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Multi-substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed beta-cyclodextrin (beta-CD) to maltose and glucose. It exhibited substrate preference for beta-CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an alpha-amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 6(2)-O-alpha-maltosyl-maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer-dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association-dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards beta-CD decreased to 80%. Therefore, multi-substrate specificity of neopullulanase was likely to be modulated by the shift of monomer-dimer association equilibrium.

Aims:  Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro‐organism Enterobacter sp. FMB‐1. Methods and Results:  A cosmid clone containing c. 6 kbp region encoding an SIase gene was identified. The 5969‐bp chromosomal DNA fragment covering the SIase (esi) gene in Enterobacter sp. FMB‐1 was sequenced. Although this DNA fragment contained several open reading frames other than esi, only the presence of esi was sufficient to produce isomaltulose in recombinant Escherichia coli. The esi gene was expressed in E. coli, leading to the characterization of its SIase activity. Conclusions:  The Enterobacter sp. FMB‐1 esi gene was successfully cloned and expressed in E. coli. This gene encoded a functional SIase that produced isomaltulose from sucrose. Significance and Impact of the Study:  This is the first molecular analysis of an SIase gene in an Enterobacter strain. The functional expression of the Enterobacter sp. FMB‐1 esi gene in E. coli offers an alternative choice for the industrial production of isomaltulose.

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-alpha-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.

2′-Fucosyllactose (2′-FL), a major component of fucosylated human milk oligosaccharides, is beneficial to human health in various ways like prebiotic effect, protection from pathogens, anti-inflammatory activity and reduction of the risk of neurodegeneration. Here, a whole-cell fluorescence biosensor for 2′-FL was developed. Escherichia coli (E. coli) was engineered to catalyse the cleavage of 2′-FL into l-fucose and lactose by constitutively expressing α-l-fucosidase. Escherichia coli ∆L YA, in which lacZ is deleted and lacY is retained, was employed to disable lactose consumption. E. coli ∆L YA constitutively co-expressing α-l-fucosidase and a red fluorescence protein (RFP) exhibited increased fluorescence intensity in media containing 2′-FL. However, the presence of 50 g/L lactose reduced the RFP intensity due to lactose-induced cytotoxicity. Preadaptation of bacterial strains to fucose alleviated growth hindrance by lactose and partially recovered the fluorescence intensity. The fluorescence intensity of the cell was linearly proportional to 1-5 g/L 2′-FL. The whole-cell sensor will be versatile in developing a 2′-FL detection system. Human milk oligosaccharides (HMOs) are present in human breast milk and are closely associated with health benefits. HMOs act as decoys for pathogens (e.g., virus, bacteria, and protozoa) by inhibiting their ability to bind to the surface of epithelial cells 1-4. Fucosylated oligosaccharides which are not found in bovine milk account for 50% of total HMOs. The HMOs 2′-fucosyllactose (2′-FL; Fuc-α1,2-Gal-β1,4-Glc) and 3-fucosyllactose (3-FL; Gal-β1,4-Fuc-α1,3-Glc), with a concentration range of 0.5-2 g/L, account for the largest portion of fucosylated HMOs 5,6. Fucosylated HMOs circulate systemically, affecting the host immune response, the regulation of tumour metastasis, and resistance to bacteria, fungi, and other pathogens 2,7,8. The concentration of 2′-FL in breast milk affects the ability to protect against vital systemic infections in nursing infant 8. While low content of 2′-FL in breast milk has been associated with a higher rate of diarrhoea during lactation 9,10 , about 20% of human milk do not contain 2′-FL 11. For these reasons, 2′-FL and 3-FL are spotlighted as nutraceutical and pharmaceutical ingredients; thus, a simple and visually measurable method is indispensable in evaluating their level in breast milk. Various methods have been developed for the production of 2′-FL, including whole-cell biocatalysis 12-20 , enzymatic synthesis 21,22 , and chemical synthesis 23,24. Regardless of the method used, a simple detection and quantification method is indispensable for the development of 2′-FL production. Quantification of 2′-FL has been mostly done using high-performance liquid chromatography (HPLC), high-pH anion exchange chromatography, and liquid chromatography-mass spectrometry (LC-MS) 15,25-28. However, those types of equipment are time-consuming, labour-intensive, and expensive. Therefore, those methods are unlikely to be used in a high-throughput manner or for ...