TY - JOUR
T1 - Levan-type fructooligosaccharides synthesis by novel levansucrase-inulosucrase fusion enzyme
AU - Charoenwongpaiboon, Thanapon
AU - Wangpaiboon, Karan
AU - Klaewkla, Methus
AU - Kuttiyawong, Kamontip
AU - Field, Robert A.
AU - Pichyangkura, Rath
N1 - Funding Information: This work (Grant No. RGNS 64-215) was supported by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI) and Silpakorn University, Thailand. We would like to thank Department of Biochemistry, Faculty of Science, Chulalongkorn University , Thailand; Department of Chemistry, Faculty of Science, Silpakorn University, Thailand for the use of experimental facilities.
PY - 2022/7
Y1 - 2022/7
N2 - A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.
AB - A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.
KW - Fructan sucrase
KW - Fructooligosaccharide
KW - Fusion enzyme
UR - http://www.scopus.com/inward/record.url?scp=85132863444&partnerID=8YFLogxK
U2 - 10.1016/j.bej.2022.108524
DO - 10.1016/j.bej.2022.108524
M3 - Article
AN - SCOPUS:85132863444
VL - 185
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
SN - 1369-703X
M1 - 108524
ER -