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An Approach for Lactulose Production Using the CotX-Mediated Spore-Displayed β-Galactosidase as a Biocatalyst
1State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China, 2Jiyang College, Zhejiang Agriculture and Forestry University, Zhuji 311800, P.R. China, 3School of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China
J. Microbiol. Biotechnol. 2016; 26(7): 1267-1277
Published July 28, 2016 https://doi.org/10.4014/jmb.1602.02036
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
References
- Adamczak M, Charubin D, Bednarski W. 2009. Influence of reaction medium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared from whey permeate. Chem. Pap. 63: 111-116.
- Aider M, de Halleux D. 2007. Isomerization of lactose and lactulose production: review. Trends Food Sci. Technol. 18: 356-364.
- Chen W, Chen H, Xia Y, Zhao J, Tian F, Zhang H. 2008. Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. J. Dairy Sci. 91: 1751-1758.
- Cutting SM, Vander-Horn PB. 1990. Genetic analysis, pp. 27-74. In Harwood CR, Cutting SM (eds.). Molecular Biological Methods for Bacillus. John Wiley & Sons, Chichester.
- Driks A. 1999. Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63: 1-20.
- Duetz WA, Van Beilen JB, Witholt B. 2001. Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis. Curr. Opin. Biotechnol. 12: 419-425.
- Gao C, Xu X, Zhang X, Che B, Ma C, Qiu J, et al. 2011. Chemoenzymatic synthesis of N-acetyl-D-neuraminic acid from N-acetyl-D-glucosamine by using the spore surfacedisplayed N-acetyl-D-neuraminic acid aldolase. Appl. Environ. Microbiol. 77: 7080-7083.
- Guerrero C, Vera C, Plou F, Illanes A. 2011. Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases. J. Mol. Catal. B Enzym. 72: 206-212.
- Henriques AO, Moran CP. 2000. Structure and assembly of the bacterial endospore coat. Methods 20: 95-110.
- Hinc K, Isticato R, Dembek M, Karczewska J, Iwanicki A, Peszy ska-Sularz G, et al. 2010. Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores. Microb. Cell Fact. 9: 2.
- Hinc K, Iwanicki A, Obuchowski M. 2013. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12: 22.
- Hwang BY, Pan JG, Kim BG, Kim JH. 2013. Functional display of active tetrameric β-galactosidase using Bacillus subtilis spore display system. J. Nanosci. Nanotechnol. 13:2313-2319.
- Imamura D, Kuwana R, Takamatsu H, Watabe K. 2010. Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J. Bacteriol. 192: 518-524.
- Imamura D, Kuwana R, Takamatsu H, Watabe K. 2011. Proteins involved in formation of the outermost layer of Bacillus subtilis spores. J. Bacteriol. 193: 4075-4080.
- Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D, Oggioni MR, et al. 2001. Surface display of recombinant proteins on Bacillus subtilis spores. J. Bacteriol. 183: 62946301.
- Khatami S, Ashtiani FZ, Bonakdarpour B, Mehrdad M. 2014. The enzymatic production of lactulose via transglycosylation in conventional and non-conventional media. Int. Dairy J. 34: 74-79.
- Kim JH, Lee CS, Kim BG. 2005. Spore-displayed streptavidin:a live diagnostic tool in biotechnology. Biochem. Biophys. Res. Commun. 331: 210-214.
- Kim YS, Park CS, Oh DK. 2006. Lactulose production from lactose and fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme Microb. Technol. 39: 903-908.
- Kwon SJ, Jung HC, Pan JG. 2007. Transgalactosylation in a water-solvent biphasic reaction system with β-galactosidase displayed on the surfaces of Bacillus subtilis spores. Appl. Environ. Microbiol. 73: 2251-2256.
- Lee SY, Choi JH, Xu Z. 2003. Microbial cell-surface display. Trends Biotechnol. 21: 45-52.
- Lee YJ, Kim CS, Oh DK. 2004. Lactulose production by βgalactosidase in permeabilized cells of Kluyveromyces lactis. Appl. Microbiol. Biotechnol. 64: 787-793.
- Li Q, Ning D, Wu C. 2010. Surface display of GFP using CotX as a molecular vector on Bacillus subtilis spores. Chin. J. Biotechnol. 26: 264-269.
- Liu Y, Li S, Xu H, Wu L, Xu Z, Liu J, Feng X. 2014. Efficient production of D-tagatose using a food-grade surface display system. J. Agric. Food Chem. 62: 6756-6762.
- Mauriello EMF, Duc LH, Isticato R, Cangiano G, Hong HA, De Felice M, et al. 2004. Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine 22: 1177-1187.
- McKenney PT, Driks A, Eskandarian HA, Grabowski P, Guberman J, Wang KH, et al. 2010. A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Curr. Biol. 20: 934-938.
- Negri A, Potocki W, Iwanicki A, Obuchowski M, Hinc K. 2013. Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores. J. Med. Microbiol. 62: 1379-1385.
- Nguyen QA, Schumann W. 2014. Use of IPTG-inducible promoters for anchoring recombinant proteins on the Bacillus subtilis spore surface. Protein Expr. Purif. 95: 67-76.
- Nicholson WL, Setlow P. 1990. Sporulation, germination, and outgrowth, pp. 391-450. In Harwood CR, Cutting SM (eds.). Molecular Biological Methods for Bacillus. John Wiley & Sons, Chichester.
- Qu Y, Wang J, Zhang Z, Shi S, Li D, Shen W, et al. 2014. Catalytic transformation of HODAs using an efficient metacleavage product hydrolase-spore surface display system. J. Mol. Catal. B Enzym. 102: 204-210.
- Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
- Sitanggang AB, Drews A, Kraume M. 2014. Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis. Bioresour. Technol. 16: 108-115.
- Song YS, Lee HU, Park C, Kim SW. 2013. Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase. Food Chem. 136: 689-694.
- Ståhl S, Uhlén M. 1997. Bacterial surface display: trends and progress. Trends Biotechnol. 15: 185-192.
- Tavassoli S, Hinc K, Iwanicki A, Obuchowski M, Ahmadian G. 2013. Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst. Arch. Microbiol. 195: 197-202.
- Vaheri M, Kaupinnen V. 1978. The formation of lactulose (4O-β-galactopyranosylfructose) by β-galactosidase. Acta Pharm. Fenn. 87: 75-83.
- Wang H, Yang R, Hua X, Zhao W, Zhang W. 2013. Enzymatic production of lactulose and 1-lactulose: current state and perspectives. Appl. Microbiol. Biotechnol. 97: 61676180.
- Wang H, Yang R, Hua X, Zhao W, Zhang W. 2015. Functional display of active β-galactosidase on Bacillus subtilis spores using crust proteins as carriers. Food Sci. Biotechnol. 24: 17551759.
- Wang H, Yang R, Jiang X, Hua X, Zhao W, Zhang W, Chen X. 2015. Expression and characterization of two β-galactosidases from Klebsiella pneumoniae 285 in Escherichia coli and their application in the enzymatic synthesis of lactulose and 1lactulose. Z. Naturforsch. C 69: 479-487.
- Wang M, Yang R, Hua X, Shen Q, Zhang W, Zhao W. 2015. Lactulose production from lactose by recombinant cellobiose 2-epimerase in permeabilised Escherichia coli cells. Int. J. Food Sci. Technol. 50: 1625-1631.
- Zilhão R, Serrano M, Isticato R, Ricca E, Moran CP, Henriques AO. 2004. Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat. J. Bacteriol. 186: 1110-1119.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2016; 26(7): 1267-1277
Published online July 28, 2016 https://doi.org/10.4014/jmb.1602.02036
Copyright © The Korean Society for Microbiology and Biotechnology.
An Approach for Lactulose Production Using the CotX-Mediated Spore-Displayed β-Galactosidase as a Biocatalyst
He Wang 1, 2, Ruijin Yang 3*, Xiao Hua 3, Wenbin Zhang 3 and Wei Zhao 3
1State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China, 2Jiyang College, Zhejiang Agriculture and Forestry University, Zhuji 311800, P.R. China, 3School of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China
Abstract
Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly
performed with glycosyl hydrolases. In this work, a new type of lactulose-producing
biocatalyst was developed by displaying β-galactosidase from Bacillus stearothermophilus
IAM11001 (Bs-β-Gal) on the surface of Bacillus subtilis 168 spores. Localization of β-Gal on the
spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence
microscopy, and flow cytometry. The optimum pH and temperature for the resulting sporedisplayed
β-Gal was 6.0 and 75oC, respectively. Under optimal conditions, it showed
maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-β-
Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from
200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-β-
Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These
results suggest that the CotX-mediated spore-displayed β-Gal may provide a promising
strategy for lactulose production.
Keywords: surface display, spore, Bacillus subtilis, CotX, β-galactosidase, lactulose
References
- Adamczak M, Charubin D, Bednarski W. 2009. Influence of reaction medium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared from whey permeate. Chem. Pap. 63: 111-116.
- Aider M, de Halleux D. 2007. Isomerization of lactose and lactulose production: review. Trends Food Sci. Technol. 18: 356-364.
- Chen W, Chen H, Xia Y, Zhao J, Tian F, Zhang H. 2008. Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. J. Dairy Sci. 91: 1751-1758.
- Cutting SM, Vander-Horn PB. 1990. Genetic analysis, pp. 27-74. In Harwood CR, Cutting SM (eds.). Molecular Biological Methods for Bacillus. John Wiley & Sons, Chichester.
- Driks A. 1999. Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63: 1-20.
- Duetz WA, Van Beilen JB, Witholt B. 2001. Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis. Curr. Opin. Biotechnol. 12: 419-425.
- Gao C, Xu X, Zhang X, Che B, Ma C, Qiu J, et al. 2011. Chemoenzymatic synthesis of N-acetyl-D-neuraminic acid from N-acetyl-D-glucosamine by using the spore surfacedisplayed N-acetyl-D-neuraminic acid aldolase. Appl. Environ. Microbiol. 77: 7080-7083.
- Guerrero C, Vera C, Plou F, Illanes A. 2011. Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases. J. Mol. Catal. B Enzym. 72: 206-212.
- Henriques AO, Moran CP. 2000. Structure and assembly of the bacterial endospore coat. Methods 20: 95-110.
- Hinc K, Isticato R, Dembek M, Karczewska J, Iwanicki A, Peszy ska-Sularz G, et al. 2010. Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores. Microb. Cell Fact. 9: 2.
- Hinc K, Iwanicki A, Obuchowski M. 2013. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12: 22.
- Hwang BY, Pan JG, Kim BG, Kim JH. 2013. Functional display of active tetrameric β-galactosidase using Bacillus subtilis spore display system. J. Nanosci. Nanotechnol. 13:2313-2319.
- Imamura D, Kuwana R, Takamatsu H, Watabe K. 2010. Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J. Bacteriol. 192: 518-524.
- Imamura D, Kuwana R, Takamatsu H, Watabe K. 2011. Proteins involved in formation of the outermost layer of Bacillus subtilis spores. J. Bacteriol. 193: 4075-4080.
- Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D, Oggioni MR, et al. 2001. Surface display of recombinant proteins on Bacillus subtilis spores. J. Bacteriol. 183: 62946301.
- Khatami S, Ashtiani FZ, Bonakdarpour B, Mehrdad M. 2014. The enzymatic production of lactulose via transglycosylation in conventional and non-conventional media. Int. Dairy J. 34: 74-79.
- Kim JH, Lee CS, Kim BG. 2005. Spore-displayed streptavidin:a live diagnostic tool in biotechnology. Biochem. Biophys. Res. Commun. 331: 210-214.
- Kim YS, Park CS, Oh DK. 2006. Lactulose production from lactose and fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme Microb. Technol. 39: 903-908.
- Kwon SJ, Jung HC, Pan JG. 2007. Transgalactosylation in a water-solvent biphasic reaction system with β-galactosidase displayed on the surfaces of Bacillus subtilis spores. Appl. Environ. Microbiol. 73: 2251-2256.
- Lee SY, Choi JH, Xu Z. 2003. Microbial cell-surface display. Trends Biotechnol. 21: 45-52.
- Lee YJ, Kim CS, Oh DK. 2004. Lactulose production by βgalactosidase in permeabilized cells of Kluyveromyces lactis. Appl. Microbiol. Biotechnol. 64: 787-793.
- Li Q, Ning D, Wu C. 2010. Surface display of GFP using CotX as a molecular vector on Bacillus subtilis spores. Chin. J. Biotechnol. 26: 264-269.
- Liu Y, Li S, Xu H, Wu L, Xu Z, Liu J, Feng X. 2014. Efficient production of D-tagatose using a food-grade surface display system. J. Agric. Food Chem. 62: 6756-6762.
- Mauriello EMF, Duc LH, Isticato R, Cangiano G, Hong HA, De Felice M, et al. 2004. Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine 22: 1177-1187.
- McKenney PT, Driks A, Eskandarian HA, Grabowski P, Guberman J, Wang KH, et al. 2010. A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Curr. Biol. 20: 934-938.
- Negri A, Potocki W, Iwanicki A, Obuchowski M, Hinc K. 2013. Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores. J. Med. Microbiol. 62: 1379-1385.
- Nguyen QA, Schumann W. 2014. Use of IPTG-inducible promoters for anchoring recombinant proteins on the Bacillus subtilis spore surface. Protein Expr. Purif. 95: 67-76.
- Nicholson WL, Setlow P. 1990. Sporulation, germination, and outgrowth, pp. 391-450. In Harwood CR, Cutting SM (eds.). Molecular Biological Methods for Bacillus. John Wiley & Sons, Chichester.
- Qu Y, Wang J, Zhang Z, Shi S, Li D, Shen W, et al. 2014. Catalytic transformation of HODAs using an efficient metacleavage product hydrolase-spore surface display system. J. Mol. Catal. B Enzym. 102: 204-210.
- Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
- Sitanggang AB, Drews A, Kraume M. 2014. Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis. Bioresour. Technol. 16: 108-115.
- Song YS, Lee HU, Park C, Kim SW. 2013. Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase. Food Chem. 136: 689-694.
- Ståhl S, Uhlén M. 1997. Bacterial surface display: trends and progress. Trends Biotechnol. 15: 185-192.
- Tavassoli S, Hinc K, Iwanicki A, Obuchowski M, Ahmadian G. 2013. Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst. Arch. Microbiol. 195: 197-202.
- Vaheri M, Kaupinnen V. 1978. The formation of lactulose (4O-β-galactopyranosylfructose) by β-galactosidase. Acta Pharm. Fenn. 87: 75-83.
- Wang H, Yang R, Hua X, Zhao W, Zhang W. 2013. Enzymatic production of lactulose and 1-lactulose: current state and perspectives. Appl. Microbiol. Biotechnol. 97: 61676180.
- Wang H, Yang R, Hua X, Zhao W, Zhang W. 2015. Functional display of active β-galactosidase on Bacillus subtilis spores using crust proteins as carriers. Food Sci. Biotechnol. 24: 17551759.
- Wang H, Yang R, Jiang X, Hua X, Zhao W, Zhang W, Chen X. 2015. Expression and characterization of two β-galactosidases from Klebsiella pneumoniae 285 in Escherichia coli and their application in the enzymatic synthesis of lactulose and 1lactulose. Z. Naturforsch. C 69: 479-487.
- Wang M, Yang R, Hua X, Shen Q, Zhang W, Zhao W. 2015. Lactulose production from lactose by recombinant cellobiose 2-epimerase in permeabilised Escherichia coli cells. Int. J. Food Sci. Technol. 50: 1625-1631.
- Zilhão R, Serrano M, Isticato R, Ricca E, Moran CP, Henriques AO. 2004. Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat. J. Bacteriol. 186: 1110-1119.