1 Introduction

Isomaltulose (or Palatinose), as an isomer of sucrose, has similar physical properties and taste to sucrose [47]. In the 1957s, Weiden Hagen. R and Lorenz. S first discovered isomaltulose [66]. Compared with sucrose, isomaltulose has a broad market application prospect in food due to its good acid stability, very low hygroscopicity, and high safety [2, 52, 65]. As a new sweetener, isomaltulose has the advantages of low sweetness (approximately 50% of the relative sweetness of sucrose), non-caries, and low calorie, which is especially suitable for diabetes and obesity [11, 51]. Moreover, isomaltulose is also a kind of reducing sugar, which can be used to produce new functional edible sugar alcohols as a precursor [31]. Given the excellent properties [57], isomaltulose has been approved as a safe sucrose substitute and attracted the whole world’s attention, leading to a market growth rate of more than 10%. Isomaltulose has been used as a sucrose substitute and approved as food for specified health uses due to its dental properties in Japan since 1985. In the USA, isomaltulose is approved as “generally regarded as safe” (GRAS) by Food and Drug Administration (GRN No.184), and used in a range of dietary supplements and foods. It was authorized by the Commission of the European Communities as a novel food or novel food ingredient (notified under document number C (2005) 2776). With the rapid growth of market demand, the production of isomaltulose has gained considerable research attention around the globe.

At present, the biochemical conversion of sucrose to isomaltulose is the common means for isomaltulose production [63], reaching a scale of more than 10,000 tons annually worldwide. Bioconversion methods mainly include single enzyme conversion [4], free cell conversion [1, 79], and immobilized cell conversion [24]. The critical factor in bioconversion is sucrose isomerase (SIase) (EC 5.4.99.11), also known as isomaltulose synthase, α-glucosyltransferase, or trehalulose synthase. In nature, isomaltulose, a reserve material during periods of low carbon availability, is present in low quantities in many bacteria [48]. Numerous microorganisms have been recognized for their ability to produce SIase with different levels of isomaltulose production. Currently, SIase is mainly obtained by microbial fermentation. Escherichia coli (E. coli) is one of the ideal industrial strains for producing target products by microbial fermentation [17, 46]. With the rapid development of genetic engineering technology, SIases from different sources were heterogeneously expressed in E. coli, which significantly increased the enzyme's production level [28]. Owing to its wide involvement in the food industry [35], the heterologous expression of SIase in food-safe grade strains has gained increasing interest.

Based on the literature reported during the last decade and the recent advance, this review systematically describes the source, catalysis mechanism, and heterogeneous expression of SIase. Among them, the protein engineering of SIase, the heterologous expression in food-grade safety strains, and the fermentation optimization strategies to improve exocrine levels were mainly presented (Fig. 1).

Fig. 1
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Summary of biological isomaltulose production from sucrose catalyzed by SIase. The highlights of the review are indicated in red

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2 The Information of SIase

2.1 Source and Structure

In the 1950s, Protaminobacter rubrum (P. rubrum) producing SIase was firstly screened and isolated from wastewater discharged from the beet plant. In recent decades, researchers have found that most of SIase are derived from bacteria [39]. The representative SIase production strains, include P. rubrum [39], Erwinia rhapontici (E. rhapontici) [27], Serratia plymuthica (S. plymuthica) [22], Klebsiella planticola (K. planticola) [19], Enterobacter sp.[24], Klebsiella sp.[76, 78], Pantoea dispersa (P. dispersa) [68], and Klebsiella pneumonia (K. pneumonia) [58]. The formation of isomaltulose (6-o-α-D-glucopyranosyl-D-fructose) and trehalulose (1-o-α-D-glucopyranosyl-D-fructose) was obtained from sucrose by isomerization of SIase [18] (Fig. 2). During the isomerization of sucrose to isomaltulose and trehalulose, small amounts of D-glucose and D-fructose can also be produced as by-products by hydrolysis. According to the main product catalyzed by the enzyme, the SIase production strains were categorized into two types: isomaltulose production strains and trehalulose production strains [41]. The isomaltulose production strains include E. rhapontici NX-5 [27], Enterobacter sp. FMB-1 [24], P. dispersa UQ68J [68], P. rubrum CBS574.77 [68], E. rhapontici WAC2928 [68], S. plymuthica ATCC15928 [22], Klebsiella sp. LX3 [22], K. planticola CCRC19112 [19], and K. planticola MX-10 (Zhng et al. 2004), from which isomaltulose catalyzed by SIase accounted for 60–90% of all products.

Fig. 2
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Hydrolysis and isomerization of sucrose catalyzed by SIase from P. rubrum CBS574.77

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In Protein Data Bank (PDB), the crystal structure of SIase from many sources has been resolved by X-ray diffraction (Table 1). The native SIases from Klebsiella sp. LX3, P. mesoacidophila MX-45, P. rubrum CBS574.77, and E. rhapontici NX-5 are representative (Table 1). SIase is a single subunit molecule (Fig. 3), which belongs to the glycosidase 13 (GH13) family. Similar to enzymes from the GH13 family, the tertiary structure of SIase from different sources has high homology and is composed of three domains (N-terminal domain, C-terminal domain, and sub-domain). The N-terminal domain is an (β/α)8-barrel super-secondary structure, which is the most important part of SIase (the enzyme's catalytic center). Multiple amino acid sequence comparisons of various SIase revealed that the N-terminal domain contained five highly conserved amino acids, Asp241, Glu295, Asp369, His145, and His368, which are located in the catalytic pocket of SIase and involved in the catalysis of the substrate. The sub-domain is a short loop rich structure, which is involved in substrate (sucrose) binding [48]. The C-terminal domain consists of two antiparallel β-sheets, which are related to the structural stability of SIase [15]. Unlike the GH13 family of enzymes, an RLDRD sequence consisting of five amino acids (Arg, Leu, Asp, Arg, Asp) in the proximity of the active site is unique to SIase, which is mainly responsible for sucrose isomerization [39] (Fig. 3). The RLDRD sequence (e.g., in SIase from Klebsiella sp.) is highly conserved in enzymes. In the process of sucrose isomerization, this sequence plays a significant role, which can ultimately affect the specificity and proportion of products [76, 78].

Table 1 The information on the representative crystal structure of SIase
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Fig. 3
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The tertiary structure of SIase from P.rubrum CBS574.77

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2.2 Reaction Mechanism

The SIase from S. plymuthica ATCC 15,928 was purified, and the reaction mechanism was studied by Véronèse and Perlot [59]. The reaction mechanism was based on the intramolecular rearrangement of sucrose that simultaneously produces two different isomerization products (isomaltulose and trehalulose) [54]. After the cleavage of glucoside bond (α1−β1) of sucrose, glucose moiety remains tightly bound to the active site of the enzyme, while, the fructosyl group is loosely attached. In this case, the fructose moiety is allowed to rotate and change its position freely, and then two different sucrose isomers are formed by intramolecular rearrangement.

By studying the catalytic mechanism of SIase, Ravaud et al. [48] speculated that the SIase proceed using a two-step double-displacement mechanism, which is similar to other GH13 enzymes. In the process of SIasecatalyzing sucrose to form isomaltulose, the formation and breakdown of a covalent glucosyl-enzyme intermediate were completed through the transition state of carboxyl ions from Glu at the enzyme active site. To complete the hydrolysis of sucrose, Glu acts as an acid catalyst, protonizing the oxygen on the substrate glycoside bond by providing a proton. Then, the Asp of the active site attacks the C1 hydrogen atoms on sucrose by the nucleophilic attack, deprotonizing the C1 hydrogen atoms and resulting in the formation of the β-glucosyl-enzyme intermediate. The free fructosyl group’s tautomerism leads to the formation of sucrose isomer (isomaltulose or trehalulose).

SIase contains a unique sequence, RLDRD, which is closely related to isomerization (Fig. 3). The conservation of this sequence affects the specificity and proportion of the final product [37]. Site-directed mutants of Arg325 and Arg328 were used for enzyme transformation, and found that the amount of isomaltulose in the end product was reduced, while the yield of trehalulose was slightly increased [25].

2.3 Protein Engineering

Although natural SIase can be used for isomaltulose biosynthesis, some problems still resisted in industrial production, such as poor thermolability or low conversion rate. Given that protein engineering based on structure information can improve the application performance of wild enzyme [14, [74], various protein engineering approaches have been developed (Table 2). The main purpose of protein modification of SIase is mainly reflected in three aspects: improving enzyme thermostability, increasing product ratio of isomaltulose, and reducing by-products. For example, eplacement of Glu498 and Arg310 with proline of SIase (PalI) from Klebsiella sp. strain LX3 by site-directed mutagenesis resulted in an 11-fold increase in the half-life of PalI at 50 °C, which significantly increased the thermostability of PalI and slightly improved the product ratio of isomaltulose [77]. Given that S. plymuthica AS9 SIase was unstable, B-factor-based site selection combined with site-directed mutagenesis was employed to improve its thermostability by constructing three mutants (E175N, K576D, E175N/K576D) [15]. The mutants exhibited increased optimal temperature (from 30 °C to 35 °C) and displayed improved isomaltulose yield as compared to the wild-type enzyme. In order to reduce by-product formation during the isomaltulose biosynthesis catalyzed by SIase from S. plymuthica, a semi-ration protein-engineering strategy resembling a “Battleship strategy” was employed, resulting in a high active SIase with enhanced product specificity [45]. Given the low product specificity of isomaltulose by SIase from P. dispersa UQ68 J (Sim1), residues of Tyr296 and Gln299 of Sim1, which were located close to the substrate-binding site, were changed. The results showed that the mutant of Q299E significantly improved the conversion rate of isomaltulose from 90.28 to 94.16% [33]. Moreover, to explain the underlying molecular mechanisms of SIase from Rhizobium sp, the variant F164L was obtained using a random-mutagenesis approach, which improved the product ratio of isomaltulose by 55.1% [32].

Table 2 SIase variants with improved isomaltulose production
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3 Heterogenic Expression of SIase

3.1 In E. coli

Given that E.coli is one of the ideal industrial strains for the production of target products, SIase genes from different microorganisms (such as K. planticola, P. dispersa, K. pneumoniae, Enterobacter sp, E. rhapontici, and S. plymuthica) have been successfully cloned and expressed in E.coli (Table 3). The biochemical properties of SIase were shown in Table 3. Most of the optimum pH values ranged from 5.0–6.0. It was reported that pH is related to SIase enzyme activity and product specificity. For example, the activity of SIase form Enterobacter sp. FMB-1 is significantly reduced outside the pH (5.0–6.0) [4]. SIase form K. planticola UQ14 S shows no isomerization activity and a little hydrolysis activity at pH 3.0. However, when the value of pH increased, the hydrolysis activity is decreased, and the isomaltulose becomes the main product [69]. Most SIases exhibit lower thermal stability, except the SIase from Enterobacter sp. FMB-1 showing an optimal temperature at 50 °C (Table 3). Temperature also affects the ratio of product composition. As for P. dispersa UQ68 J SIase, isomaltulose production is significantly inhibited above 40 °C [69]. In addition, SIases from different sources exhibit different enzymatic properties (Table 3). The Michaelis constant (Km) has a wide range of values from 30.1 mM [15] to 257 mM [27], depending on different sources of SIases.

Table 3 SIase with different general characteristics
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3.2 In food-Grade Safety Strains

Given that the application of isomaltulose in the food industry and E. coli is unsuitable for commercial isomaltulose biosynthesis due to the presence of cell wall pyrogens and endotoxin synthesis [56], the heterogeneous expression of SIase in safe food-safety grade hosts, such as Lactococcus lactis (L. lactis), Yarrowia lipolytica (Y. lipolytica), Bacillus subtilis (B. subtilis), and Saccharomyces cerevisiae (S. cerevisiae), attracts more and more attention (Table 4). Like the recombinant SIases in E. coli, most recombinant SIases in food-grade safety strains own weakly acidic optimal pH and low thermal stability (Table 4). Depending on the microbial sources, the conversion rate of isomaltulose varies significantly (Table 4). The bacterial system of L. lactis, a non-pathogenic, non-invasive Gram-positive bacterium, has been examined as an efficient expression system for recombinant protein production and secretion [38]. Park et al. [44] first reported the heterologous expression of SIase from Enterobacter sp. FMB-1 in L. lactis MG1363. The recombinant SIase from L. lactis was successfully secreted extracellularly by using an auto-inducible promoter (P170) and an optimized signal peptide (SP310mut2). The recombinant L. lactis MG1363 was employed for isomaltulose, with a conversion yield of 72%.

Table 4 Heterogeneous expression of SIase in safe food-grade microbial
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Like L. lactis, B. subtilis is also considered a suitable candidate for expressing industrially relevant proteins [16]. Using shuttle plasmid pHA01, SIase from E. rhapontici NX-5 was overexpressed in an engineered B. subtilis WB800, which effectively solves the food safety problem of isomaltulose production [71]. Moreover, SIase from P. dispersa UQ68J was heterologously expressed in the yeast Y. lipolytica, a “generally recognized as safe” (GRAS) microbe, by employing the expression vector pINA1312 [81].

Except for intracellular expression, SIase could also be displayed on the surface of cells or spores in food-grade safety strains for isomaltulose production. Protein display technology has made great progress in recent years. Compared to intracellular expression of enzyme, the living whole-cell biocatalyst is easily produced by microbial cultivation techniques when enzyme molecules are simultaneously synthesized and self-immobilized on the cell surface [7]. In addition to prokaryotic expression systems, yeast expression systems, such as S. cerevisiae and Y. lipolytica, are also used to produce heterogeneous expression proteins [64]. SIase from Enterobacter sp. FMB-1 has been successfully displayed on the cell surface of S. cerevisiae EBY100, using a glycosylphosphatidylinositol (GPI) anchor attachment signal sequence for anchoring protein [24]. Using the cell wall protein Pir1 as an anchor protein [26] or a constructed GPI (a cell wall protein anchor signal sequence) [82], P. dispersa UQ68 J SIase was displayed on the Y. lipolytica cell surface. Unlike cell surface display, SIase from E. rhapontici NX-5 was displayed on B. subtilis 168 spores using CotX as the anchoring protein. The anchored SIase showed relatively high bioactivity for the economical production of isomaltulose from agricultural residues [75].

4 Optimization Strategies for Improving SIase Expression

The expression level of wild-type SIase from different microbial sources is often low, so various optimization strategies were carried out for enhancing the expression of SIase (Table 5). Optimization of fermentation medium composition is a common method to improve enzyme activity or expression level, and the key points of optimization are the composition and content of carbon sources, nitrogen sources and inorganic salts. For instance, the fermentation medium component of recombinant E. coli was optimized through response surface method (RSM). The results showed that the optimal media contained: cane molasses 10.65 g/L, corn steep 22.22 g/L, NaCl 7.57 g/L, MgSO4·7H2O 0.52 g/L, KH2PO4 4.46 g/L. Among the components, cane molasses, corn steep, and NaCl are the most useful carbon sources, nitrogen sources, and inorganic salt. Under optimal fermentation conditions, the final SIase activity was 29.1 U/ml, increasing 21.4 times compared to that of the original strain (1.3 U/ml) [62]. To develop an economical industrial medium, the concentration of untreated cane molasses, yeast extract, and corn steep liquor was optimized for SIase production, leading to a relatively high SIase activity of 14.5 U mg−1 DCW−1 in the recombinant E. coli [29]. The inducer lactose is also often used as an optimization factor during the fermentation process. For example, the rate of lactose induction was optimized, and the results showed that the enzyme level was the highest under the rate of 0.4 g/(L·h) lactose.

Table 5 Optimization strategies for improving the expression level of SIase
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In addition to medium composition optimization, signal peptide, or promoter optimization is also a common method to improve enzyme expression or secretion [34, 83]. For instance, to improve the expression level of SIase from P. dispersa UQ68J, optimizing signal peptide and fermentation conditions were employed. The results showed that Pel B signal peptide, glycine concentration (3 g/L), and lactose concentration (0.5%) were key factors. The total enzyme activity and extracellular enzyme activity were up to 2640 and 1981 U/mL, respectively, which were the highest expression level of SIase [34]. To enhance the expression of SIase from Brevibacillus choshinensis, Papr-E signal peptide and optimal medium composition (glucose 10 g/L, polypeptone 15 g/L, and beef extract 15 g/L) were used, leading to SIase activity of 485.5 U/mL [83]. In addition, a modified nucleotide sequence encoding SIase from P. dispersa UQ68 J was heterologous expression in E.coli JM109 using expression plasmid pET11. The modification of the native gene increased expression level (14–19%) of the total cellular protein [50].

5 Optimizing Reaction Conditions for Isomaltulose Biosynthesis by SIase or Cells Harboring Siase Gene

Free SIase or cells harboring the SIase gene can be used for converting sucrose to isomaltulose. Application condition optimization, including substrate concentration, temperature, pH, enzyme amount, and reaction time, is a common method for isomaltulose production (Table 6). In an enzyme-catalyzed reaction system, substrate (sucrose) concentration is the key point of optimization. For example, K. pneumoniae NK33 SIase, the conversion rate of isommalone was 76.8%, with a sucrose concentration of 40% using K. pneumoniae NK33 SIase [3]. The recombinant cells harboring SIase from Enterobacter sp. FMB-1 were implemented for catalysis reaction, resulting in the conversion rate of isomaltulose by 78% with sucrose concentration of 6% [4]. To maintain the stability of enzymatic properties during the reaction, the optimum reaction temperature and pH of the enzyme reaction system are often the same or adjusted slightly according to the optimum conditions of SIase itself. At the optimum temperature and pH of SIase, the conversion rate of isomaltulose by 7.4% was obtained [24]. In addition to the above factors, the amount of enzyme added and reaction time also affect the final product's yield. As the amount of enzyme increased, the conversion rate of isomaltose also increased,when the amount of enzyme was 20 U/g, the conversion rate of isomaltulose reached a maximum of 86.9%. After that, the conversion rate is slightly decreased with the increase of enzyme amount (Cheng 2015. By overexpressing SIase from P. dispersa UQ68J in yeast Y. lipolytica, an engineered strain S47 was constructed, leading to a 95% conversion rate of isomaltulose with sucrose concentration of 600 g L−1 after 72 h [81]. Moreover, E. rhapontici NX-5 SIase displayed on the surface of B. subtilis 168 spores was applied for isomaltulose production, and the productive spores converted 92% of sucrose after 6 h under the optimal conditions [75].

Table 6 Reaction condition optimization for isomaltulose production
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6 Immobilization Technologies for Isomaltulose Production

6.1 Cell Immobilization

Immobilized cells or enzymes have a wide range of applications, and a variety of immobilized cells or enzyme systems have been successfully used in industrial production [53, 60, 73]. In the last few decades, most of the works report the isomaltulose production by immobilization of microbial cells, such as E. rhapontici, Erwinia sp, and S. plymuthica, which have been immobilized using different strategies to improve isomaltulose productivity or enhance the application performance of the immobilized cells (Table 7). Compared to isomaltulose production by free cells, cell immobilization shows several potential advantaged, including minimum downstream processing, reusability of biocatalyst, and improved operational stability [6]. In the production of isomaltulose, there are two main methods for cell immobilization: alginate entrapment and chitosan cross-linking with glutaraldehyde (Table 7). By using columns of E. rhapontici NCPPB 1578 cells entrapped in alginate gel pellets, isomaltulose production was achieved, obtaining a yield of 0.2 g isomaltulose per gram wet weight of cells per hour and an improved operational stability of the immobilized cells (8,625 h of half-life) [5]. Mundra et al. [40] increased the isomaltulose yield by 40% and achieved a maximum isomaltulose production of 140 g L−1 h−1 by RSM optimization of the immobilized E. rhapontici NCPPB 1578 cells. The optimized immobilization process by RSM and calcium alginate was implemented in Erwinia sp. D12 cell immobilization for isomaltulose production, leading to an isomaltulose conversion rate of 50–60% by using transglutaminase cross-linking in cell immobilization [20]. By comparing with different treatments of Erwinia sp. D12 strain, a maximum conversion (53–59%) was obtained using granules of sonicated immobilized cells [21]. In addition to the abovementioned microorganisms, some other microorganisms were also used for isomaltulose production by cell immobilization utilizing alginate entrapment, such as Klebsiella sp [30, 42], P. rubrum [12] and recombinant E. coli [27, 29]. By using immobilized Klebsiella sp. LX3 cells, more than 87% of isomaltulose, was obtained [30]. Immobilized pellets of P. rubrum CBS574.77 in calcium alginate led to a higher isomaltulose conversion rate (more than 90%) [12]. In addition, recombinant E. coli cells producing SIase from E. rhapontici NX-5 were also immobilized, which showed productivity of 0.45 g isomaltulose g pellet−1 h−1 under 30 batches [29].

Table 7 Isomaltulose production by immobilized cells or enzymes
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S. plymuthica ATCC15928 has also been widely employed to produce isomaltulose. Chitosan cross-linking with glutaraldehyde, another simple and effective cell immobilization method, was developed for forming immobilized S. plymuthica ATCC15928 cells, which resulted in the highest conversion rate of isomaltulose (94%) [23]. The perfect operational stability of immobilized cells was also achieved with a half-life of 155 days. Immobilized S. plymuthica ATCC15928 cells in hollow fiber bioreactors were used to produce isomaltulose, leading to specific productivity of 16.8 g m−2 h−1 and a half-life (42 days) of immobilized enzymes [22]. Furthermore, immobilized cells of S. plymuthica ATCC15928 in calcium alginate were studied for isomaltulose production in a packed bed bioreactor, and the highest conversion rate of isomaltulose (81.26%) was obtained during seven days in a continuous process [43].

6.2 SIase Immobilization

Although cell immobilization has many advantages, it also has some disadvantages compared with enzyme immobilization, such as cell autolysis and lower product purity caused by cell metabolism. Immobilized SIases from different microbial sources have also been carried out to enhance the production efficiency of isomaltulose in recent years (Table 7). The commonly used methods of SIase immobilization, include crosslinking, embedding, and adsorption (Table 7). Contesini et al. developed two different techniques to immobilize the crude SIase from Erwinia sp. D12 by entrapment (low methoxyl pectin microcapsules) and adsorption (Celite), achieving isomaltulose conversion rate of 30% and 60%, respectively [10]. By immobilizing SIase from Erwinia sp 12 onto Celite, Contesini et al. [9] obtained an isomaltulose conversion rate of 65% by immobilized SIase under the optimal reaction conditions. With a strong constitutive promoter, a high level of secreted SIase was obtained by overexpressing the SIase gene from P. dispersa UQ68J in Y. lipolytica. Then by immobilizing the recombinant SIase onto polyvinyl alcohol-alginate, the stability of the SIase was significantly improved, resulting in isomaltulose production of 620.7 g L−1 with a yield of 0.96 g g−1. The conversion rate of sucrose remained above 90% after 13 batches under optimal conditions [80]. In addition to the immobilization methods of entrapment and adsorption, an innovative approach was developed for immobilizing SIase on ε-poly-L-lysine modified mesoporous [70]. The immobilized SIase displayed good operational stability with a half-life period up to 114 h under continuous reaction, and the conversion rate of sucrose remained at around 95% after 16 batches. The stability and reusability of SIase PalI from Klebsiella sp. LX3 were studied by sodium alginate embedding and glutaraldehyde crosslinking. The results showed that the residual activity of the immobilized enzyme by sodium alginate embedding and glutaraldehyde crosslinking was more than 95% and 60%, respectively, compared with that of the free enzyme (below 30%) after stored at 4 oC for 20 days. In addition, the residual enzyme activity by glutaraldehyde crosslinking was 80% after 12 batches for reuse [63]. SIase from recombinant B. pumilus was immobilized by adsorption crosslinking with chitosan as carrier and glutaraldehyde as a cross-linking agent. The maximum conversion rate of isomaltulose reached 87.8% under optimal conversion conditions [36].

7 Conclusions and Future Prospects

Nowadays, the market demand for isomaltulose, as a safe sucrose substitute, is increasing rapidly. SIase is responsible for the commercial biological production of isomaltulose from sucrose. The transformation from sucrose to isomaltulose by SIase or microbial cells harboring SIase gene has received extensive attention in the industry. Significant progress has been made in the biological production of isomaltose; however, some problems still exist in industrial production, such as the low secretion level from food safety grade strain, weak thermal stability, and poor application performance of SIase. To solve the above problems, attempts can be made from two aspects of strain and enzyme. In consideration of the high protein expression capacity, approximately one-third of protein for therapeutics is produced in E. coli. Given contamination with large amounts of endotoxins by E. coli [13] and the application characteristics of isomaltulose in the food industry, E. coli-based cell-free protein synthesis, a promising platform for protein expression, can be used [67]. In addition to E. coli, studies on the heterogeneous expression and secretion of SIase have been tried in food-grade safety microorganisms, but the secretion levels of SIase are still low, requiring urgent improvement in genetic engineering or fermentation condition optimization. For example, the recombinant proteins in corynebacterium glutamicum were enhanced by constructing a bicistronic gene expression system [55].

Compared to the immobilized cell, the immobilization technologies of SIase for isomaltulose production are relatively immature, which need to be improved. Moreover, some SIase possess poor thermal stability, and although some SIases were modified with related protein engineering, the improvement was not noticeable. To improve the application performance of SIase in industrial production, key sites of SIase need to be further explored and modified.