Abstract
BackgroundL-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values.ResultsThe gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C.ConclusionsHere we reported the purification and the biochemical characterization of the novel Shewanella sp. ANA-3 L-arabinose isomerase. Determination of the biochemical properties demonstrated that this enzyme was highly active at low temperatures. The generated T268K mutant displays an increase of the enzyme stability essentially at low pH. These features seem to be very attractive for the bioconversion of D-galactose into D-tagatose at low temperature which is very interesting from industrial point of view.
Highlights
L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems
Gene, cloning and over-expression A DNA fragment of approximately 1.5 kb was obtained by polymerase chain reaction (PCR) amplification using Shewanella sp
DNA was ligated into the pGEMT-Easy vector and transferred into the Escherichia coli BL21 (DE3) host strain
Summary
L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. Rare sugars have made their way to the market and the demands have been increased as in agro-food industry rare natural sugars such as D-tagatose have become of a great interest as sweeteners [4] This natural sugar has the taste and natural properties of sucrose but it is an antihyperglycemiant factor known to reduce symptoms associated with type 2 diabetes [5,6]. D-Tagatose has a very low caloric value of 1.5 kcal/g (only 30% of the energy content of sucrose), but has been reported to promote weight loss [5] All these properties are very interesting for industrials, yet the use of this sugar is quite limited due to its cost [7]. Biological methods using Larabinose isomerase as a catalyst are being developed due to its low cost [8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
