Recombinant Expression and Biochemical Characterization of Levansucrase from Halophilic Bacteria Bacillus licheniformis BK1 and BK2

Abstract:

Levansucrase was an extracellular polysacharride (EPS) which has a role in synthesizing levans by transferring fructose moiety from sucrose to acceptor molecules. In the previous study, we have successfully cloned the levansucarese gene from two Bacillus licheniformis strains of BK1 and BK2 labeled as lsbl-bk1 and lsbl-bk2. The present study aims to optimize the expression level of both genes in E. coli expression system and also to obtain the optimum conditions for the recombinant enzymes activity by applying the response surface methodology (RSM). The optimization result found that the highest Lsbl-bk1 production in E. coli expression system was occurred when the recombinant cells grown in the medium containing 0.6% (w/v) NaCl at 42°C, and induced by 0.6 mM IPTG. Different optimum conditions were found for Lsbl-bk2 production. It was achieved when 1.1% (w/v) NaCl added to the production medium and induced by 0.7 mM IPTG at 40°C. RSM optimization result for biochemical characterization of Lsbl-bk1 levansucrase showed the highest specific activity achieved at 56°C and pH 7.5, whereas for the Lsbl-bk2 levansucrase reached the highest specific activity at 50°C and pH 7.5. The addition of Co²⁺, Ti²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Fe³⁺, Ca²⁺ metal ion to both levansucrases solution did not significantly altered their specific activity, indicating that both levansucrases are not metalo enzymes. Furthermore, the specific activity of levansucrase was also not affected by the addition of 1-25% (w/v) NaCl, suggesting that the variation of ionic strength did not alter the native state of both enzymes. The plot results of levansucrase specific activities toward sucrose concentration showed that both levansucrases follow Michaelis-Menten profile with k_cat/K_M values ​​about 3.8 and 3.6 s^-1/mM respectively. These data indicated that the recombinant levansucrases from halophilic bacteria B. licheniformis BK1 and BK2 are a non metaloenzyme with high affinity and binding rate to sucrose substrate, in which the catalytic efficiency on hydrolysis reactions is relatively low.

• Periodical: Key Engineering Materials (Volume 874)
• Pages: 96-106
• DOI: https://doi.org/10.4028/www.scientific.net/KEM.874.96
• Online since: January 2021
• Authors: Nur Umriani Permatasari, Enny Ratnaningsih, Rukman Hertadi*
• Keywords: E. Coli BL21 (DE3)pLysS, Exopolysccaharides, Full Factorial CCD, Levansucrase, Moderately Halophilic Bacteria

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