ZHU Yanbing, GAO He, LI Hebin, NI Hui, JIANG Zedong, LI Lijun, XIAO Anfeng. Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp. AG1[J]. Acta Oceanologica Sinica, 2019, 38(2): 96-106. doi: 10.1007/s13131-019-1349-y
Citation: ZHU Yanbing, GAO He, LI Hebin, NI Hui, JIANG Zedong, LI Lijun, XIAO Anfeng. Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp. AG1[J]. Acta Oceanologica Sinica, 2019, 38(2): 96-106. doi: 10.1007/s13131-019-1349-y

Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp. AG1

doi: 10.1007/s13131-019-1349-y
  • Received Date: 2017-01-14
  • An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21 (DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively. Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.
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  • Allouch J, Helbert W, Henrissat B, et al. 2004. Parallel substrate binding sites in a β-agarase suggest a novel mode of action on double-helical agarose. Structure, 12(4):623-632, doi: 10.1016/j.str.2004.02.020
    Altschul S F, Madden T L, Schäffer A A, et al. 1997. Gapped BLAST and PSI-BLAST:a new generation of protein database search programs. Nucleic Acids Research, 25(17):3389-3402, doi: 10.1093/nar/25.17.3389
    Araki T, Lu Z, Morishita T. 1998. Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). Journal of Marine Biotechnology, 6(3):193-197
    Ardestani A, Yazdanparast R. 2007. Antioxidant and free radical scavenging potential of Achillea santolina extracts. Food Chemistry, 104(1):21-29, doi: 10.1016/j.foodchem.2006.10.066
    Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2):248-254
    Chen Haimin, Yan Xiaojun. 2005. Antioxidant activities of agaro-oligosaccharides with different degrees of polymerization in cell-based system. Biochimica et Biophysica Acta, 1722(1):103-111, doi: 10.1016/j.bbagen.2004.11.016
    Chi W J, Chang Y K, Hong S K. 2012. Agar degradation by microorganisms and agar-degrading enzymes. Applied Microbiology and Biotechnology, 94(4):917-930, doi: 10.1007/s00253-012-4023-2
    Chi W J, Park J S, Kang D K, et al. 2014b. Production and characterization of a novel thermostable extracellular agarase from Pseudoalteromonas hodoensis newly isolated from the West Sea of South Korea. Applied Biochemistry and Biotechnology, 173(7):1703-1716, doi: 10.1007/s12010-014-0958-3
    Chi W J, Park da Y, Seo Y B, et al. 2014a. Cloning, expression, and biochemical characterization of a novel GH16β-agarase AgaG1 from Alteromonas sp. GNUM-1. Applied Microbiology and Biotechnology, 98(10):4545-4555, doi: 10.1007/s00253-014-5510-4
    Cui Fangyuan, Dong Sujie, Shi Xiaocheng, et al. 2014. Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01T. Marine Drugs, 12(5):2731-2747, doi: 10.3390/md12052731
    Duckworth M, Yaphe W. 1971. The structure of agar:Part I. Fractionation of a complex mixture of polysaccharides. Carbohydrate Research, 16(1):189-197, doi: 10.1016/S0008-6215(00)86113-3
    Fu Xiaoting, Kim S M. 2010. Agarase:review of major sources, categories, purification method, enzyme characteristics and applications. Marine Drugs, 8(1):200-218, doi: 10.3390/md8010200
    Fu Xiaoting, Pan C H, Lin Hong, et al. 2009. Gene cloning, expression, and characterization of a β-agarase, AgaB34, from Agarivorans albus YKW-34. Journal of Microbiology and Biotechnology, 19:257-264
    Hamer G K, Bhattacharjee S S, Yaphe W. 1977. Analysis of the enzymic hydrolysis products of agarose by 13C-n.m.r. spectroscopy. Carbohydrate Research, 54(1):C7-C10, doi: 10.1016/S0008-6215(00)80567-4
    Hehemann J H, Michel G, Barbeyron T, et al. 2010. Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a β-agarase from the flavobacterium Zobellia galactanivorans. Acta Crystallographica, 66(4):413-417
    Hou Yanping, Chen Xinglin, Chan Zhuhua, et al. 2015. Expression and characterization of a thermostable and pH-stable β-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1. Process Biochemistry, 50(7):1068-1075, doi: 10.1016/j.procbio.2015.04.005
    Hu Bin, Gong Qianhong, Wang Ye, et al. 2006. Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe, 12(5-6):260-266
    Kim H T, Yun E J, Wang Damao, et al. 2013. High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass. Bioresource Technology, 136:582-587, doi: 10.1016/j.biortech.2013.03.038
    Kobayashi R, Takisada M, Suzuki T, et al. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Bioscience, Biotechnology, and Biochemistry, 61(1):162-163, doi: 10.1271/bbb.61.162
    Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259):680-685, doi: 10.1038/227680a0
    Lee D G, Jang M K, Lee O H, et al. 2008. Over-production of a glycoside hydrolase family 50β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnology Letters, 30(5):911-918, doi: 10.1007/s10529-008-9634-4
    Lee D G, Jeon M J, Lee S H. 2012. Cloning, expression, and characterization of a glycoside hydrolase family 118β-agarase from Agarivorans sp. JA-1. Journal of Microbiology and Biotechnology, 22(12):1692-1697, doi: 10.4014/jmb
    Letunic I, Doerks T, Bork P. 2012. SMART 7:recent updates to the protein domain annotation resource. Nucleic Acids Research, 40(D1):D302-D305, doi: 10.1093/nar/gkr931
    Li Jiang, Sha Yujie, Seswita-Zilda D, et al. 2014. Purification and characterization of thermostable agarase from Bacillus sp. BI-3, a thermophilic bacterium isolated from hot spring. Journal of Microbiology and Biotechnology, 24(1):19-25, doi: 10.4014/jmb.1308.08055
    Liang S S, Chen Y P, Chen Y H, et al. 2014. Characterization and overexpression of a novel β-agarase from Thalassomonas agarivorans. Journal of Applied Microbiology, 116(3):563-572, doi: 10.1111/jam.2014.116.issue-3
    Lin Bokun, Lu Guoyong, Zheng Yandan, et al. 2012. Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105. World Journal of Microbiology and Biotechnology, 28(4):1691-1697, doi: 10.1007/s11274-011-0977-y
    Liu Nan, Mao Xiangzhao, Yang Meng, et al. 2014. Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801. World Journal of Microbiology and Biotechnology, 30(6):1691-1698, doi: 10.1007/s11274-013-1591-y
    Marchler-Bauer A, Derbyshire M K, Gonzales N R, et al. 2015. CDD:NCBI's conserved domain database. Nucleic Acids Research, 43(D1):D222-D226, doi: 10.1093/nar/gku1221
    Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3):426-428, doi: 10.1021/ac60147a030
    Minegishi H, Shimane Y, Echigo A, et al. 2013. Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A. Extremophiles, 17(6):931-939, doi: 10.1007/s00792-013-0575-z
    Moreno C, Romero J, Espejo R T. 2002. Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio. Microbiology, 148(4):1233-1239, doi: 10.1099/00221287-148-4-1233
    Oh C, Nikapitiya C, Lee Y, et al. 2010. Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4. Journal of Industrial Microbiology & Biotechnology, 37(5):483-494, doi: 10.1007/s10295-010-0694-9
    Ohta Y, Hatada Y, Nogi Y, et al. 2004a. Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea Microbulbifer. Applied Microbiology and Biotechnology, 64(4):505-514, doi: 10.1007/s00253-004-1573-y
    Ohta Y, Nogi Y, Miyazaki M, et al. 2004b. Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from the novel marine isolate, JAMB-A94. Bioscience, Biotechnology, and Biochemistry, 68(5):1073-1081, doi: 10.1271/bbb.68.1073
    Sahreen S, Khan M R, Khan R A. 2010. Evaluation of antioxidant activities of various solvent extracts of Carissa opaca fruits. Food Chemistry, 122(4):1205-1211, doi: 10.1016/j.foodchem.2010.03.120
    Saitou N, Nei M. 1987. The neighbor-joining method:a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4):406-425
    Takagi E, Hatada Y, Akita M, et al. 2015. Crystal structure of the catalytic domain of a GH16β-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94. Bioscience, Biotechnology, and Biochemistry, 79(4):625-632, doi: 10.1080/09168451.2014.988680
    Tamura K, Stecher G, Peterson D, et al. 2013. MEGA6:Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30(12):2725-2729, doi: 10.1093/molbev/mst197
    Tawara M, Sakatoku A, Tiodjio R E, et al. 2015. Cloning and characterization of a novel agarase from a newly isolated bacterium Simiduia sp. strain TM-2 able to degrade various seaweeds. Applied Biochemistry and Biotechnology, 177(3):610-623, doi: 10.1007/s12010-015-1765-1
    Temuujin U, Chi W J, Chang Y K, et al. 2012. Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo-and endo-type β-agarase-producing neoagarobiose. Journal of Bacteriology, 194(1):142-149, doi: 10.1128/JB.05978-11
    Temuujin U, Chi W J, Lee S Y, et al. 2011. Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2):an endo-type β-agarase producing neoagarotetraose and neoagarohexaose. Applied Microbiology and Biotechnology, 92(4):749-759, doi: 10.1007/s00253-011-3347-7
    Vieille C, Zeikus G J. 2001. Hyperthermophilic enzymes:sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews, 65(1):1-43, doi: 10.1128/MMBR.65.1.1-43.2001
    Xie Wei, Lin Bokun, Zhou Zhengrong, et al. 2013. Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3. Applied Microbiology and Biotechnology, 97(11):4907-4915, doi: 10.1007/s00253-012-4385-5
    Yu Ran, Graf J, Smets B F. 2008. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments. Journal of Microbiological Methods, 72(3):235-240, doi: 10.1016/j.mimet.2007.12.001
    Yun E J, Shin M H, Yoon J J, et al. 2011. Production of 3,6-anhydro-L-galactose from agarose by agarolytic enzymes of Saccharophagus degradans 2-40. Process Biochem, 46(1):88-93, doi: 10.1016/j.procbio.2010.07.019
    Zhou Kequan, Yu Liangli. 2004. Antioxidant properties of bran extracts from Trego wheat grown at different locations. Journal of Agricultural and Food Chemistry, 52(5):1112-1117, doi: 10.1021/jf030621m
    Zhu Kexue, Lian Caixia, Guo Xiaona, et al. 2011. Antioxidant activities and total phenolic contents of various extracts from defatted wheat germ. Food Chemistry, 126(3):1122-1126, doi: 10.1016/j.foodchem.2010.11.144
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