Biosynthesis and Structural Characterization of Levan by a Recombinant Levansucrase from Bacillus subtilis ZW019

Abstract

Purpose: The yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan.Methods: A recombinant plasmid pET-28a-AcmA-Z constructed in the previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, NMR techniques.Results: The recombinant levansucrase could be easily puri ed in one step and the puri ed enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K + and Ca 2+ . The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×10 6 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan.Conclusions: The results of this study provide a basis for large-scale production of levan by enzymatic method.

Multifunctional Biotechnological Lip Moisturizer for Lip Repair and Hydration: Development, In Vivo Efficacy Assessment and Sensory Analysis

“…LEV production in the present study was high due to previous studies carried out by our research group related to fermentative parameters [53,54] and the use of the enzyme levansucrase for its production. Other studies that used levansucrase for the production of LEV presented higher production than those that used only the microorganism, such as reported by Ko et al [55], with a production of 76 g•L −1 , and Wang et al [56], with a production of 30.6 g•L −1 . As seen, enzymes derived from microorganisms are important tools in the biotechnological process, being more useful than naturally occurring enzymes in microorganisms [57], probably because in microbial cells there are other enzymatic routes that interfere in the best yield of the levansucrase route.…”

Valorization of Using Agro-Wastes for Levan through Submerged Fermentation and Statistical Optimization of the Process Variables Applying Response Surface Methodology (RSM) Design

“…Levan’s typical molecular weight was found to be 7.6 × 10 6 KDa. Wang et al [ 25 ] found the average molecular weight of levan to be 1.56 × 10 6 Da. A study by Carbajal et al [ 26 ] found that high-molecular-weight levan (2.3 × 10 6 Da) and low-molecular-weight levan (7.2 × 10 3 Da) were both present in levan generated by Bacillus subtilis.…”