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Protein & Peptide Letters

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Research Article

Construction and Evaluation of Peptide-Linked Lactobacillus brevis β-Galactosidase Heterodimers

Author(s): Yuan-Yuan Han, Hai-Yun Yue, Xiao-Yang Zhang, Yong-Mei Lyu, Li Liu* and Josef Voglmeir*

Volume 28, Issue 2, 2021

Published on: 13 August, 2020

Page: [221 - 228] Pages: 8

DOI: 10.2174/0929866527666200813201242

Price: $65

Abstract

Background: β-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry.

Objective: We describe the recombinant expression of an unstudied, heterodimeric β-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated.

Methods: The heterodimeric subunits of the β-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits.

Results: The co-expression in two independent expression vectors only resulted in low β-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the β-galactosidase activity significantly. The recombinant β-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-β-D-galactoside.

Conclusion: The usability of the recombinant L. brevis β-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused β-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.

Keywords: β-galactosidase, lactobacillus, fusion protein, peptide linkers, lactose hydrolysis, subunit co-expression.

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[1]
Deng, Y.; Misselwitz, B.; Dai, N.; Fox, M. Lactose intolerance in adults: Biological mechanism and dietary management. Nutrients, 2015, 7(9), 8020-8035.
[http://dx.doi.org/10.3390/nu7095380] [PMID: 26393648]
[2]
Mattar, R.; de Campos Mazo, D.F.; Carrilho, F.J. Lactose intolerance: diagnosis, genetic, and clinical factors. Clin. Exp. Gastroenterol., 2012, 5, 113-121.
[http://dx.doi.org/10.2147/CEG.S32368] [PMID: 22826639]
[3]
Misselwitz, B.; Butter, M.; Verbeke, K.; Fox, M.R. Update on lactose malabsorption and intolerance: pathogenesis, diagnosis and clinical management. Gut, 2019, 68(11), 2080-2091.
[http://dx.doi.org/10.1136/gutjnl-2019-318404] [PMID: 31427404]
[4]
Shaukat, A.; Levitt, M.D.; Taylor, B.C.; MacDonald, R.; Shamliyan, T.A.; Kane, R.L.; Wilt, T.J. Systematic review: effective management strategies for lactose intolerance. Ann. Intern. Med., 2010, 152(12), 797-803.
[http://dx.doi.org/10.7326/0003-4819-152-12-201006150-00241] [PMID: 20404262]
[5]
Silanikove, N.; Leitner, G.; Merin, U. The interrelationships between lactose intolerance and the modern dairy industry: Global perspectives in evolutional and historical backgrounds. Nutrients, 2015, 7(9), 7312-7331.
[http://dx.doi.org/10.3390/nu7095340] [PMID: 26404364]
[6]
Meena, G.S.; Singh, A.K.; Panjagari, N.R.; Arora, S. Milk protein concentrates: opportunities and challenges. J. Food Sci. Technol., 2017, 54(10), 3010-3024.
[http://dx.doi.org/10.1007/s13197-017-2796-0] [PMID: 28974785]
[7]
Dekker, P.J.T.; Koenders, D.; Bruins, M.J. Lactose-free dairy products: Market developments, production, nutrition and health benefits. Nutrients, 2019, 11(3), 11.
[http://dx.doi.org/10.3390/nu11030551] [PMID: 30841534]
[8]
Dutra Rosolen, M.; Gennari, A.; Volpato, G.; Volken de Souza, C.F. Lactose hydrolysis in milk and dairy whey using microbial β-galactosidases. Enzyme Res., 2015, 2015, 806240.
[http://dx.doi.org/10.1155/2015/806240] [PMID: 26587283]
[9]
Miqdady, M.; Al Mistarihi, J.; Azaz, A.; Rawat, D. Prebiotics in the infant microbiome: The past, present, and future. Pediatr. Gastroenterol. Hepatol. Nutr., 2020, 23(1), 1-14.
[http://dx.doi.org/10.5223/pghn.2020.23.1.1] [PMID: 31988871]
[10]
Maischberger, T.; Leitner, E.; Nitisinprasert, S.; Juajun, O.; Yamabhai, M.; Nguyen, T-H.; Haltrich, D. β-galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides. Biotechnol. J., 2010, 5(8), 838-847.
[http://dx.doi.org/10.1002/biot.201000126] [PMID: 20669255]
[11]
Juajun, O.; Nguyen, T-H.; Maischberger, T.; Iqbal, S.; Haltrich, D.; Yamabhai, M. Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13. Appl. Microbiol. Biotechnol., 2011, 89(3), 645-654.
[http://dx.doi.org/10.1007/s00253-010-2862-2] [PMID: 20852995]
[12]
Iqbal, S.; Nguyen, T-H.; Nguyen, H.A.; Nguyen, T.T.; Maischberger, T.; Kittl, R.; Haltrich, D. Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides. J. Agric. Food Chem., 2011, 59(8), 3803-3811.
[http://dx.doi.org/10.1021/jf103832q] [PMID: 21405014]
[13]
Nguyen, T-T.; Nguyen, H.A.; Arreola, S.L.; Mlynek, G.; Djinović-Carugo, K.; Mathiesen, G.; Nguyen, T-H.; Haltrich, D. Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization. J. Agric. Food Chem., 2012, 60(7), 1713-1721.
[http://dx.doi.org/10.1021/jf203909e] [PMID: 22283494]
[14]
Yin, H.; Dijkhuizen, L.; van Leeuwen, S.S. Synthesis of galacto-oligosaccharides derived from lactulose by wild-type and mutant β-galactosidase enzymes from Bacillus circulans ATCC 31382. Carbohydr. Res., 2018, 465, 58-65.
[http://dx.doi.org/10.1016/j.carres.2018.06.009] [PMID: 29944997]
[15]
Fortina, M.G.; Ricci, G.; Mora, D.; Guglielmetti, S.; Manachini, P.L. Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus. Appl. Environ. Microbiol., 2003, 69(6), 3238-3243.
[http://dx.doi.org/10.1128/AEM.69.6.3238-3243.2003] [PMID: 12788721]
[16]
Fred, E.B.; Peterson, W.H.; Davenport, A. Acid fermentation of xylose. J. Biol. Chem., 1919, 39, 347-384.
[17]
Honda, H.; Yajima, N.; Saito, T. Characterization of lactose utilization and β-galactosidase in Lactobacillus brevis KB290, the hetero-fermentative lactic acid bacterium. Curr. Microbiol., 2012, 65(6), 679-685.
[http://dx.doi.org/10.1007/s00284-012-0216-2] [PMID: 22936499]
[18]
Li, G.; Huang, Z.; Zhang, C.; Dong, B-J.; Guo, R-H.; Yue, H-W.; Yan, L-T.; Xing, X-H. Construction of a linker library with widely controllable flexibility for fusion protein design. Appl. Microbiol. Biotechnol., 2016, 100(1), 215-225.
[http://dx.doi.org/10.1007/s00253-015-6985-3] [PMID: 26394862]
[19]
Chen, X.; Zaro, J.L.; Shen, W.C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev., 2013, 65(10), 1357-1369.
[http://dx.doi.org/10.1016/j.addr.2012.09.039] [PMID: 23026637]
[20]
Shamriz, S.; Ofoghi, H.; Moazami, N. Effect of linker length and residues on the structure and stability of a fusion protein with malaria vaccine application. Comput. Biol. Med., 2016, 76, 24-29.
[http://dx.doi.org/10.1016/j.compbiomed.2016.06.015] [PMID: 27393958]
[21]
Huang, K.; Wang, M.M.; Kulinich, A.; Yao, H.L.; Ma, H.Y.; Martínez, J.E.R.; Duan, X.C.; Chen, H.; Cai, Z.P.; Flitsch, S.L.; Liu, L.; Voglmeir, J. Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila. Carbohydr. Res., 2015, 415, 60-65.
[http://dx.doi.org/10.1016/j.carres.2015.08.001] [PMID: 26340137]
[22]
Datsenko, K.A.; Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645.
[http://dx.doi.org/10.1073/pnas.120163297] [PMID: 10829079]
[23]
Li, Q.; Huang, Y.Y.; Conway, L.P.; He, M.; Wei, S.; Huang, K.; Duan, X.C.; Flitsch, S.L.; Voglmeir, J. Discovery and biochemical characterization of a thermostable glucose-1-phosphate nucleotidyltransferase from Thermodesulfatator indicus. Protein Pept. Lett., 2017, 24(8), 729-734.
[http://dx.doi.org/10.2174/0929866524666170724110408] [PMID: 28741459]
[24]
Wei, S.; Zhang, X.Y.; Sun, Y.; Conway, L.P.; Liu, L. Discovery and biochemical characterization of UDP-glucose dehydrogenase from Akkermansia muciniphila. Protein Pept. Lett., 2017, 24(8), 735-741.
[http://dx.doi.org/10.2174/0929866524666170724111147] [PMID: 28741460]
[25]
Cai, Z.P.; Wang, W.L.; Conway, L.P.; Huang, K.; Awad, F.N.; Liu, L.; Voglmeir, J. 1,3-Di(2-dipyridyl)propan-1,3-dione – a new fluorogenic labeling reagent for milk oligosaccharides. Pure Appl. Chem., 2017, 89, 921-930.
[http://dx.doi.org/10.1515/pac-2016-0914]
[26]
Dereeper, A.; Audic, S.; Claverie, J-M.; Blanc, G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol. Biol., 2010, 10, 8.
[http://dx.doi.org/10.1186/1471-2148-10-8] [PMID: 20067610]
[27]
Nguyen, T.H.; Splechtna, B.; Yamabhai, M.; Haltrich, D.; Peterbauer, C. Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli. J. Biotechnol., 2007, 129(4), 581-591.
[http://dx.doi.org/10.1016/j.jbiotec.2007.01.034] [PMID: 17360065]
[28]
Fowler, A.V.; Zabin, I. Amino acid sequence of beta-galactosidase. XI. Peptide ordering procedures and the complete sequence. J. Biol. Chem., 1978, 253(15), 5521-5525.
[29]
O’Connell, S.; Walsh, G. Purification and properties of a beta-galactosidase with potential application as a digestive supplement. Appl. Biochem. Biotechnol., 2007, 141(1), 1-14.
[http://dx.doi.org/10.1007/s12010-007-9206-4] [PMID: 17625262]
[30]
Hu, X.; Robin, S.; O’Connell, S.; Walsh, G.; Wall, J.G. Engineering of a fungal beta-galactosidase to remove product inhibition by galactose. Appl. Microbiol. Biotechnol., 2010, 87(5), 1773-1782.
[http://dx.doi.org/10.1007/s00253-010-2662-8] [PMID: 20496147]
[31]
Chantarangsee, M.; Tanthanuch, W.; Fujimura, T.; Fry, S.C.; Cairns, J.K. Molecular characterization of beta-galactosidases from germinating rice (Oryza sativa). Plant Sci., 2007, 173, 118-134.
[http://dx.doi.org/10.1016/j.plantsci.2007.04.009]
[32]
Konno, H.; Nakashima, S.; Nakato, T.; Katoh, K. Pectin-bound beta-galactosidase present in cell walls of carrot cells under the different calcium status. Physiol. Plant., 2002, 114(2), 213-222.
[http://dx.doi.org/10.1034/j.1399-3054.2002.1140207.x] [PMID: 11903968]
[33]
Gantulga, D.; Turan, Y.; Bevan, D.R.; Esen, A. The Arabidopsis At1g45130 and At3g52840 genes encode beta-galactosidases with activity toward cell wall polysaccharides. Phytochemistry, 2008, 69(8), 1661-1670.
[http://dx.doi.org/10.1016/j.phytochem.2008.01.023] [PMID: 18359051]
[34]
Iqbal, S.; Nguyen, T.H.; Nguyen, T.T.; Maischberger, T.; Haltrich, D. beta-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydr. Res., 2010, 345(10), 1408-1416.
[http://dx.doi.org/10.1016/j.carres.2010.03.028] [PMID: 20385377]
[35]
Nguyen, T.H.; Splechtna, B.; Steinböck, M.; Kneifel, W.; Lettner, H.P.; Kulbe, K.D.; Haltrich, D. Purification and characterization of two novel beta-galactosidases from Lactobacillus reuteri. J. Agric. Food Chem., 2006, 54(14), 4989-4998.
[http://dx.doi.org/10.1021/jf053126u] [PMID: 16819907]
[36]
Hung, M.N.; Lee, B.H. Purification and characterization of a recombinant beta-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96. Appl. Microbiol. Biotechnol., 2002, 58(4), 439-445.
[http://dx.doi.org/10.1007/s00253-001-0911-6] [PMID: 11954789]
[37]
Stephens, R.; DeBusk, A.G. Beta-galactosidases from Neurospora crassa. Methods Enzymol., 1975, 42, 497-503.
[http://dx.doi.org/10.1016/0076-6879(75)42158-9] [PMID: 237204]
[38]
Tello-Solís, S.R.; Jiménez-Guzmán, J.; Sarabia-Leos, C.; Gómez-Ruíz, L.; Cruz-Guerrero, A.E.; Rodríguez-Serrano, G.M.; García-Garibay, M. Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH. J. Agric. Food Chem., 2005, 53(26), 10200-10204.
[http://dx.doi.org/10.1021/jf051480m] [PMID: 16366715]
[39]
Lee, D.H.; Kang, S.G.; Suh, S.G.; Byun, J.K. Purification and characterization of a beta-galactosidase from peach (Prunus persica). Mol. Cells, 2003, 15(1), 68-74.
[PMID: 12661763]
[40]
Kim, J.W.; Rajagopal, S.N. Isolation and characterization of beta-galactosidase from Lactobacillus crispatus. Folia Microbiol. (Praha), 2000, 45(1), 29-34.
[http://dx.doi.org/10.1007/BF02817446] [PMID: 11200668]
[41]
de Macías, M.E.; Manca de Nadra, M.C.; Strasser de Saad, A.M.; Pesce de Ruiz Holgado, A.A.; Oliver, G. Isolation and properties of beta-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter. J. Appl. Biochem., 1983, 5(4-5), 275-281.
[PMID: 6434507]
[42]
Liao, X.; Huang, J.; Zhou, Q.; Guo, L.; Lin, J.; You, L.; Liu, S.; Yang, J. Designing of a novel β-galactosidase for production of functional oligosaccharides. Eur. Food Res. Technol., 2017, 243, 979-986.
[http://dx.doi.org/10.1007/s00217-016-2813-y]
[43]
Ornelas, A.P.; Silveira, W.B.; Sampaio, F.C.; Passos, F.M.L. The activity of β-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate. J. Appl. Microbiol., 2008, 104(4), 1008-1013.
[http://dx.doi.org/10.1111/j.1365-2672.2007.03622.x] [PMID: 17976174]
[44]
Todorova-Balvay, D.; Stoilova, I.; Gargova, S.; Vijayalakshmi, M.A. An efficient two step purification and molecular characterization of β-galactosidases from Aspergillus oryzae. J. Mol. Recognit., 2006, 19(4), 299-304.
[http://dx.doi.org/10.1002/jmr.788] [PMID: 16865665]
[45]
Otieno, D.O.; Shah, N.P. Endogenous β-glucosidase and β-galactosidase activities from selected probiotic micro-organisms and their role in isoflavone biotransformation in soymilk. J. Appl. Microbiol., 2007, 103(4), 910-917.
[http://dx.doi.org/10.1111/j.1365-2672.2007.03438.x] [PMID: 17897193]
[46]
Rhimi, M.; Aghajari, N.; Jaouadi, B.; Juy, M.; Boudebbouze, S.; Maguin, E.; Haser, R.; Bejar, S. Exploring the acidotolerance of beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus: an attractive enzyme for lactose bioconversion. Res. Microbiol., 2009, 160(10), 775-784.
[http://dx.doi.org/10.1016/j.resmic.2009.09.004] [PMID: 19786095]
[47]
Halbmayr, E.; Mathiesen, G.; Nguyen, T.H.; Maischberger, T.; Peterbauer, C.K.; Eijsink, V.G.; Haltrich, D. High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system. J. Agric. Food Chem., 2008, 56(12), 4710-4719.
[http://dx.doi.org/10.1021/jf073260+] [PMID: 18512940]
[48]
Reddy Chichili, V.P.; Kumar, V.; Sivaraman, J. Linkers in the structural biology of protein-protein interactions. Protein Sci., 2013, 22(2), 153-167.
[http://dx.doi.org/10.1002/pro.2206] [PMID: 23225024]
[49]
Tollefsen, S.; Hotta, K.; Chen, X.; Simonsen, B.; Swaminathan, K.; Mathews, I.I.; Sollid, L.M.; Kim, C-Y. Structural and functional studies of trans-encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) protein molecule. J. Biol. Chem., 2012, 287(17), 13611-13619.
[http://dx.doi.org/10.1074/jbc.M111.320374] [PMID: 22362761]
[50]
Eldridge, B.; Cooley, R.N.; Odegrip, R.; McGregor, D.P.; Fitzgerald, K.J.; Ullman, C.G. An in vitro selection strategy for conferring protease resistance to ligand binding peptides. Protein Eng. Des. Sel., 2009, 22(11), 691-698.
[http://dx.doi.org/10.1093/protein/gzp052] [PMID: 19755412]
[51]
Horner, T.W.; Dunn, M.L.; Eggett, D.L.; Ogden, L.V. β-Galactosidase activity of commercial lactase samples in raw and pasteurized milk at refrigerated temperatures. J. Dairy Sci., 2011, 94(7), 3242-3249.
[http://dx.doi.org/10.3168/jds.2010-3742] [PMID: 21700008]

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