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Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

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Abstract

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein−1 under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.

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References

  • Basaveswara Rao V, Sastri NVS, Subba Rao PV (1981) Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus. Biochem J 193:379–387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cherry HM, Hossain MT, Anwar MN (2004) Extracellular glucoamylase from the isolate Aspergillus fumigatus. Pak J Biol Sci 7:1988–1992

    Article  Google Scholar 

  • Coutinho PM, Reilly PJ (1997) Glucoamylase structural, functional, and evolutionary relationships. Proteins 29:334–347

    Article  CAS  PubMed  Google Scholar 

  • Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS (2003) Microbial conversion of steroid compounds: recent developments. Enzyme Microb Technol 32:688–705

    Article  CAS  Google Scholar 

  • Feng B, Ma BP, Kang LP, Xiong CQ, Wang SQ (2005) The microbiological transformation of steroidal saponins by Curvularia lunata. Tetrahedron 61:11758–11763

    Article  CAS  Google Scholar 

  • Feng B, Kang LP, Ma BP, Quan B, Zhou WB, Zhao Y, Liu YX, Wang SQ (2007) The substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata. Tetrahedron 63:6796–6812

    Article  CAS  Google Scholar 

  • Hata Y, Kitamoto K, Gomi K, Kumagai C, Tamura G, Hara S (1991a) The glucoamylase cDNA from Aspergillus oryzae: its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae. Agric Biol Chem 55(4):941–949

    CAS  PubMed  Google Scholar 

  • Hata Y, Tsuchiya K, Kitamoto K, Gomi K, Kumagai C, Tamura G, Hara S (1991b) Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae. Gene 108:145–150

    Article  CAS  PubMed  Google Scholar 

  • Kanlayakrit W, Ishiatsu K, Nakao M, Hayshida S (1987) Characteristics of raw starch degrading glucoamylase from thermophilic Rhizomucor pusilus. J Ferment Technol 65:379–385

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lowry OH, Rosebroough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Manzanares P, Graaff LH, Visser J (1997) Purification and characterization of an α-L-rhamnosidase from Aspergillus niger. FEMS Microbiol Lett 157:279–283

    Article  CAS  PubMed  Google Scholar 

  • Manzanares P, Broeck HCVD, Graaff LH, Visser J (2001) Purification and characterization of two different α-L-rhamnosidases, Rha-A and Rha-B, from Aspergillus aculeatus. Appl Environ Microbiol 67:2230–2234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manzanares P, Orejas M, Gil JV, Graaff LH, Visser J, Ramon D (2003) Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rha-A gene, encoding an α-L-Rhamnosidase of enological interest. Appl Environ Microbiol 69:7558–7562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mateo JJ, Stefano RD (1997) Description of the β-glucosidase activity of wine yeasts. Food Microbiol 14:583–591

    Article  CAS  Google Scholar 

  • McCann K, Barnett JA (1986) The utilization of starch by yeasts. Yeast 2:109–115

    Article  CAS  PubMed  Google Scholar 

  • Monti D, Pivejcová A, Ken V, Lama M, Riva S (2004) Generation of an α-L-Rhamnosidase library and its application for the selective Derhamnosylation of natural products. Biotechnol Bioeng 87:763–771

    Article  CAS  PubMed  Google Scholar 

  • Pan F, Yang SL, Shi XL, Bao XF, Sun DP (2001) Purification and properties of cellulase activity from Aspergillus niger. Biotechnology 11:7–9

    CAS  Google Scholar 

  • Paraszkiewicz K, Dlugonski J (1998) Cortexolone 11β-hydrozylation in protoplasts of Curvularia lunata. J Biotechnol 65:217–224

    Article  CAS  Google Scholar 

  • Riou C, Salmon JM, Vallier MJ, Günata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microb 64:3607–3614

    Article  CAS  Google Scholar 

  • Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B (2000) Glucoamylase: structure/function relationship and protein engineering. Biochim Biophys Acta 1543:275–293

    Article  CAS  PubMed  Google Scholar 

  • Shelley DC (2003) Enzymes with extra talents: moonlighting functions and catalytic promiscuity. Curr Opin Chem Biol 7:265–272

    Article  Google Scholar 

  • Sue M, Ishihara A, Iwamura H (2000) Purification and characterization of a β-glucosidase from rye (Secale cereale L.) seedlings. Plant Sci 155:67–74

    Article  CAS  PubMed  Google Scholar 

  • Tani Y, Voungsuvaneert V, Kumunantu J (1986) Raw cassava starch digestive glucoamylase of Aspergilli sp. N-2 isolated from cassava starch. J Ferment Technol 64:405–412

    Article  CAS  Google Scholar 

  • Tripathi P, Lo Leggio L, Mansfeld J, Ulbrich-Hofmann R, Kayastha AM (2007) a. -Amylase from mung beans (Vigna radiata)—correlation of biochemical properties and tertiary structure by homology modeling. Photochemistry 68:1623–1631

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Beijing Institute of Radiation Medicine, Center for Research of proteome (Beijing 100850, China).

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Authors and Affiliations

  1. Beijing Institute of Radiation Medicine, Beijing, 100850, China

    Bing Feng, Bai-ping Ma, Yong-ze Wang, Hong-ze Huang, Sheng-qi Wang & Xiao-hong Qian

  2. Life Science School of Shandong University of Technology, Zibo, 255029, China

    Wei Hu

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  1. Bing Feng
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  2. Wei Hu
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  3. Bai-ping Ma
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  4. Yong-ze Wang
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  5. Hong-ze Huang
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  7. Xiao-hong Qian
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Corresponding author

Correspondence to Bing Feng.

Additional information

This project was supported by the National Natural Science Foundation of China (NSFC; 30572333).

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Feng, B., Hu, W., Ma, Bp. et al. Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata . Appl Microbiol Biotechnol 76, 1329–1338 (2007). https://doi.org/10.1007/s00253-007-1117-3

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  • DOI: https://doi.org/10.1007/s00253-007-1117-3

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