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.
  • 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
  • Relative Articles

  • Cited by

    Periodical cited type(5)

    1. Yafang Shi, Yuanfan Yang, Hebin Li, et al. Effects of neoagaroligosaccharides on quality characteristics and antioxidant activity of set yogurt. LWT, 2025. doi:10.1016/j.lwt.2025.117638
    2. Weimao Zhong, Vinayak Agarwal. Polymer degrading marine Microbulbifer bacteria: an un(der)utilized source of chemical and biocatalytic novelty. Beilstein Journal of Organic Chemistry, 2024, 20: 1635. doi:10.3762/bjoc.20.146
    3. Lixing Liu, Lixi Cai, Yunmeng Chu, et al. Thermostability mechanisms of β-agarase by analyzing its structure through molecular dynamics simulation. AMB Express, 2022, 12(1) doi:10.1186/s13568-022-01394-x
    4. Zhi-peng Li, Qing-song Hu, Jin-fang Chen, et al. Optimized strategy for simultaneous recovering bioactive oligosaccharides and reusable perlite from agar industrial waste residues. Journal of Cleaner Production, 2022, 378: 134631. doi:10.1016/j.jclepro.2022.134631
    5. Chunsheng Li, Chi Li, Laihao Li, et al. Comparative Genomic and Secretomic Analysis Provide Insights Into Unique Agar Degradation Function of Marine Bacterium Vibrio fluvialis A8 Through Horizontal Gene Transfer. Frontiers in Microbiology, 2020, 11 doi:10.3389/fmicb.2020.01934

    Other cited types(0)

  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Article Metrics

    Article views (678) PDF downloads(359) Cited by(5)
    Proportional views
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return