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Molecular Cloning, Expression, and Functional Characterization of the Β-Agarase Agab-4 from Paenibacillus Agarexedens

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Molecular Cloning, Expression, and Functional Characterization of the Β-Agarase Agab-4 from Paenibacillus Agarexedens Chen et al. AMB Expr (2018) 8:49 https://doi.org/10.1186/s13568-018-0581-8 ORIGINAL ARTICLE Open Access Molecular cloning, expression, and functional characterization of the β‑agarase AgaB‑4 from Paenibacillus agarexedens Zeng‑Weng Chen1, Hui‑Jie Lin1, Wen‑Cheng Huang1, Shih‑Ling Hsuan2, Jiunn‑Horng Lin1 and Jyh‑Perng Wang1* Abstract In this study, a β-agarase gene, agaB-4, was isolated for the frst time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883- amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purifed from the soluble frac‑ tion of E. coli cell lysate through immobilized metal ion afnity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specifcity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confrmed to be neoagarotetraose. Purifed rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degrada‑ tion for the production of neoagarotetraose. Keywords: β-Agarase, Paenibacillus agarexedens, Next-generation sequencing, Neoagarotetraose Introduction with α-L-galactose-6-sulfate (Knutsen et al. 1994; Chi Agar is a hydrophilic colloid extracted from the cell walls et al. 2012). of red algae (Rhodophyceae), such as Gelidium spp., Agarases are enzymes that catalyze the hydrolysis of Gracilaria spp., and Porphyra spp. It is a heterogeneous agar into oligosaccharides; these enzymes cleave glyco- polysaccharide which consists of agarose and porphy- sidic bonds at diferent positions. Tus, agarases can be ran (Chi et al. 2012). Agarose is a neutral polysaccharide classifed into α-agarases (EC 3.2.1.158), β-agarases (EC that forms a gel; its molecular weight is approximately 3.2.1.81), and β-porphyranases (EC 3.2.1.178) according 120 kDa; and it consists of alternating β-d-galactose and to the cleavage pattern (Chi et al. 2012). α-Agarases act on 3,6-anhydro-α-l-galactopyranose linked by α-1,3 and the α-1,3 glycosidic bonds of agarose, producing agaro- β-1,4 glycosidic bonds (Armisén 1991; Yun et al. 2017). oligosaccharides with a 3,6-anhydro-α-l-galactose resi- Porphyran, the non-gelling fraction, is a linear sulfated due at the reducing end. β-agarases, on the other hand, galactan; its composition is similar to that of agarose, act on the β-1,4 glycosidic bonds of agarose, producing except that some 3,6-anhydro-α-l-galactose are replaced neoagaro-oligosaccharides with a d-galactose residue at the reducing end (Fu and Kim 2010). β-porphyranases act on the β-1,4 glycosidic bonds of porphyran, produc- ing oligosaccharides with a d-galactose residue at the *Correspondence: [email protected] reducing end. 1 Animal Technology Laboratories, Agricultural Technology Research Various microbes from seawater, marine sediments, Institute, No.52, Kedong 2nd Rd., Zhunan Township, Miaoli County 350, Taiwan, ROC seaweed, marine mollusks, soil, solar salt, city drain Full list of author information is available at the end of the article water, and hot spring produce agarases. Seawater © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Chen et al. AMB Expr (2018) 8:49 Page 2 of 10 isolates, including Alteromonas agarlyticus GJ1B (Potin Materials and methods et al. 1993) and Talassomonas sp. JAMB-A33 (Ohta Bacterial strains, plasmids, and culture condition et al. 2005), produce α-agarases. Based on the amino Paenibacillus agarexedens BCRC 17346 was purchased acid sequence similarity, known α-agarases belong to the from BCRC (Bioresource Collection and Research glycoside hydrolase (GH) family GH96. Compared with Center, Hsinchu, Taiwan) and was used for the isolation the source of α-agarases, more bacterial strains produce of genomic DNA. Escherichia coli ECOS™ 9-5 (Yeastern, β-agarases, such as Vibrio sp. JT0107 (Sugano et al. 1993) Taipei, Taiwan) was used for the propagation and manip- and Catenovulum sp. X3 (Xie et al. 2013) from seawater; ulation of recombinant DNA. E. coli BL21(DE3) (Merck Vibrio sp. PO-303 (Dong et al. 2007) and Agarivorans sp. Millipore, Darmstadt, Germany) was used as the expres- HZ105 (Hu et al. 2009) from marine sediments; Vibrio sion host. pJET1.2 (Fermentas, Maryland, USA) and sp. AP-2 (Aoki et al. 1990) and Pseudoalteromonas ant- pET-29a(+) (Merck Millipore) were used as cloning and arctica N-1 (Vera et al. 1998) from seaweed; Agarivorans expression vectors, respectively. P. agarexedens BCRC albus YKW-34 (Fu et al. 2008) from marine mollusks; 17346 was cultured at 30 °C in nutrient broth medium Paenibacillus sp. SSG-1 (Song et al. 2014) and Altero- supplemented with 0.1% (w/v) urea and 1% (w/v) glucose. monas sp. E-1 (Kirimura et al. 1999) from soil; Halococcus E. coli was grown at 37 °C in Luria–Bertani (LB) medium sp. 197A (Minegishi et al. 2013) from solar salt; Alcali- (Difco, Detroit, USA) containing 30 μg/mL kanamycin, genes sp. Yen (Sie et al. 2009) from city drain water; and when required. Bacillus sp. BI-3 (Li et al. 2014) from hot spring. Based on the amino acid sequence similarity, known β-agarases General DNA techniques are classifed into the four GH families of GH16, GH50, Bacterial genomic DNA was isolated using the DNeasy GH86, and GH118 (Lombard et al. 2014). Unlike the Blood & Tissue Kit (Qiagen, Hilden, Germany). Plasmid source of β-agarases, only two bacterial strains, includ- DNA was isolated using the Plasmid Miniprep Purifca- ing Zobellia galactanivorans DSM 12802 from the red tion Kit II (GMbiolab, Taichung, Taiwan) according to alga and Bacteroides plebeius DSM 17135 from Japa- the manufacturer’s instructions. DNA fragments were nese individuals (Hehemann et al. 2010, 2012), produce amplifed using GDP-HiFi DNA Polymerase (Genedirex, β-porphyranases. Based on the amino acid sequence sim- Las Vegas, USA) according to the manufacturer’s rec- ilarity, known β-porphyranases belong to the GH families ommendations. All polymerase chain reactions (PCRs) of GH16 and GH86. were performed on a TProfessional TRIO thermocycler Previous studies reported that α-agarases and (Biometra GmbH, Göttingen, Germany). PCR products β-agarases have various applications, for example, those were purifed using a PCR Clean-Up Kit (GMbiolab). in the recovery of DNA from agarose gel (Finkelstein Restriction enzyme digestions were performed according and Rownd 1978), preparation of seaweed protoplasts to the supplier’s recommendations (Termo Fisher Sci- (Araki et al. 1998), and production of agar-derived oligo- entifc, Waltham, USA). DNA fragments were recovered saccharides (Fu and Kim 2010). Studies have shown that from gels by using a Gel Elution Kit (GMbiolab). DNA the oligosaccharides generated by the hydrolysis of agar ligation reactions were performed using a DNA Liga- or seaweed polysaccharide crude extracts by agarases tion Kit (Yeastern). Te purifed PCR product was cloned have numerous biological activities, such as antioxidative into pJET1.2 by using a CloneJET PCR Cloning Kit (Fer- activity (Wu and Pan 2004), hepatoprotective potential mentas) according to the manufacturer’s recommenda- (Chen et al. 2006), immunostimulatory activity (Lee et al. tions. Plasmids were introduced into E. coli through heat 2017), antiobesity efect (Hong et al. 2017a), whitening shock transformation according to the manufacturer’s efect on melanoma cells (Jang et al. 2009), moisturizing instructions. efect on skin (Kobayashi et al. 1997), and prebiotic efect (Hu et al. 2006). Hence, agar-derived oligosaccharides Whole‑genome sequencing of P. agarexedens can be used as new-generation, high-value functional oli- Te genomic DNA of P. agarexedens was isolated and gosaccharides in cosmetic, health food, and pharmaceu- quantifed using a Quant-iT dsDNA BR assay (Termo tical industries. Fisher Scientifc). Te quality of the extracted genomic In the present study, the newly identifed β-agarase DNA was verifed on a 0.6% agarose gel. DNA librar- gene agaB-4 from Paenibacillus agarexedens BCRC ies were constructed using an Illumina TruSeq DNA LT 17346 was cloned and expressed in the cytoplasm of Sample Prep Kit. Mate-pair libraries were constructed Escherichia coli BL21(DE3). Te characteristics and using an Illumina Mate Pair Library Prep Kit v2. For potential applications of the purifed recombinant the de novo assembly of the complete genome, we used enzyme were also analyzed. Velvet (Zerbino and Birney 2008) to assemble paired- end and mate-pair reads to scafolds. Genes on these Chen et al. AMB Expr (2018) 8:49 Page 3 of 10 assembled scafolds were predicted using GeneMark. (soluble fraction) was collected, and the remaining pel- hmm (Besemer and Borodovsky 1999) and were anno- let (insoluble fraction) was resuspended in an equal vol- tated using a BLAST (blastp) search against the NCBI nr ume of lysis bufer. Equal volumes of the total cell lysate, protein database, with an e-value cutof of 0.00001. For soluble fraction, and insoluble fraction were analyzed the functional annotation of predicted genes, the acces- through 12.5% sodium dodecyl sulfate polyacrylamide sion numbers from BLAST hits were mapped to GO gel electrophoresis (SDS-PAGE) (Laemmli 1970), using terms by querying the GO database (Ashburner et al.
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