Abstract
A multimodular pectinase of glycoside hydrolase family 28, S6A, was identified in Penicillium oxalicum SX6 that consists of an N-terminal catalytic domain of pectin methylesterase, a Thr/Ser-rich linker region, and a C-terminal catalytic domain of polygalacturonase. Recombinant S6A and its two derivatives, S6PE (the catalytic domain of pectin methylesterase) and S6PG (the catalytic domain of polygalacturonase), were produced in Pichia pastoris. S6A was a bifunctional protein and had both pectin methylesterase and polygalacturonase activities. Three enzymes showed similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 50 °C) and excellent stability at pH 3.5–6.0 and 40 °C. Most metal ions tested (Na+, K+, Ca2+, Li+, Co2+, Cr3+, Ni2+, Cu2+, Mn2+,Mg2+, Fe3+, Zn2+, and Pb2+) enhanced the pectin methylesterase activities of S6PE and S6A, but had little or inhibitory effects on the polygalacturonase activities of S6A and S6PG. In comparison with most fungal pectin methylesterases, S6A had higher specific activity (271.1 U/mg) towards 70 % DM citrus pectin. When S6PE and S6PG were combined at the activity ratio of 1:4, the most significant synergistic effect was observed in citrus pectin degradation and degumming of sisal fiber, which is comparable with the performance of S6A (95 v.s. 100 % and 16.9 v.s. 17.2 %, respectively). To the best of our knowledge, this work represents the first report of gene cloning, heterologous expression, and biochemical characterization of a bifunctional pectinase with separate catalytic domains.
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References
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Anbarasan S, Jänis J, Paloheimo M, Laitaoja M, Vuolanto M, Karimäk J, Vainiotalo P, Leisola M, Turunen O (2010) Effect of glycosylation and additional domains on the thermostability of a family 10 xylanase produced by Thermopolyspora flexuosa. Appl Environ Microbiol 76:356–360
Basu S, Saha M, Chattopadhyay D, Chakrabarti K (2009) Degumming and characterization of ramie fiber using pectate lyase from immobilized Bacillus pumilus DKS1. Lett Appl Microbiol 48:593–597
Berensmeier S, Singh SA, Meens J, Buchholz K (2004) Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase. Appl Microbiol Biotechnol 64:560–567
Borin MDF, Said S, Fonseca MJV (1996) Purification and biochemical characterization of an extracellular endopolygalacturonase from Penicillium frequentans. J Agric Food Chem 44:1616–1620
Caffall KH, Mohnen D (2009) The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr Res 344:1879–1900
Cho SW, Lee S, Shin W (2001) The X-ray structure of Aspergillus aculeatus polygalacturonase and a modeled structure of the polygalacturonase-octagalacturonate complex. J Mol Biol 314:863–878
Cho KM, Hong SY, Lee SM, Kim YH, Kahng GG, Kim H, Yun HD (2006) A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains. Appl Microbiol Biotechnol 73:618–630
Christgau S, Kofod L, Halkier T, Andersen L, Hockauf M, Dorreich K, Dalboge H, Kauppinen S (1996) Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme. Biochem J 319:705–712
Edwards MC, Doran-Peterson J (2012) Pectin-rich biomass as feedstock for fuel ethanol production. Appl Microbiol Biotechnol 95:565–575
Federici L, Caprari C, Mattei B, Savino C, Di Matteo A, De Lorenzo G, Cervone F, Tsernoglou D (2001) Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). Proc Natl Acad Sci U S A 98:13425–13430
Flint H, Martin J, McPherson C, Daniel A, Zhang J (1993) A bifunctional enzyme, with separate xylanase and β (1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens. J Bacteriol 175:2943–2951
Fonseca-Maldonado R, Vieira DS, Alponti JS, Bonneil E, Thibault P, Ward RJ (2013) Engineering the pattern of protein glycosylation modulates the thermostability of a GH11 xylanase. J Biol Chem 288:25522–25534
Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE (2010) Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol 101:2770–2781
Gibbs MD, Reeves RA, Farrington GK, Anderson P, Williams DP, Bergquist PL (2000) Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1. Curr Microbiol 40:333–340
Harholt J, Suttangkakul A, Scheller HV (2010) Biosynthesis of pectin. Plant Physiol 153:384–395
Jiang X, Chen P, Yin M, Yang Q (2012) Constitutive expression, purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry. J Sci Food Agric 93:375–381
Jolie RP, Duvetter T, van Loey AM, Hendrickx ME (2010) Pectin methylesterase and its proteinaceous inhibitor: a review. Carbohydr Res 345:2583–2595
Khandeparker R, Numan MT (2008) Bifunctional xylanases and their potential use in biotechnology. J Ind Microbiol Biotechnol 35:635–644
Kitamoto N, Okada H, Yoshino S, Ohmiya K, Tsukagoshi N (1999) Pectin methylesterase gene (pmeA) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene product. Biosci Biotechnol Biochem 63:120–124
Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, Dörreich K, Dalbøge H (1994) Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. J Biol Chem 269:29182–29189
Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681
Liu G, Zhang L, Wei X, Zou G, Qin Y, Ma L, Li J, Zheng H, Wang S, Wang C (2013) Genomic and secretomic analyses reveal unique features of the lignocellulolytic enzyme system of Penicillium decumbens. PLoS ONE 8:e55185
Lu G, Moriyama EN (2004) Vector NTI, a balanced all-in-one sequence analysis suite. Brief Bioinform 5:378–388
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Öberg F, Sjöhamn J, Fischer G, Moberg A, Pedersen A, Neutze R, Hedfalk K (2011) Glycosylation increases the thermostability of human aquaporin 10 protein. J Biol Chem 286:31915–31923
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786
Ribeiro LF, Furtado GP, Lourenzoni MR, Costa-Filho AJ, Santos CR, Nogueira SCP, Betini JA, Polizeli MDTM, Murakami MT, Ward RJ (2011) Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance. J Biol Chem 286:43026–43038
Semenova M, Grishutin S, Gusakov A, Okunev O, Sinitsyn A (2003) Isolation and properties of pectinases from the fungus Aspergillus japonicus. Biochemistry 68:559–569
Sharma N, Rathore M, Sharma M (2013) Microbial pectinase: sources, characterization and applications. Rev Environ Sci Biotechnol 12:45–60
Shental-Bechor D, Levy Y (2008) Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc Natl Acad Sci U S A 105:8256–8261
Shi P, Tian J, Yuan T, Liu X, Huang H, Bai Y, Yang P, Chen X, Wu N, Yao B (2010) Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain. Appl Environ Microbiol 76:3620–3624
Sila DN, Smout C, Satara Y, Truong V, Loey AV, Hendrickx M (2007) Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity. J Food Eng 78:755–764
Toda H, Nagahata N, Amano Y, Nozaki K, Kanda T, Okazaki M, Shimosaka M (2008) Gene cloning of cellobiohydrolase II from the white rot fungus Irpex lacteus MC-2 and its expression in Pichia pastoris. Biosci Biotechnol Biochem 72:3142–3147
Tomme P, Warren RAJ, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–81
Torres S, Sayago JE, Ordoñez RM, Isla MI (2011) A colorimetric method to quantify endo-polygalacturonase activity. Enzym Microbiol Technol 48:123–128
Tu T, Meng K, Bai Y, Shi P, Luo H, Wang Y, Yang P, Zhang Y, Zhang W, Yao B (2013) High-yield production of a low-temperature-active polygalacturonase for papaya juice clarification. Food Chem 141:2974–2981
van den Brink J, de Vries RP (2011) Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol Biotechnol 91:1477–1492
Yadav S, Yadav PK, Yadav D, Yadav KDS (2009) Pectin lyase: a review. Process Biochem 44:1–10
Zhang C, Yao J, Zhou C, Mao L, Zhang G, Ma Y (2013) The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber. BMC Biotechnol 13:26
Zhao J, Shi P, Li Z, Yang P, Luo H, Bai Y, Wang Y, Yao B (2012) Two neutral thermostable cellulases from Phialophora sp. G5 act synergistically in the hydrolysis of filter paper. Bioresour Technol 121:404–410
Acknowledgments
This study was supported by the National Natural Science Foundation of China (no. 31271937) and the National Science and Technology Support Program of China (no. 2013BAD10B01-2) and the 948 Program of the Ministry of Agriculture of China (no. 2011-G7-4) and China Modern Agriculture Research System (CARS-42).
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Tu, T., Bai, Y., Luo, H. et al. A novel bifunctional pectinase from Penicillium oxalicum SX6 with separate pectin methylesterase and polygalacturonase catalytic domains. Appl Microbiol Biotechnol 98, 5019–5028 (2014). https://doi.org/10.1007/s00253-014-5533-x
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DOI: https://doi.org/10.1007/s00253-014-5533-x