Molecular Cloning and Characterization of a Novel Exo-β-1,3-Galactanase from Penicillium oxalicum sp. 68
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Arabinogalactans have diverse biological properties and can be used as pharmaceutical agents. Most arabinogalactans are composed of β-(1→3)-galactan, so it is particularly important to identify β-1,3-galactanases that can selectively degrade them. In this study, a novel exo-β-1,3-galactanase, named PoGal3, was screened from Penicillium oxalicum sp. 68, and hetero-expressed in P. pastoris GS115 as a soluble protein. PoGal3 belongs to glycoside hydrolase family 43 (GH43) and has a 1,356- bp gene length that encodes 451 amino acids residues. To study the enzymatic properties and substrate selectivity of PoGal3, β-1,3-galactan (AG-P-I) from larch wood arabinogalactan (LWAG) was prepared and characterized by HPLC and NMR. Using AG-P-I as substrate, purified PoGal3 exhibited an optimal pH of 5.0 and temperature of 40°C. We also discovered that Zn2+ had the strongest promoting effect on enzyme activity, increasing it by 28.6%. Substrate specificity suggests that PoGal3 functions as an exo-β-1,3-galactanase, with its greatest catalytic activity observed on AG-P-I. Hydrolytic products of AG-P-I are mainly composed of galactose and β-1,6-galactobiose. In addition, PoGal3 can catalyze hydrolysis of LWAG to produce galacto-oligomers. PoGal3 is the first enzyme identified as an exo-β-1,3-galactanase that can be used in building glycan blocks of crucial glycoconjugates to assess their biological functions.
Abstract
Arabinogalactans have diverse biological properties and can be used as pharmaceutical agents. Most arabinogalactans are composed of β-(1→3)-galactan, so it is particularly important to identify β-1,3-galactanases that can selectively degrade them. In this study, a novel exo-β-1,3-galactanase, named PoGal3, was screened from Penicillium oxalicum sp. 68, and hetero-expressed in P. pastoris GS115 as a soluble protein. PoGal3 belongs to glycoside hydrolase family 43 (GH43) and has a 1,356- bp gene length that encodes 451 amino acids residues. To study the enzymatic properties and substrate selectivity of PoGal3, β-1,3-galactan (AG-P-I) from larch wood arabinogalactan (LWAG) was prepared and characterized by HPLC and NMR. Using AG-P-I as substrate, purified PoGal3 exhibited an optimal pH of 5.0 and temperature of 40°C. We also discovered that Zn2+ had the strongest promoting effect on enzyme activity, increasing it by 28.6%. Substrate specificity suggests that PoGal3 functions as an exo-β-1,3-galactanase, with its greatest catalytic activity observed on AG-P-I. Hydrolytic products of AG-P-I are mainly composed of galactose and β-1,6-galactobiose. In addition, PoGal3 can catalyze hydrolysis of LWAG to produce galacto-oligomers. PoGal3 is the first enzyme identified as an exo-β-1,3-galactanase that can be used in building glycan blocks of crucial glycoconjugates to assess their biological functions.
참고문헌 (44)
- Goellner EM, "Structure of arabinogalactan from Larix laricina and its reactivity with antibodies directed against type-II-arabinogalactans", Carbohydr. Polym, 86, pp.1739~1744 (2011)
- Prescott JH, "New molecular weight forms of arabinogalactan from Larix occidentalis", Carbohydr. Res, 301, pp.89~93 (1997)
- Tang S, "Characterization of arabinogalactans from Larix principis-rupprechtii and their effects on NO production by macrophages", Carbohydr. Polym, 200, pp.408~415 (2018)
- Currier NL, "Effect over time of in-vivo administration of the polysaccharide arabinogalactan on immune and hemopoietic cell lineages in murine spleen and bone marrow", Phytomedicine, 10, pp.145~153 (2003)
- Beuth J, "Inhibition of liver tumor cell colonization in two animal tumor models by lectin blocking with D Galactose or arabinogalactan", Clin. Exp. Metastasis, 6, pp.115~120 (1988)
- Enriquez PM, "Conjugation of Adenine arabinoside 5’-Monophosphate to arabinogalactan : Synthesis, characterization, and antiviral activity", Bioconjug. Chem, 6, pp.195~202 (1995)
- Riede L, "Larch arabinogalactan effects on reducing incidence of upper respiratory infections", Curr. Med. Res. Opin, 29, pp.251~258 (2013)
- Scigelova M, "Glycosidases—a great synthetic tool", J. Mol. Catal. B Enzym, 6, pp.483~494 (1999)
- Müller M, "The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases", J. Biol. Chem, 296, pp.100519~ (2021)
- Yang H, "Characterization of a thermostable Endo-β-1, 4-galactanase from the Hyperthermophile Thermotoga maritima", Biosci. Biotechnol. Biochem, 70, pp.538~541 (2006)
- Wang D, "Establishment of fingerprint of Astragali Radix polysaccharides based on endo-1, 4-β-malactanase hydrolysis and identification of Astragali Radix of different germplasm resources", Zhongguo Zhong Yao Za Zhi, 43, pp.2964~2972 (2018)
- Bueren AL, "Prebiotic galactooligosaccharides activate mucin and pectic galactan utilization pathways in the human gut symbiont Bacteroides thetaiotaomicron", Sci. Rep, 7, pp.40478~ (2017)
- Ichinose H, "Characterization of an exo-β-1, 3-D-galactanase from Streptomyces avermitilis NBRC14893 acting on arabinogalactan-proteins", Biosci. Biotechnol. Biochem, 70, pp.2745~2750 (2006)
- Lemaire A, "Three novel rhamnogalacturonan I-pectins degrading enzymes from Aspergillus aculeatinus : Biochemical characterization and application potential", Carbohydr. Polym, 248, pp.116752~ (2020)
- Torpenholt S, "Structure of Aspergillus aculeatus β-1, 4-galactanase in complex with galactobiose", Acta Crystallogr. F Struct. Biol. Commun, 75, pp.399~404 (2019)
- Okemoto K, "Purification and characterization of an endo-β-(1→6)-galactanase from Trichoderma viride", Carbohydr. Re, 338, pp.219~230 (2003)
- Tsumuraya Y, "Purification of an exo-β-(1→3)-D-galactanase of Irpex lacteus(Polyporus tulipiferae)and its action on arabinogalactan-proteins", J. Biol. Chem, 265, pp.7207~7215 (1990)
- Kotake T, "Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-β-(1→3)-galactanase Gene", Biosci. Biotechnol. Biochem, 73, pp.2303~2309 (2009)
- Fujita K, "Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan", Appl. Environ. Microbiol, 80, pp.4577~4584 (2014)
- Okawa M, "Identification of an exo-β-1, 3-D-galactanase from Fusarium oxysporum and the synergistic effect with related enzymes on degradation of type II arabinogalactan", Appl. Microbiol. Biotechnol, 97, pp.9685~9694 (2013)
- Ichinose H, "An exo-beta-1, 3-galactanase having a novel beta-1, 3-galactan-binding module from Phanerochaete chrysosporium", J. Biol. Chem, 280, pp.25820~25829 (2005)
- Ishida T, "Crystallization of selenomethionyl exo-beta-1, 3-galactanase from the basidiomycete Phanerochaete chrysosporium", Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun, 65, pp.1274~1276 (2009)
- Ichinose H, "Characterization of an exo-beta-1, 3-galactanase from Clostridium thermocellum", Appl. Environ. Microbiol, 72, pp.3515~3523 (2006)
- Jiang D, "Crystal structure of 1, 3Gal43A, an exo-β-1, 3-galactanase from Clostridium thermocellum", J. Struct. Biol, 80, pp.447~457 (2012)
- Ichinose H, "Characterization of an exo-β-1, 3-D-galactanase from Streptomyces avermitilis NBRC14893 acting on arabinogalactan-proteins", J. Agric. Chem. Soc. Japan, 70, pp.6~ (2006)
- Ling NX, "An exo-β-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure", Carbohydr. Res, 352, pp.70~81 (2012)
- Yanbo Hu, "Cloning and expression of a novel α-1,3-arabinofuranosidase from Penicillium oxalicum sp. 68", AMB Express, 8(1) (2018), Springer Science and Business Media LLC[DOI]
- Li J, "Cellodextrin transporters play important roles in cellulose induction in the cellulolytic fungus Penicillium oxalicum", Appl. Microbiol. Biotechnol, 97, pp.10479~10488 (2013)
- Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp.680~685 (1970)
- Somogyi M, "Notes on sugar determination", J. Biol. Chem, 195, pp.19~23 (1952)
- Tsumuraya Y, "Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves", Carbohydr. Res, 134, pp.215~228 (1984)
- Zhang X, "Total fractionation and characterization of the water-soluble polysaccharides isolated from Panax ginseng C. A. Meyer", Carbohydr. Polym, 77, pp.544~552 (2009)
- Wu D, "Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison", Carbohydr. Polym, 108, pp.209~215 (2018)
- Shakhmatov EG, "Structure of acid-extractable polysaccharides of tree greenery of Picea abies", Carbohydr. Polym, 199, pp.320~330 (2018)
- Yao Y, "Structural elucidation and immune-enhancing activity of an arabinogalactan from flowers of Carthamus tinctorius L", Carbohydr. Polym, 202, pp.134~142 (2018)
- Kotake T, "Endo-β-1, 3-galactanase from winter mushroom flammulina velutipes", J. Biol. Chem, 286, pp.27848~27854 (2011)
- Ali N, "Purification, characterization, gene cloning and sequencing of a new β-glucosidase from aspergillus niger be-2", Appl. Biochem. Microbiol, 52, pp.564~571 (2016)
- Sakamoto T, "Identification of a GH62 alpha-L-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum", Appl. Microbiol. Biotechnol, 90, pp.137~146 (2011)
- Dragosits M, "Recombinant aspergillus β-galactosidases as a robust glycomic and biotechnological tool", Appl. Microbiol. Biotechnol, 98, pp.3553~3567 (2014)
- Kotake T, "Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene", Biochem. J, 377, pp.749~755 (2004)
- Hideaki Kato, "In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (Raphanus sativus L.) seedlings", Planta, 217(2), pp.271~282 (2003), Springer Science and Business Media LLC[DOI]
- Liu Y, "Biochemical characterization of a novel GH43 family β-xylosidase from Bacillus pumilus", Food Chem, 295, pp.653~661 (2019)
- Jordan DB, "Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates", Appl. Microbiol. Biotechnol, 97, pp.4415~4428 (2013)
- Vandermarliere E, "Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family", Biochem. J, 418, pp.39~47 (2009)