# A Novel Sucrose Isomerase Producing Isomaltulose from _Raoultella terrigena_
###### Abstract  
Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (Slase) with sucrose as substrate. In this study, a gene _pal-2_ from _Raoultella terrigena_ was predicted to produce Slase, which was subcloned into pET-28a (+) and transformed to the _E. coli_ system. The purified recombinant Slase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 \({}^{\circ}\)C and optimal pH value of 5.5. The Michaelis constant (\(K_{\text{m}}\)) and maximum reaction rate (\(V_{\text{mm}}\)) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of Slase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel Slase from the _Raoultella_ genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.  
sucrose isomerase; isomaltulose; enzymology properties; molecular modification +
Footnote †: journal: Applied Sciences  
chemical methods and so biochemical conversion is the common means for isomaltulose production [14]. The critical factor in bioconversion is sucrose isomerase (Slase) (EC 5.4.99.11) which is capable in isomerizing sucrose into isomaltulose and trehalulose, but often results in a small quantity of hydrolysis products including glucose and fructose [15]. Slase is found in many microorganisms, such as _Prottaminobacter rubrum_ sp. [16], _Erwinia rhapontici_ sp. [17], _Serratia plymuthia_ sp. [18], _Enterobacter_ sp. [19], _Klebsiella_ sp. [20], _Pantoea dispersa_ sp. [21], _Pseudomonas mesoacidophila_ sp. [22], and _Agrobacteriumrium radiobacter_ sp. [23]. The isomaltulose produced by these Slases accounted for 60-90% of all products. Therefore, it is of great significance to identify novel Slases with potential applications.  
The enzyme activity of Slase obtained from wild bacteria is generally low and the highest is only 30 U/mL, which is difficult to meet the demand of industrial production. Thus, a heterogenous expression approach was investigated. Slase genes from different microorganisms have been successfully cloned and expressed in the _E. coli_ system. The expression level of Slase is between 15-654 U/mL.  
The generation of by-products and low enzyme activity are two adverse factors in isomaltulose production. For instance, the Slases from _Erwinia_ sp. D12 [24] had 19.8 U/mg of enzymatic activity and 65.7% of conversion rate of isomaltulose. In addition, the Slases from _S. plymuthica_ ATCC159282 [25] had 120 U/mg of enzymatic activity and 72.6% of conversion rate of isomaltulose. Therefore, protein engineering approachs based on structural information can be used to improve the application performance of these Slases.  
To date, there has been no Slase explored from the _Raoultella_ genus. Therefore, to expand the microorganism sources and find a promising Slase, this work identified a putative Slase Pal-2 from _Raoultella terrigena_ and expressed this enzyme in the E. coli system. The enzymatic properties of Pal-2 and its potential application for the isomaltulose production were also investigated. Simultaneously, the mutants with high enzyme performance were obtained by the mutagenesis method, which provided a feasible approach with application in the production of isomaltulose.