Cloning of sucrose isomerase encoding gene from Klebsiella singaporensis ISB-36 and its expression in Pichia pastoris

Cao Xuan Bach, Dang Thi Kim Anh, Nguyen Thanh Thuy, Truong Tu Anh, Nguyen Thi Dieu Linh, Vu Nguyen Thanh

Abstract


Given potential health benefits including low glycemic index, tooth friendly, suitable to infants, elderly and diabetic patients, isomaltulose was considered as a promising alternative sweetener to sucrose. Due to the presence of liposaccharide endotoxin in Serratia plymuthica CBS 574.44, a Gram-negative bacterium, and minute amount of formaldehyde carried over, purification of isomaltulose requires rigorous controls in industry. To reduce the cost associated with product purification, here we propose the use of recombinant enzyme in isomaltulose production. The mature gene coding for sucrose isomerase synthase (K.SI36.PalI) from Klebsiella singarporensis ISB 36, which isolated from woodborer in Vietnam, was expressed in Pichia pastoris X33. The nucleotide sequence of K.SI36.PalI gene was similar to AY040843.1 of Klebsiella sp. LX3 except one nucleotide C1025 in AY040843.1 replaced by T1025 in K.SI36.PalI. This leads to single amino acid difference in deduced protein sequence (from 342Ser to 342Phe). Furthermore, the addition of two amino acids (Glu and Phe) was observed at N-terminus. The calculated molecular weight of sucrose isomerase from K.SI36.PalI was 67.46 kDa and the pI was 6.55. There was one potential glycosylation site at 466Asn. The maximum sucrose isomerase activity in the culture broth reached 36,6 U.mL-1in 1 L shake-flask. The purified recombinant enzyme was most active at 40°C and pH 7.0. At the optimum condition, within 6 hours, the enzyme converted 94% of sucrose in a 40% sucrose solution into isomaltulose. This was the first study on the expression of sucrose isomerase synthase gene in P. pastoris, and the results showed the efficient conversion of sucrose isomerase recombinant.


Keywords


isomaltulose, food sweetner, sucrose isomerase, recombinant enzyme, Klebsiella, Pichia pastorsis.

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References


BeMiller JN (2019) Oligosaccharides. In Carbohydrate Chemistry for Food Scientists: 49–74.

Cha J, Jung JH, Park SE, Cho M H, Seo DH, Ha SJ, Park CS (2009) Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1. J Appl Microb 107(4): 1119–1130.

Ghose TK (1987) Measurement of cellulase activities. Pure and Applied Chemistry 59(2): 257–268.

Goulter KC, Hashimi SM, Birch RG (2012) Microbial sucrose isomerases: Producing organisms, genes and enzymes. Enzyme and Microbial Technology 50(1): 57–64.

Kawaguti HY, Sato HH (2010) Isomaltulose production by free cells of Serratia plymuthica in a batch process. Food Chemistry 120(3): 789–793.

Krastanov A, Yoshida T (2003) Production of palatinose using Serratia plymuthica cells immobilized in chitosan. Journal of Industrial Microbiology and Biotechnology 30(10): 593–598.

Lee GY, Jung JH, Seo DH, Hansin J, Ha SJ, Cha J, Park CS (2011a) Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae. Bioresource Technology 102(19): 9179–9184.

Lee GY, Jung JH, Seo DH, Hansin J, Ha SJ, Cha J, Park CS (2011b) Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae. Bioresource Technology 102(19): 9179–9184.

Lina BR, Jonker D, Kozianowski G (2002) Isomaltulose (Palatinose 1): a review of biological and toxicological studies. Food and Chemical Toxicology 40: 1375–1381.

Mu W, Li W, Wang X, Zhang T, Jiang B (2014a) Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase. Applied Microbiology and Biotechnology 98(15): 6569–6582.

Mu W, Li W, Wang X, Zhang T, Jiang B (2014b) Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase. Applied Microbiology and Biotechnology 98(15): 6569–6582.

Nguyen T Thuy, Nguyen T Hoa Mai, Dinh T My Hang, Vu N Thanh (2013) Technology for isomaltulose production from sucrose using Enterobacter sp. ISB-25. Journal of Science and Technology 51: 173–184.

O’Donnell K, Kearsley M (2012) Sweeteners and sugar alternatives in food technology. John Wiley & Sons.

Orsi DC, Sato HH (2016) Isomaltulose production using free and immobilized Serratia plymuthica cells. African Journal of Biotechnology 15(20): 835–842.

Park JY, Jung JH, Seo DH, Ha SJ, Yoon JW, Kim YC, Park CS (2010) Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363. Bioresource Technology 101(22): 8828–8833.

Tiefenbacher KF (2017) Technology of Main Ingredients—Sweeteners and Lipids. In Wafer and Waffle : 123–225.

Veronese T, Perlot P (1998) Proposition for the biochemical mechanism occurring in the sucrose isomerase active site. FEBS Lett, 441(3): 348–352.

Véronèse T, Perlot P (1999) Mechanism of sucrose conversion by the sucrose isomerase of Serratia plymuthica ATCC 15928. Enzyme and Microbial Technology 24(5–6): 263–269.

Wei Y, Liang J, Huang Y, Lei P, Du L, Huang R (2013) Simple, fast, and efficient process for producing and purifying trehalulose. Food Chem 138(2–3): 1183–1188.

Xu Z, Li S, Li J, Li Y, Feng X, Wang R, Zhou J (2013) The Structural Basis of Erwinia rhapontici Isomaltulose Synthase. PLoS ONE 8(9).

Zhang D, Li N, Lok SM, Zhang LH, Swaminathan K (2003) Isomaltulose synthase (PalI) of Klebsiella sp. LX3: Crystal structure and implication of mechanism. Journal of Biological Chemistry 278(37): 35428–35434.

Zhang D, Li X, Zhang LH (2002) Isomaltulose synthase from Klebsiella sp. strain LX3: Gene cloning and characterization and engineering of thermostability. Applied and Environmental Microbiology 68(6): 2676–2682.




DOI: https://doi.org/10.15625/1811-4989/17/4/14722 Display counter: Abstract : 64 views. PDF : 34 views.