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2018 | 65 | 3 | 383-389
Article title

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs)

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An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

Physical description
  • Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China
  • State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China
  • Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China
  • Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China
  • Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China
  • State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China
  • School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
  • Andreotti G, Citro V, De Crescenzo A, Orlando P, Cammisa M, Correra A, Cubellis MV (2011) Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests. Orphanet J Rare Dis 6: doi: 10.1186/1750-1172-6-66.
  • Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, Smith E, Rahman MM (2008) Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J Nat Prod 71: 1427-1430. doi: 10.1021/np8002673.
  • Callaway JC (2004) Hempseed as a nutritional resource: An overview Euphytica 140: 65-72. doi: 10.1007/s10681-004-4811-6
  • Cao Y, Wang Y, Luo H, Shi P, Meng K, Zhou Z, Zhang Z, Yao B (2009) Molecular cloning and expression of a novel protease-resistant GH-36 alpha-galactosidase from Rhizopus sp. F78 ACCC 30795. J Microbiol Biotechnol 19: 1295-1300.
  • Celem EB, Bolle SS, Onal S (2009) Efficient and rapid purification of lentil alpha-galactosidase by affinity precipitation with alginate. Indian J Biochem Biophys 46: 366-370.
  • Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, Kelly RM (2007) Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases Biochemistry 46: 3319-3330. doi: 10.1021/bi061521n.
  • Dey PM, Patel S, Brownleader MD (1993) Induction of alpha-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum. Biotechnol Appl Biochem 17: 361-371.
  • Du F, Zhu MJ, Wang HX, Ng T (2013) Purification and characterization of an alpha-galactosidase from Phaseolus coccineus seeds showing degrading capability on raffinose family oligosaccharides. Plant Physiol Biochem 69: 49-53. doi: 10.1016/j.plaphy.2013.04.017.
  • Ferreira JG, Reis AP, Guimaraes VM, Falkoski DL, Fialho LD, de Rezende ST (2011) Purification and characterization of Aspergillus terreus alpha-galactosidases and their use for hydrolysis of soymilk oligosaccharides. Appl Biochem Biotechnol 164: 1111-1125. doi: 10.1007/s12010-011-9198-y.
  • Galasso I, Russo R, Mapelli S, Ponzoni E, Brambilla IM, Battelli G, Reggiani R (2016) Variability in seed traits in a collection of Cannabis sativa L. genotypes. Front Plant Sci 7: 688. doi: 10.3389/fpls.2016.00688.
  • Gao X, Yang J, Li SB, Liu ZP, Zhang YP (2003) Fermentation and purification of recombinant alpha-galactosidase from Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 19: 223-226.
  • Gaudreault PR, Webb JA (1983) Partial purification and properties of an alkaline alpha-galactosidase from mature leaves of Cucurbita pepo. Plant Physiol 71: 662-668.
  • Goulas T, Goulas A, Tzortzis G, Gibson GR (2009) A novel alpha-galactosidase from Bifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression. Appl Microbiol Biotechnol 82: 471-477. doi: 10.1007/s00253-008-1750-5.
  • Kim WD, Kaneko S, Park GG, Tanaka H, Kusakabe I, Kobayashi H (2003) Purification and characterization of alpha-galactosidase from sunflower seeds. Biotechnol Lett 25: 353-358.
  • Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80: 575-599.
  • Li HL (1974) Origin and use of cannabis in eastern asia linguistic-cultural implications. Econ Bot 28: 293-301. doi: 10.1007/Bf02861426
  • Liu X, Gao GF, Yang L, Xiao-Xiao HE, Meng Z, Teng LR (2007) Tryptophan modification and fluorescence spectrum of inulinase. Chem J Chinese Univ 28: 103-105.
  • Mi SJ, Bai YG, Meng K, Wang YR, Yao B, Shi XY, Huang HQ, Zhang YH, Shi PJ (2007) Purification and characterization of a novel alpha-galactosidase from penicillium sp. F63 CGMCC1669. Wei Sheng Wu Xue Bao 47: 156-160.
  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426-428. doi: 10.1021/Ac60147a030
  • Nakagawa N, Maruyama H, Ishihara T, Seino U, Kawabe J, Takahashi F, Kobayashi M, Yamauchi A, Sasaki Y, Sakamoto N, Ota H, Tanabe Y, Takeuchi T, Takenaka T, Kikuchi K, Hasebe N (2011) Clinical and genetic investigation of a Japanese family with cardiac fabry disease. Identification of a novel alpha-galactosidase A missense mutation (G195V). Int Heart J 52: 308-311.
  • Nissen L, Zatta A, Stefanini I, Grandi S, Sgorbati B, Biavati B, Monti A (2010) Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.). Fitoterapia 81: 413-419. doi: 10.1016/j.fitote.2009.11.010.
  • Patil AG, K PK, Mulimani VH, Veeranagouda Y, Lee K (2010) alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk. J Microbiol Biotechnol 20: 1546-1554.
  • Porter JE, Ladisch MR, Herrmann KM (1991) Ion exchange and affinity chromatography in the scaleup of the purification of alpha-galactosidase from soybean seeds. Biotechnol Bioeng 37: 356-363. doi: 10.1002/bit.260370409.
  • Ramalingam, Saraswathy N, Sadasivam S, Subha K, Poorani N (2007) Purification and properties of alpha-galactosidase from white-rot fungus Pleurotus florida. Indian J Biochem Biophys 44: 76-81.
  • Rezende STD, Guimarães VM, Rodrigues MDC, Felix CR (2005) Purification and characterization of an α-galactosidase from Aspergillus fumigatus. Braz Arch Biol Technol 48: 195-202. doi: 10.1590/S1516-89132005000200005
  • Rezessy-Szabo JM, Nguyen QD, Hoschke A, Braet C, Hajos G, Claeyssens M (2007) A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization. Biochim Biophys Acta 1770: 55-62. doi: 10.1016/j.bbagen.2006.06.022.
  • Shankar SK, Dhananjay SK, Mulimani VH (2009) Purification and characterization of thermostable alpha-galactosidase from Aspergillus terreus (GR). Appl Biochem Biotechnol 152: 275-285. doi: 10.1007/s12010-008-8271-7.
  • Shen WY, Jin ZY (2011) Chemical modification of α-galactosidase from germinating coffee beans. J Wuhan Polytechnic Univ 30: 1-4 (in Chinese).
  • Shibuya H, Nagasaki H, Kaneko S, Yoshida S, Park GG, Kusakabe I, Kobayashi H (1998) Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenumi. Appl Environ Microbiol 64: 4489-4494.
  • Singh N, Kayastha AM (2012) Purification and characterization of alpha-galactosidase from white chickpea (Cicer arietinum). J Agric Food Chem 60: 3253-3259. doi: 10.1021/jf204538m.
  • Spande TF, Witkop B (1967) Determination of the tryptophan content of proteins with N-bromosuccinimide. Methods Enzymol 11: 498-506. doi: 10.1016/S0076-6879(67)11060-4
  • Steggerda FR (1968) Gastrointestinal gas following food consumption. Ann Ny Acad Sci 150: 57-66.
  • Teng L, Fan H, Zhang Y, Yu Q, Huang Y, Liu L-y (2006) Chemical modification and fluorescence spectrum of tryptophan residues in pullulanase. Chem Res Chinese U 22: 61-64.
  • van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, Page JE (2011) The draft genome and transcriptome of Cannabis sativa. Genome Biol 12: doi: 10.1186/Gb-2011-12-10-R102.
  • Wang H, Luo H, Li J, Bai Y, Huang H, Shi P, Fan Y, Yao B (2010) An alpha-galactosidase from an acidophilic Bispora sp. MEY-1 strain acts synergistically with beta-mannanase. Bioresour Technol 101: 8376-8382. doi: 10.1016/j.biortech.2010.06.045.
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