Efficient Biosynthesis of Lactosucrose from Sucrose and Lactose by

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Efficient Biosynthesis of Lactosucrose from Sucrose and Lactose by the Purified Recombinant Levansucrase from Leuconostoc mesenteroides B‑512 FMC Wenjing Li,† Shuhuai Yu,† Tao Zhang,† Bo Jiang,†,‡ Timo Stressler,*,§ Lutz Fischer,§ and Wanmeng Mu*,†,‡,§ †

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China § University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstrasse 25, Stuttgart 70599, Germany ‡

S Supporting Information *

ABSTRACT: Lactosucrose, a rare trisaccharide formed from sucrose and lactose by enzymatic transglycosylation, is a type of indigestible carbohydrate with a good prebiotic effect. In this study, lactosucrose biosynthesis was efficiently carried out by a purified levansucrase from Leuconostoc mesenteroides B-512. The target gene was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity by nickel affinity and gel filtration chromatography. The effects of pH, temperature, substrate concentration, substrate ratio, and enzyme amount on lactosucrose biosynthesis were studied in detail, and the optimized conditions were determined to be pH 6.5, 50 °C, 27% (W/V) sucrose, 27% (W/V) lactose, and 5 U mL−1 of the purified recombinant enzyme. Under the optimized reaction conditions, the maximal lactosucrose yield reached 224 g L−1 after reaction for 1 h. Therefore, L. mesenteroides levansucrase could be considered a potential candidate for future industrial production of lactosucrose. KEYWORDS: lactosucrose, Leuconostoc mesenteroides, levansucrase, prebiotic, transfructosylation

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INTRODUCTION Sucrose and lactose are two of the most common and cheapest disaccharides that abundantly exist in nature, and they are both common types of sugar prominently featured in typical diets. Sucrose, as the most common sweetener in the food industry, has been recognized to induce some potential health risks. Lactose is a major component of whey, but in industry, disposal of excess whey in rivers not only wastes valuable lactose sources but also has resulted in environmental problems. Therefore, the use of sucrose and lactose as cheap sources to biologically produce valuable and healthy derivatives has aroused much interest over the past decades. The functional sucrose derivatives include isomaltulose (isomer of sucrose),1 fructooligosaccharide (polymerization product of sucrose),2 and so on. The typical bioactive lactose derivatives include galactooligosaccharide (polymerization product of lactose),3 lactulose (isomer of lactose),4 epilactose (epimer of lactose),5 and Dtagatose (isomer of D-galactose).6,7 In addition, the enzymatic transglycosylation of both sucrose and lactose may produce another functional derivative, named lactosucrose.8 Lactosucrose (4G-β-D-galactosylsucrose, O-β-D-galactopyranosyl-(1, 4)-O-α-D-glucopyranosyl-(1, 2)-β-D-fructofuranoside) is a trisaccharide composed of glucose, galactose, and fructose. It is a nondigestible carbohydrate,9 but can be selectively utilized by intestinal Bifidobacterium and promotes Bif idobacterium multiplication to contribute to intestinal microflora maintenance and intestinal protection,10,11 indicating that it is a low-caloric carbohydrate with prebiotic properties. In © XXXX American Chemical Society

addition, it has been shown by many studies that lactosucrose has many other good physiological effects. It enhances the intestinal calcium absorption,12,13 amino acid metabolism,14 immunoregulatory function,15 reduces body fat accumulation,16,17 and prevents obesity18 and IgE-mediated allergic disease.19 Importantly, lactosucrose has been approved as a functional food ingredient for foods for specified health uses (FOSHU) by the Japanese government, and it has been allowed to be produced and used commercially in Japan.8 Much attention has recently been focused on the production of lactosucrose by enzymatic and microbial reactions. Three types of enzymes with glycosyltransfer activity have been used for lactosucrose biosynthesis from sucrose and lactose as glycosyl donor or acceptor, including levansucrase (EC 2.4.1.10), β-fructofuranosidase (EC 3.2.1.26), and β-galactosidase (EC 3.2.1.23). Among them, β-galactosidase catalyzed lactosucrose production via transgalactosylation from lactose to sucrose, usually accompanied by the formation of several other oligosaccharides as byproducts. Therefore, most of the recent studies focused on lactosucrose production via transfructosylation from sucrose to lactose by levansucrase or βfructofuranosidase. Received: July 26, 2015 Revised: October 20, 2015 Accepted: October 21, 2015

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DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

L−1 tryptone, and 5 g L−1 NaCl (pH 7.5). A single colony of the transformant was inoculated in LB medium plus ampicillin (100 μg mL−1) for growth at 37 °C and 200 rpm. When the optical density at 600 nm of the culture reached 0.6, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the protein expression was performed at 28 °C for 5 h. Purification of the Recombinant Levansucrase. All purification steps were performed at 4 °C. The culture broth was centrifuged at 10 000 g for 20 min to collect the cell pellets. The pellets were washed twice with 50 mM phosphate buffer containing 100 mM NaCl (pH 7.0), and the suspension was then homogenized by sonication using a Scientz-II D ultrasonic homogenizer (Ningbo Scientz Biotechnology Co Ltd., China). Cell debris was removed by centrifugation at 20 000g for 30 min at 4 °C. The supernatant was directly loaded on a Chelating Sepharose Fast Flow resin column for nickel affinity chromatography. The purification process was carried out according to the exact manufacturer’s protocol (pET His Tag System, Novagen). The collected proteins from affinity chromatography were then loaded onto a Superdex 200 column (HiLoad 10/300 prep grade; GE Healthcare, Sweden), pre-equilibrated with 50 mM phosphate buffer (pH 6.5) containing 150 mM NaCl, at a flow rate of 0.5 mL min−1. All purification steps were carried out using an Ä kta Purifier System (GE Healthcare, Sweden) at 4 °C. The purified enzymes were dialyzed against 50 mM phosphate (pH 6.5). Analytical Methods. The purity and integrity of the enzyme were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 5% stacking gel and 12% separating gel. Coomassie brilliant blue R250 was used for protein staining. According to a previously reported method,25 the concentrations of various sugars were analyzed using an HPLC system (Agilent 1260, CA, U.S.A.) equipped with a refractive index detector and an Asahipak NH2P-50−4E column (4.6 mm id × 250 mm, Shodex, Tokyo, Japan). The column was eluted at room temperature with 75% (V/V) acetonitrile at a flow rate of 1 mL min−1. Identification of Reaction Products. The purification and preparation of produced oligosaccharide were performed by HPLC (Waters 1525 system, Waters Corporation, MA, U.S.A.) equipped with a refractive index detector and a preparative column, XBridge Prep Amide (5 μm, 10 mm id × 250 mm, Waters, MA, U.S.A.). The column was eluted at room temperature with 75% (V/V) acetonitrile at a flow rate of 2.5 mL min−1. The produced oligosaccharide was separated and collected. The purified oligosaccharide was freeze-dried in the 4.5 L FreeZone freeze-dry system (Labconco Corp, MO, U.S.A.) for 24 h and was then subjected to NMR measurement. The purified carbohydrate (25 mg) was dissolved in 0.5 mL of D2O. NMR spectra were recorded at 27 °C with a Bruker Avance III 400 MHz Digital NMR Spectrometer (Bruker, Karlsruhe, Germany) with standard pulse sequences operating at 400 and 100 MHz. The 1H NMR, 13C NMR, and 2D NMR spectra, including two-dimensional 1H shift correlated spectroscopy ( 1H 1H COSY), 1H detected heteronuclear single-quantum coherence (1H13C HSQC), and 1H detected heteronuclear multiple-bond correlation (1H13C HMBC), were recorded to identify the structure of the product. The chemical shifts were determined with respect to the signals for sodium 4,4dimethyl-4-silapentane-1-sulfonate (DSS) (δH = δC = 0.00 ppm) dissolved in the samples. Measurement of Enzyme Activity. The levansucrase activity could be assayed by either sucrose hydrolysis or transfructosylation activity. The sucrose hydrolysis activity was determined by measuring the release of glucose from sucrose. A reaction mixture of 1 mL contained sucrose (10%, W/V), sodium phosphate buffer (50 mM, pH 6.5), and 10 μL of purified enzyme at a final concentration of 10 μg mL−1. The reactions were performed at 50 °C for 20 min and were stopped after 10 min by boiling. One unit of sucrose hydrolysis was defined as the amount of enzyme catalyzing the release of 1 μmol glucose per min at pH 6.5 and 50 °C. In this article, the quantification of enzyme activity was mainly based on the sucrose hydrolysis activity. The transfructosylation activity could be measured as well. All the reactions were exactly the same as above but in the presence of lactose (10%, W/V) as the transfructosyl acceptor. One unit of trans-

In 1975, Avigad first reported lactosucrose production by Aerobacter levanicum levansucrase.20 And in 1991, purified levansucrase from Bacillus natto was reported to yield efficient production of lactosucrose from sucrose and lactose.9 Now, increasingly more lactosucrases or lactosucrase-containing bacteria have been isolated and characterized for lactosucrose biosynthesis. Whole cells harboring intracellular levansucrase have been widely used to produce lactosucrose from sucrose and lactose, such as Bacillus subtilis KCCM 32835,21 Paenibacillus polymyxa IFO 3020,22 Sterigmatomyces elviae ATCC 18894,23,24 and so on. In our recent studies, the crude enzyme from Bacillus methylotrophicus SK 21.002 was used to study lactosucrose production.25 In addition, lactosucrose biosynthesis was studied using the purified recombinant levansucrase from Bacillus licheniformis 8−37−0−126 and the crude recombinant levansucrases from B. subtilis NCIMB 1187127 and Zymomonas mobiliz.28 The transfructosylation reaction to produce lactosucrose was also studied using extracellular β-fructofuranosidase from Microbacterium saccharophilum K-1.29 β-Fructofuranosidase (β-D-fructofuranoside fructohydrolase, saccharase, invertase, EC 3.2.1.26) mainly catalyzes the irreversible hydrolysis of sucrose into glucose and fructose.30 The strain K-1 that produces lactosucrose was isolated more than 20 years ago and was classified as Arthrobacter sp..9,31 However, recently, the strain has been reclassified as M. saccharophilum through phylogenetic analysis based on 16S rDNA sequence.32 So far, lactosucrose-producing β-fructofuranosidase has been exclusively characterized from only M. saccharophilum K-1, although β-fructofuranosidase widely exists in various microorganisms. As mentioned above, there were only three studies reporting the lactosucrose production by recombinant levansucrases. Only one of them used the purified recombinant enzyme, and its lactosucrose production was performed without optimization of the reaction conditions. In this study, the lactosucrose production was studied by the purified recombinant levansucrase from Leuconostoc mesenteroides B-512 FMC. The conditions of lactosucrose production from sucrose and lactose were investigated. The lactosucrose productivity was measured and compared with other reports. In addition, the structure of the produced lactosucrose was identified by nuclear magnetic resonance (NMR) analysis.

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MATERIALS AND METHODS

Chemicals and Reagents. Standards, including lactosucrose, lactose, sucrose, glucose, and fructose, were purchased from Sigma (St. Louis, MO, U.S.A.) for high-performance liquid chromatography (HPLC) analysis. HPLC grade acetonitrile was from Tedia Company Inc. (Fairfield, OH, U.S.A.). Chelating Sepharose Fast Flow, the resin for affinity chromatography, was obtained from Amersham Biosciences (Arlington Heights, IL, U.S.A.). Other chemicals were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Gene Cloning and Expression. The gene sequence determination and the expression of L. mesenteroides levansucrase have been reported by Kang et al.33 In this article, the full length of the encoding gene (GenBank accession No. AY665464) was synthesized by Shanhai Generay Biotech Co., Ltd. (Shanghai, China). The synthesized gene was designed to contain an in-frame 6× histidine-tag sequence before the stop codon, and the restriction enzyme sites NdeI and XhoI were designed at the 5′- and 3′-terminus. Then, the synthesized gene was inserted into pET-22b(+) vector with the same restriction sites. The recombinant plasmid was then transformed into Escherchia coli BL21(DE3) for expression. The basic medium for culture was the Luria−Bertani (LB) medium composed of 5 g L−1 yeast extract, 10 g B

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry fructosylation activity was defined as the amount of enzyme producing 1 μmol lactosucrose per min at pH 6.5 and 50 °C. Effects of pH and Temperature on Lactosucrose Production. Three buffer systems (50 mM), sodium acetate buffer (4.0−5.5), sodium phosphate buffer (6.0−7.5), and Tris-HCl buffer (8.0−9.0), were used to study the effect of pH on lactosucrose production at 40 °C. The effect of temperature was investigated in sodium phosphate buffer (50 mM, pH 6.5) by measuring the lactosucrose production by varying the temperatures from 30−70 °C. All reactions were performed with 5 U mL−1 (hydrolysis activity) of the purified recombinant levansucrase for 1 h in 1.0 mL of a reaction mixture containing 21% (W/V) sucrose and 21% (W/V) lactose. Effect of Substrate Concentration and Ratio on Lactosucrose Production. To study the effect of substrate concentration, different concentrations (6%, 9%, 12%, 15%, 18%, 21%, 24%, 27%, and 30%, W/V) of both sucrose and lactose (equal concentration) were used to measure lactosucrose production. To determine the effect of substrate ratio, the ratios of sucrose (W/V) to lactose (W/V) were set as 9%: 27%, 18%: 27%, 27%: 27%, 30%: 27%, 27%: 9%, 27%: 18%, and 27%: 30%. All reactions were performed with 5 U mL−1 (hydrolysis activity) of the recombinant levansucrase at pH 6.5 and 50 °C for 1 h. Effect of Enzyme Amount on Lactosucrose Production. The reaction mixtures were prepared with 27% (W/V) sucrose and 27% (W/V) lactose and different enzyme amounts of 2−8 U mL−1 (hydrolysis activity). All reactions were performed at pH 6.5 and 50 °C for 1 h. Production of Lactosucrose under the Optimized Conditions. The lactosucrose biosynthesis from sucrose and lactose was carried out with 5 U mL−1 (hydrolysis activity) of the purified recombinant levansucrase in 1 L of reaction mixture composed of 27% (W/V) sucrose and 27% (W/V) lactose at pH 6.5 and 50 °C. The study of kinetic conversion of lactosucrose was investigated over 2 h.

Figure 1. SDS-PAGE analysis of the recombinant L. mesenteroides levansucrase. Lane 1, molecular standard marker; lane 2, eluted proteins by nickel affinity chromatography; lane 3, purified enzyme after Superdex 200 gel filtration; and lane 4, the soluble crude enzymes after disruption.

crude enzymes or whole cells (Table 1). Usually, crude enzymes or whole cells are recognized as a complicated and multicomponent mixture of various biocatalysts, and these biocatalysts might interfere with the experimental results, especially common enzymes acting on sucrose and lactose, such as β-galactosidase, β-fructofuranosidase, invertase, and fructosyltransferase. In the past, only the purified native B. natto levansucrase35 and the purified recombinant B. licheniformis levansucrase26 were used to produce lactosucrose. Unfortunately, their lactosucrose productivities were both relatively low (less than 60 g L−1) and had no competitive advantage compared with other reports (Table 1). However, the heterogeneous expression of levansucrase in E. coli possibly forms inclusion bodies.36 In this article, actually several different levansucrases were attempted for recombinant enzyme production by heterogeneous expression (data not shown); however, only the L. mesenteroides levansucrase was effectively expressed in a soluble form using the pET-22(+) plasmid under the T7 promoter (Figure 1, lane 4). Lactosucrose production has been studied by recombinant levansucrases from B. subtilis NCIMB 1187127 and Z. mobiliz,28 but the biocatalyst used for these studies were both crude enzymes. Identification of Reaction Products. The reaction products were analyzed by HPLC using an Asahipak NH2P50-4E column. The standards, including fructose, glucose, sucrose, lactose, and lactosucrose, were completely separated, with retention times at 7.3, 8.9, 11.6, 13.8, and 18.5 min, respectively (data not shown). On the basis of the HPLC profile, the reaction products by the recombinant levansucrase from sucrose and lactose exhibited an obvious eluted peak at approximately 18 min, which was close to the retention time of the standard lactosucrose. This carbohydrate was then purified and prepared by a preparative column, XBridge Prep Amide and was freeze-dried for NMR analysis. The NMR data as shown in the spectra were analyzed and compared with the results of those reported previously. The chemical shifts in 13C and 1H NMR spectra of the produced carbohydrate were shown in Table 2, which were obtained from

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RESULTS AND DISCUSSION Purification of the Recombinant L. mesenteroides Lactosucrase. The cloning and expression of levansucrase from L. mesenteroides B-512 FMC has been reported by Kang et al.33 However, they focused on the fructooligosaccharide and levan production by the recombinant enzyme from sucrose, and did not evaluate the possibility of lactosucrose production. In this article, the exact gene was successfully cloned and expressed in E. coli, and the recombinant L. mesenteroides levansucrase was purified to homogeneity by two steps, including nickel affinity and gel filtration chromatography (Figure 1). The molecular mass of the protein was estimated to be 45 kDa based on SDS-PAGE analysis, which agreed with the 46 958 Da calculated based on the deduced amino acid sequence. The gel filtration showed a single protein peak at approximately 100 kDa (data not shown), indicating that the purified enzyme should be a homodimer. These results were the same as that found in the previous report by Kang et al.33 The specific activities were calculated to be 437 ± 15 and 791 ± 23 U mg−1 for sucrose hydrolysis and transfructosylation with lactose as an acceptor, respectively, indicating the enzyme prefers to produce lactosucrose rather than hydrolyzing sucrose in the presence of lactose. It is well-known that levansucrase catalyzes three distinct reactions from sucrose, including polymerization (forming levan and releasing of glucose), hydrolysis (liberating glucose and fructose), and transfructosylation (forming fructosylated compounds by using other compounds as glycosyl acceptors).34 Lactosucrose formation is catalyzed by the transfructosylation of levansucrase using sucrose and lactose as a glycosyl transfer donor and acceptor, respectively. The lactosucrose production by levansucrase has been widely studied; however, in most of the reports, the lactosucrose formation was catalyzed by the C

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Lactosucrose Production Using Enzymes from Various Microorganisms strains

responsible enzyme

L. mesenteroides B-512 FMC

levansucrase

B. licheniformis 8−37−0−1

levansucrase

B. natto

levansucrase

P. polymyxa IFO 3020 B. subtilis KCCM 32835 B. subtilis NCIMB 11871

levansucrase levansucrase levansucrase

P. aurantiaca Z. mobiliz

levansucrase levansucrase

S. elviae mutant S. elviae mutant

levansucrase levansucrase

M. saccharophilum K-1

βfructofuranosidase β-D-galactosidase

B. circulans

biocatalyst

optimum pH

optimum temperature (°C)

substrate (g L−1) (sucrose + lactose)

lactosucrose produced

reference

purified recombinant enzyme purified recombinant enzyme purified enzyme

6.5

50

270 + 270

224 g L−1

6.5

40

171 + 171a

yield of 25.8%b

26

6.2

35

85.5 + 85.5a

35

free whole cells free whole cells crude recombinant enzyme NR crude recombinant enzyme free whole cells immobilized whole cells crude enzyme

6.0 6.0

55 55 NR

225 + 225 225 + 225 205 + 410a

31% of total sugars 170 g L−1 183 g L−1 68 g L−1d

4.0 7.0

45 23

510 + 360 180 + 180

285 g L−1 28.5% of total sugars

38 28

NR 6.0

NR 50

250 + 250 250 + 250

183.78 g L−1 192 g L−1

23 24

6.0

55

6.0

40

NRc

commercial crude enzyme

200 + 200 300 + 300

this study

22 21 27

135 g L−1

9

56 g L−1

44

a

The values were converted from the original references after exchanging the unit. bYield was calculated from sucrose. cNR, not reported. dThe lactosucrose yield was 34% of initial sucrose concentration.

Table 2. Chemical Shifts in 1H and 13C Spectra of the Carbohydrate Produced from Sucrose and Lactose by the Purified Recombinant L. mesenteroides Levansucrase group

carbon atoms

δ Ca

δ Ha

galactose

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

102.94 71.08 72.59 68.61 75.39 61.07 91.92 70.77 71.25 78.12 71.03 59.58 61.38 103.72 76.46 74.05 81.41 62.38

4.47 3.55 3.66 3.93 3.72 3.80/3.75 5.41 3.60 3.89 3.70 3.98 3.91/3.86 3.66

glucose

fructose

was indicated that the compound contained two protons on anomeric sugar carbons (δH, 5.39 and 4.46 ppm). The three sugar moieties were assigned as D-galactose, D-glucose, and Dfructose by 1H1H COSY (Figure S2A) and 1H13C HSQC (Figure S2B) spectra. As shown in Figure 2, the quaternary carbon (δC, 103.71 ppm) having a HMBC correlation with GlcH-1′ (δH, 5.41 ppm) was confirmed as Fru-C-2″, indicating a Glu (1 → 2) Fru connectivity; the connectivity of Gal (1 → 4) Glc was deduced by the HMBC correlation between Gal-H-1 (δH, 4.47 ppm) and Glc-C-4′ (δC, 78.12 ppm). Therefore, the structure of the product was inferred to be O-β- D galactopyranosyl-(1, 4)-O-α-D-glucopyranosyl-(1, 2)-β-D-fructofuranoside, i.e., lactosucrose. These data were almost identical to the reported values from the previously reported 13C and 1H NMR spectra of lactosucrose.28,37 Effect of pH and Temperature on Lactosucrose Production. The effect of pH on lactosucrose production was investigated at 40 °C (Figure 3A). Lactosucrose could be effectively produced by the purified recombinant L. mesenteroides levansucrase at a pH range from 4.5−9.0, with the optimum pH being 6.5. By comparison, with the exception of P. aurantiaca levansucrase which produced lactosucrose maximally at pH 4.0,38 all reported lactosucrose-producing enzymes exhibited optimum pH between 6.0−7.0 (Table 1). Temperature is an important factor for industrial production of lactosucrose from sucrose and lactose. Increased temperature is helpful for increasing the substrate solubility and improving transfructosylation rate.22 Herein, the temperature exhibited significant effect on lactosucrose production by the purified recombinant L. mesenteroides levansucrase. The optimum temperature for lactosucrose production was measured to be 50 °C (Figure 3B), and the biological lactosucrose production at 55 °C was close to that at 50 °C, but the production significantly decreased above 55 °C. By comparison, almost all of the reported lactosucrose-producing enzymes from various

4.20 4.03 3.88 3.80

a Chemical shifts (δ) in ppm were determined relative to the internal standard sodium 4,4-dimethyl-4-silapentane-1-sulfonate (δH = δC = 0.00 ppm).

1

H NMR, 13C NMR, 1H1H COSY, and 1H13C HSQC spectra. The 13C NMR spectrum (Figure S1A) showed 18 carbons with three anomeric sugar carbons (δC, 102.94, 91.92, and 103.71 ppm) with a large downfield and four methylene oxylated carbons (δC, 61.07, 59.58, 61.35, and 62.34 ppm) with relative upfield shifts. The 1H NMR spectrum (Figure S1B) indicated that the produced carbohydrate was composed of three hexoses in the structure. From the 1H NMR spectrum, it D

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Identification of the structure of the carbohydrate produced from sucrose and lactose by the purified recombinant L. mesenteroides levansucrase. A shows the 1H13C HMBC spectrum of the produced compound. Connectivity of Gal (1 → 4) Glc and Glc (1 → 2) Fru was deduced by the correlation between Glc-C-4′ and Gal-H-1 and that between Fru-C-2″ and Glc-H-1′, which were obviously shown in the regions marked a and b, respectively. B shows the chemical structure of the produced compound based on the NMR spectra.

highest sucrose hydrolysis activity at 45−50 °C but exhibit maximal polymerization activity at a lower temperature (30 °C). In a previous study, the purified recombinant L. mesenteroides levansucrase showed the highest levan formation activity at 30 °C and lost half of its maximal activity above 40 °C.33 Herein, the optimum temperature of the exact enzyme for both lactosucrose production and sucrose splitting (data not shown) was determined to be 50 °C, which was much higher than that for the polymerization to produce levan. Effect of Substrate Concentration and Ratio on Lactosucrose Production. Different concentrations of both

microorganisms showed the optimum temperature at the range of 35−55 °C; however, the Z. mobiliz levansucrase showed the lowest optimum temperature (23 °C).28 In general, levansucrase displays a lower optimum temperature for the polymerization than sucrose hydrolysis.34 The optimum temperature for Bacillus amyloliquefaciens levansucrase is 4 °C for levan formation but 30 °C for sucrose hydrolysis.39 That for Pseudomonas syringae pv. phaseolicola exhibits maximal activity at 18 and 60 °C for levan synthesis and sucrose hydrolysis, respectively.40 And those for Zymomonas mobiliz41 and Microbacterium laevaniformans ATCC 1595342 show the E

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Effect of pH (A) and temperature (B) on lactosucrose production. All reactions were performed with 5 U mL−1 (hydrolysis activity) of the purified recombinant levansucrase for 1 h in 1.0 mL of a reaction mixture containing 21% (W/V) sucrose and 21% (W/V) lactose. Values are the means of three replications ± standard deviation.

Figure 4. Effect of substrate concentration (A) and ratio (B) on lactosucrose production. All reactions were performed with 5 U mL−1 (hydrolysis activity) of the purified recombinant levansucrase at pH 6.5 and 50 °C for 1 h. Values are means of three replications ± standard deviation.

sucrose and lactose at an equal concentration were used to optimize the lactosucrose production. Shown in Figure 4A, the lactosucrose concentration was steadily improved by increasing each substrate concentration up to 27% (W/V). The effect of substrate ratio was also studied, and it was found that the highest lactosucrose production was realized by using 27% (W/ V) sucrose and 27% (W/V) lactose (Figure 4B). Optimization of the substrate concentration and ratio on lactosucrose production was also studied for the levansucrases from Z. mobiliz,28 P. polymyxa,22 and B. methylotrophicus;25 they all showed the best substrate ratio at 1:1; however, each optimum concentration was determined as 18%, 22.5%, and 20% (W/V), respectively. Herein, the optimum concentration for each substrate reached 27% (W/V), which was much higher than that of other levansucrases, and it was suggested that a higher substrate concentration was helpful for the practical application in industry. Effect of Enzyme Amount on Lactosucrose Production. When lactosucrose production was performed from 27% (W/V) sucrose and 27% (W/V) lactose, the optimum enzyme

amount was measured to be 5 U mL−1 (hydrolysis activity), and the lactosucrose production was slightly reduced by the addition of more enzyme amount (Figure 5). However, the release of fructose increased steadily but very slowly, with increasing enzyme amount, indicating that the sucrose hydrolysis activity increased. Lactosucrose Production under the Optimal Conditions. Lactosucrose biosynthesis was investigated under the optimized conditions, in which the pH, temperature, sucrose concentration, lactose concentration, and enzyme amount were pH 6.5, 50 °C, 27% (W/V), 27% (W/V), and 5 U mL−1 (hydrolysis activity), respectively (Figure 6). The production of lactosucrose increased with increasing reaction time up to 1 h. The maximal lactosucrose yield reached 224 g L−1 after reaction for 1 h and then the lactosucrose concentration slightly dropped, probably because of the lasting hydrolysis reaction toward both substrate sucrose and product lactosucrose after a long-term reaction. The steady and slowly released fructose F

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

depended on the enzyme or microbial strain. Among these reports, M. saccharophilum K-1 β-fructofuranosidase and B. circulans β-galactosidase produced 135 and 56 g L −1 lactosucrose, respectively; by comparison, the biosynthesis by levansucrase had a higher turnover yield of lactosucrose. From an International Conference Abstract, Han et al. reported the highest yield of lactosucrose at 285 g L−1 produced by P. aurantiaca levansucrase, however, they used very high concentrations of substrates (510 g L−1 sucrose and 360 g L−1 lactose) and did not give more detailed information, especially the biocatalyst form that they used.38 It was interesting that, with the exception of crude B. methylotrophicus levansucrase,25 all the purified or crude levansucrases produced lactosucrose with yields less than 100 g L−1,27,28,35 which were less than those by most of whole cells harboring levansucrase activity. The majority of levansucrase-producing microorganisms could produce 100−200 g L−1 lactosucrose by transfructoylation from various concentrations of sucrose and lactose (Table 1). Herein, the purified recombinant L. mesenteroides levansucrase produced 244 g L−1 lactosucrose from 270 g L−1 sucrose and 270 g L−1 lactose, showing a significantly competitive productivity. Therefore, it was suggested that L. mesenteroides levansucrase could be used as a good producer of lactosucrose. A promising advantage for the recombinant enzyme study is the convenience of performing the molecular modification. Recently, the thermostability of the lactosucrose-producing βfructofuranosidase from M. saccharophilum K-1 has been significantly improved through site-directed mutagenesis.29 In addition, the enzyme immobilization24 and simulated moving bed reactor43 have been studied for the continuous lactosucrose production with higher productivity. These techniques may increase the industrialization feasibility of lactosucrose biosynthesis. In the near future, various techniques will be attempted to improve lactosucrose production by L. mesenteroides levansucrase.

Figure 5. Effect of enzyme amount on lactosucrose production. The enzyme quantification is based on the sucrose hydrolysis activity. All reactions were performed in 1.0 mL of a reaction mixture containing 27% (W/V) sucrose and 27% (W/V) lactose at pH 6.5 and 50 °C for 1 h. Values are the means of three replications ± standard deviation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03648. (A) 13C NMR and (B) 1H NMR spectra of the carbohydrate produced from sucrose and lactose from the purified recombinant L. mesenteroides levansucrase (Figure S1) and (A) the 1H1H COSY (A) and (B) 1 H13C HSQC spectra of the carbohydrate produced from sucrose and lactose by the purified recombinant L. mesenteroides levansucrase (PDF)

Figure 6. Bioconversion of sucrose and lactose into lactosucrose under the optimized conditions. The enzymatic reaction was performed with 5 U mL−1 (hydrolysis activity) of the purified recombinant levansucrase from 27% (W/V) sucrose and 27% (W/V) lactose at pH 6.5 and 50 °C. Values are the means of three replications ± standard deviation.

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AUTHOR INFORMATION

Corresponding Authors

demonstrated the constant occurrence of the hydrolysis reaction. Generally, a reaction time of 1 h could be relatively short when operating in large-scale because at the industrial level, the filling and discharge of the reactor might take a similar or even greater time. In this article, the optimization of lactosucrose production was conducted based on the single factor experiment method and the maximal production was reached after reaction for only 1 h. However, for the practical application, it would require further optimization to obtain the maximal productivity after a relatively longer reaction time. Table 1 summarizes the biological lactosucrose production ever reported, and it shows that the productivity remarkably

*Phone: +49 711 459 22576. Fax: +49 711 459 24267. E-mail: [email protected] (T.S.). *Phone: +86 510 85919161. Fax: +86 510 85919161. E-mail: [email protected] (W.M.). Funding

This work was supported by the NSFC Project (No. 21276001), the 863 Project (No. 2013AA102102), Fundamental Research Funds for the Central Universities (No. JUSRP51304A), the Support Project of Jiangsu Province (No. BK20130001), and the project of outstanding scientific and technological innovation group of Jiangsu Province (J.W.). G

DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography; IPTG, isopropyl-β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DSS, 4,4-dimethyl-4-silapentane-1-sulfonate

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DOI: 10.1021/acs.jafc.5b03648 J. Agric. Food Chem. XXXX, XXX, XXX−XXX