerase chain reaction (PCR), using TaKaRa La Taq DNA polymerase. Sequences of forward and reverse synthetic primers containing XhoI and NheI recognition sities were designed as 5’- CCA TAG CTA GCA TGT GGT TGG 3’ and 5’ -CGT CTC GAG ACC TCA GGA AGT ATT GG -3’ respectively. A expression vector was constructed using the plasmid pET28b(+) (Novagen, Germany), which was digested with NdeI/XhoI (New England Biolabs Inc.) The ligation of digested PCR amplicon and plasmid was performed using T4 DNA Ligase (Promega Inc.) The resulting recombinant plasmid was called pET-malA.

Protein expression and purification

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The recombinant plasmid (pET-malA) was transformed into E.coli Rosetta™. Transformed E. coli cells were grown in 4L of Luria Bertani Broth (containing 34 μg/ml chloramphenicol and 30 μg/ml kanamycin) at 37°C. After an OD­₆₀₀ 0.6-0.8, 1mM isopropyl-D-thiogalactopyranoside (IPTG) was added to the culture. Following an induction period of 3h, the cells were harvested by centrifugation (4,000 rpm x 20 min, at 4ºC), resuspended in sodium phosphate buffer (NaH­₂PO₄, pH 8.0; 3M NaCl, 10mM imidazole), and lysed by sonication on ice (100W, 1s of sonication vs. 2s pause, 500 cycles). The cellular extract was centrifugated (13,000rpm, 15min, 4ºC) and the obtained supernatant was loaded into a chromatography column filled with Ni-NTA agarose (Qiagen, Germany), followed by an extensive wash with sodium phosphate and an increased imidazole concentration (≤80mM) to remove… The -glucosidase containing fractions were detected by SDS-PAGE, 10% polyacrylamide gels, which were stained with Bio-Safe™ Coomasie Stain (BioRad Inc.). Protein concentration was determined using the Pierce BCA Protein Assay (ThermoScientific, Inc), using bovine serum albumin (BSA) as standard.

Enzymatic assays

The α-glucosidase activity was determined by measuring the formation of p-nitrophenol (pNP) from the hydrolysis of p-nitrophenyl α-D-glucopyranoside (PNPG, the typical substrate for α-glucosidase) at 40ºC. It was monitored at 420 nm using a spectrophotometer. The reaction mixture consisted of the substrate prepared at 10mM, and it was added in a reaction buffer consisting of 50mM MES, 3M KCl (pH 6.0). Reactions were initiated by the addition of the enzyme to the reaction mixture and terminated after 30mins by the addition of 500μL 1M Na₂CO₃.

Effects of salinity, pH and Temperature

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The effects of salinity were determined in KCl (0-4M) and NaCl (0-5M) as salt variables in a reaction buffer 50mM MES (pH 6.0) in 40°C. Optimal temperature was determined as described before in the following temperatures (°C): 10, 25, 30, 40, 50, 60. Effects of pH were determined at 40°C with 3M KCl in the following buffers: citric acid (pH 2.5-3.5), Sodium acetate (pH 4.0), MES (pH 5-6), Tris-HCl (pH 7-8), Na₂HPO₄ (pH 9.0), CAPS (pH 10.0). All enzyme assays were performed as described above in the standard PNPG assay.

Sequence handling

Sequences encoding for archaea α-glucosidases were retrieved from the Pfam protein families database (Finn, et al. 2014), except Haloquadratum walsbyi C23 that was obtained from KEGG Genes Database. Sequence alignment and phylogenetic tree was performed using MEGA6.

Results and discussion

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Results

In silico analysis of the Haloquadratum walsbyi genome at the Kegg database, (http://www.genome.jp/kegg/pathway.html), revealed an open reading frame potentially coding for a putative alpha glucosidase (Fig. 16). With the use of different databases (Pfam database, Expasy Proteomics Server, PROSITE, Inter Pro Scan, NCBI Conserverd Domains, “DAS –Transmenbranal Predictor Server, TMpred, HMMTOP) and primary annotation, we determined the position of the gene in the chromosome of H. walsbyi, the predicted molecular weight of the coded protein, and its biochemical characterization, which includes the presence of two putative transmembrane helices (Fig. 18). The coordinates of the gene in the chromosome are between 1108309-1105961 and the ORF codifies for a polypeptide of 782 aa (Fig. 17).

The three strains of Haloquadratum walsbyi that were kindly provided by Dr. Mike Dyall-Smith (Martinsried, Germany.) were used to extract their genomic DNA (Fig. 19). To confirm the nature of the strains provided, the genomic DNA extraction was used as template for 16s rDNA amplification by PCR (Fig. 20).

Figure. Evolutionary relationships of taxa

The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 4.81604479 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method [3] and are in the units of the number of amino acid differences per site. The analysis involved 24 amino acid sequences. All positions with less than 100% site coverage were eliminated. That is, fewer than 0% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 475 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [4].

1. Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees.Molecular Biology and Evolution4:406-425.
2. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap.Evolution39:783-791.
3. Nei M. and Kumar S. (2000).Molecular Evolution and Phylogenetics. Oxford University Press, New York.
4. Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.Molecular Biology and Evolution30: 2725-2729.

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