Enzyme Activity Measurement for Levansucrase

Creative Enzymes is an industrial biotech company specialized in enzymes and their activity measurement. We partner with our diverse and unique customers to support them in their process optimization needs. Capabilities of Creative Enzymes are extensive, covering all aspects of the discovery, development, and optimization of enzyme assays. Creative Enzymes is competent to offer the most reliable activity assay for levansucrase.

Levansucrases (EC 2.4.1.10) have been identified in a number of Gram-negative species such as Pantoea agglomerans, Zymomonas mobilis, Gluconobacter oxydans, and a Rhanella aquatilis isolate as well as in Gram-positive bacteria such as Bacillus subtilis, Streptococcus mutans, and Actinomyces viscosus. The enzyme catalyzes three characteristic reactions: (i) the synthesis of levan from sucrose by transfructosylation while releasing glucose, (ii) the hydrolysis of levan to monosaccharides of fructose, (iii) exchange of [¹⁴C]glucose in the reaction: fructose-2,1-glucose + [¹⁴C]glucose → fructose-2,1-[¹⁴C]glucose + glucose. Levansucrases join the fructosyl units through the β(2,6)-linkage, while the related enzymes inulosucrases (EC 2.4.1.9) do so through the β(2,1)-linkage. According to the linkage type, the fructan polymers are either called levan or inulin. The enzymes are members of the glycoside hydrolases (GH), with bacterial enzymes being grouped into family 68, and structurally related plant and fungal enzymes into family 32. An interesting aspect of levansucrases from different bacteria is their specificity in the formation of either fructooligosaccharides (FOS) or high molecular weight polymers. Generally, levansucrases from Gram-positive bacteria produce polymers and those from Gram-negative bacteria produce FOS.

Levansucrases are useful in levan production for application in the food and pharmaceutical industries. For instance, levan could be important in the pathogenesis of periodontal disease, because levan may be an important structural polysaccharide in plaque, and provides a reservoir of carbohydrate for the growth of oral microbes. Besides, levan may trigger immunological injury, since levan from other organisms has been shown to be mitogenic for human B-lymphocytes and to activate complement by the alternative pathway. Additionally, levansucrases can also be used in the production of the artificial sweetener and lactosucrose. Therefore, to investigate the functional significance of levansucrases successfully, it is important to be able to monitor the activity of this enzyme. However, the levansucrases described until now differ widely in their properties, e.g., molecular weight, donor, acceptor, and inhibitor specificity, which represents a hindrance to performing the enzyme assay smoothly on an industrial scale. Fortunately, Creative Enzymes makes the accurate activity assay available for levansucrases, based on years of experiences on discovering enzymes and optimizing methods.

The spectrophotometric levansucrase assay is based upon an ultraviolet glucose assay kit measuring the amount of glucose liberated during incubation with sucrose. The amount of glucose in the sample is proportional to the amount of reduced NADPH generated. To convert this assay kit into a continuous spectrophotometric assay for levansucrase, sucrose is added and the absorbance at 340 nm is recorded by a spectrophotometer. Regardless of the challenges, Creative Enzymes will always meet the requirements for the next development stage and will be your best choice with satisfying services. The highest quality standard for our products is one of the pillars of our global success and the basis of our high-level customer satisfaction.

Figure: The crystal structure of Bacillus subtilis levansucrase.
PDB: 1OYG