Levansucrase Production of Bacillus siamensis KKSB4 and Bacillus velezensis KKSB6 in Fed-Batch Culture

Abstract

Background and Objectives : Levan is a natural homopolymer of fructose from both plants and microorganisms. Levan structure consists of D-fructofuranosyl groups linked to each other by β-(2, 6) bonds and D-glucosyl at the end of chain. Microbial levans have wide range of applications in food, pharmaceutical, cosmetic and commercial industries; they are produced by levansucrase with sucrose as a substrate. Bacillus siamensis KKSB4 and Bacillus velezensis KKSB6 were isolated from fermented soybeans and contained the ability to produce levan by levansucrase. Therefore, these strains were investigated the optimal culture, sucrose concentration and fed-batch cultivation for growth and levansucrase production.

Methodology : The optimal culture conditions of B. siamensis KKSB4 and B. velezensis KKSB6 were determined for suitable growth and maximizing enzyme production by various parameters of temperature, initial pH and sucrose concentration. The levansucrase activity was determined by dinitrosalicylic acid (DNS) method. The optical density (OD600 nm) and the amount of sucrose were also detected. The conditions of fed-batch cultivation were investigated by the addition of LB medium, sucrose solution or LB with sucrose during the cultivation period.

Main Results : The results showed that B. siamensis KKSB4 and B. velezensis KKSB6 had high growth and levansucrase activity when cultured in Luria-Bertani (LB) at 37°C, pH 7 with 30 g/L and 20 g/L of sucrose, respectively. The fed-batch cultivation was studied to increase enzyme production; B. siamensis KKSB4 gave the highest enzyme activity by the addition of LB medium at 24 and 48 hours of cultivation. Whereas, B. velezensis KKSB6 indicated high activity by the addition of LB medium with sucrose as a substrate at 24 and 48 hours of cultivation.

Conclusions  : B. siamensis KKSB4 and B. velezensis KKSB6 showed enzyme activity at 5.06 U/ml and  4.03 U/ml, respectively. The optimal condition of fed-batch cultivation was high sucrose medium (30 g/L) with adding only nutrient at 24 and 48 hours for B. siamensis KKSB4. On the other hand, B. velezensis KKSB6 used the condition of the medium with low amount of sucrose (10 g/L) and adding nutrient with sucrose during cultivation.