Article pubs.acs.org/JAFC
Hidden Reaction: Mesophilic Cellobiose 2‑Epimerases Produce Lactulose Beatrice Kuschel,† Ines Seitl,† Claudia Glück,† Wanmeng Mu,‡ Bo Jiang,‡ Timo Stressler,*,† and Lutz Fischer† †
Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, D-70599, Stuttgart, Germany ‡ State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China S Supporting Information *
ABSTRACT: Lactulose (4-O-β-D-galactopyranosyl-D-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4O-β-D-galactopyranosyl-D-glucopyranose). In our study we observed for the first time that known cellobiose 2-epimerases (CEs; EC 5.1.3.11) from mesophilic microorganisms were generally able to catalyze the isomerization reaction of lactose into lactulose. Commonly, CEs catalyze the C2-epimerization of D-glucose and D-mannose moieties at the reducing end of β-1,4-glycosidiclinked oligosaccharides. Thus, epilactose (4-O-β-D-galactopyranosyl-D-mannopyranose) is formed with lactose as substrate. So far, only four CEs, exclusively from thermophilic microorganisms, have been reported to additionally catalyze the isomerization reaction of lactose into lactulose. The specific isomerization activity of the seven CEs in this study ranged between 8.7 ± 0.1 and 1300 ± 37 pkat/mg. The results indicate that very likely all CEs are able to catalyze both the epimerization as well as the isomerization reaction, whereby the latter is performed at a comparatively much lower reaction rate. KEYWORDS: cellobiose 2-epimerase, isomerization, epimerization, lactulose, lactose, RaCE
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INTRODUCTION Lactulose (4-O-β-D-galactopyranosyl-D-fructofuranose) is a nondigestible disaccharide and a lactose (4-O-β-D-galactopyranosyl-D-glucopyranose) derivative, which displays prebiotic properties.1 Lactulose has been reported to improve the calcium absorption in adult men and the growth of some bifidobacterial species.2,3 As a pharmaceutical, it is used to treat chronic constipation and hepatic encephalopathy. Lactulose is currently produced for commercial purposes by alkaline isomerization of lactose via the Lobry−de-Bruyn−Alberda van Ekenstein rearrangement.4 However, the usage of inorganic catalysts leads to various side products, which, in turn, make further elaboration and cost-intensive purification steps necessary.5 Enzymatically, lactulose can be formed from lactose when fructose is added as a cosubstrate by employing the transgalactosylation reaction of β-glucosidases (EC 3.2.1.21) and β-galactosidases (EC 3.2.1.23).4−7 However, a single substrate reaction, such as the isomerization of lactose using cellobiose 2-epimerases (CEs; EC 5.1.3.11), would be preferable in order to simplify the lactulose production process and increase its cost-effectiveness. Commonly, CEs catalyze the C2-epimerization of the β-1,4glycosidic-bound reducing glucose and mannose moieties in disaccharides, such as cellobiose (4-O-β-D-glucopyranosyl-Dglucopyranose), mannobiose (4-O-β-D-mannopyranosyl-D-mannopyranose), and lactose, leading to the production of 4-O-β-Dglucopyranosyl-D-mannopyranose, 4-O-β-D-mannopyranosyl-Dglucopyranose, and epilactose (4-O-β-D-galactopyranosyl-Dmannopyranose), respectively.8,9 The physiological function of CEs was proposed to be within the mannan pathway, facilitating the usage of the environmental polysaccharide β© XXXX American Chemical Society
mannan for bacterial growth. There, the CE catalyzes the C2epimerization of mannobiose into 4-O-β-D-mannopyranosyl-Dglucopyranose, which is phosphorylized into α-D-mannosyl 1phosphate and D-glucose by another enzyme called mannosylglucose phosphorylase (EC 2.4.1.281).8,10 Entering the Embden−Meyerhof−Parnas pathway, the two monosaccharides are then further catabolized.8 In addition to the epimerization activity, four CEs have been reported to catalyze an additional isomerization reaction on the same moieties of their substrates.11−14 Lactulose is formed when using lactose as a substrate.12 The reaction mechanism of the epimerization was recently proposed to occur via a cis-enediol intermediate through ring opening, deprotonation, and reprotonation; carbon−carbon bond rotation; and ring closure.8,15 The aldose−ketose isomerase of Salmonella enterica (YihS) was described previously to catalyze the isomerization of D-mannose into D-fructose via a cis-enediol intermediate as well.16 Both enzymes, CE and YihS, show similarities in their substrates, amino acid sequence, and structure ((α/α)6-barrel).15 Thus, it was inferred that the isomerization reaction of CE might occur in a similar way as described for YihS. However, the structural data available so far were deemed not conclusive enough to verify the presumption.8 The first CE was discovered by Tyler and Leatherwood in 1967 and belonged to the Gram-positive bacterium Ruminococcus albus (RaCE).17 Since then, more than 15 CEs from Received: Revised: Accepted: Published: A
December 14, 2016 February 28, 2017 March 2, 2017 March 2, 2017 DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
respective CE-like genes (National Center for Biology Information: NCBI accession number WP_022776249 and NCBI accession number WP_02235242), carried out as previously described,25 and resulted in the constructs pET20bB15CE and pET-20bFBCE. For the expression of the respective CE, each construct was transformed in E. coli BL21 (DE3) cells. The sequences of the codon-optimized genes for the expression of B15CE, Bf CE, FbCE, FjCE, and RmCE are deposited at GenBank with the accession numbers KX792148, KX792149, KX792150, KX792151, and KX792152, respectively. Cultivation. The production of the respective CE was carried out as previously reported for another enzyme.26 The E. coli BL21 (DE3) cells containing either pET-20bB15CE, pET20bBFCE, pET-20bDFCE, pET-20bFBCE, pET-20bRACE, or pET-20bRMCE were cultivated separately in 0.6 L of 2-yeasttryptone medium containing 16 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl, 20 g/L of glucose, and 100 μg/mL of ampicillin in a stirred tank reactor (Multifors, Infors, Bottmingen, Switzerland) for 11 h, respectively. The expression of the respective CE was induced with 0.5 mM isopropyl-β-Dthiogalactopyranoside (IPTG) after 2.5 h of cultivation at 37 °C. The cultivation was continued for 8.5 h at 30 °C. The pH was regulated to 7.0 by adding 2 M NaOH and 0.6 M H3PO4. The recombinant E. coli cells were harvested by centrifugation (8,000g, 15 min, 4 °C) and washed twice with 0.9% (w/v) NaCl solution. In addition, E. coli BL21 (DE3) pET-20bFJCE was cultivated in 35 L of the same medium as described above in a 70-L stirred tank reactor (Techfors, Infors, Bottmingen, Switzerland) for 13 h. The expression of the FjCE was induced with 0.5 mM IPTG after 4 h of cultivation at 37 °C. The cultivation was continued for 9 h at 20 °C. The pH regulation and cell harvesting were performed as described above. Purification. For enzyme purification a 30% (w/v) cell suspension of the particular cells in 10 mM 1,4-piperazinediethanesulfonic acid (PIPES) buffer, pH 7.5, containing 300 mM NaCl was used. The cell disruption was performed by a ten cycle sonication program on ice with each cycle consisting of 1 min of sonication (95% amplitude, 0.5 cycles; UP200S/S3, Hielscher Ultrasonics, Teltow, Germany) and a 1 min break. Cell debris was removed subsequently by centrifugation (8,000g, 15 min, 4 °C), and the particular supernatant containing CE was filtered (0.45 μm) and further purified using an Ä KTA FPLC system (GE Healthcare, Little Chalfont, UK) equipped with a Ni2+-charged iminodiacetic acid column (column volume: CV = 6.4 mL; Biofox 40/1200 IDAlow IMAC, KNAUER, Berlin). After equilibration with binding buffer (BB; 10 mM PIPES buffer, pH 7.5, containing 300 mM NaCl), an appropriate amount of sample was applied to the column. After elution of unbound protein with BB and a subsequent washing step with 25 mM imidazole in BB, target protein was eluted using a step to 125 mM imidazole over 3 CV, followed by a gradient to 250 mM imidazole over 3 CV. The flow rate was kept constant at 1 mL/min. Eluted protein was detected at 280 nm during chromatography, and fractions of 2.5 mL were collected. The active fractions were desalted into 10 mM PIPES buffer with the respective optimal pH of the particular CE, containing 100 mM NaCl using PD-10 columns (GE Healthcare, Little Chalfont, UK). All enzyme preparations were stored on ice until use. Protein quantification and polyacrylamide gels. Author: The protein concentration of the samples was determined by the method of Bradford27 using bovine serum
various microorganisms of, so far, six different phyla have been described, most of them originating from mesophilic microorganisms.8,18−20 However, only four CEs that were capable of catalyzing the isomerization reaction were published, and all of them belong to thermophilic microorganisms, namely Caldicellulosiruptor saccharolyticus (CsCE), Caldicellulosiruptor obsidiansis (CoCE), Dictyoglomus turgidum (DtCE), and Spirochaeta thermophila (StCE). Although it has been shown recently that CsCE and DtCE can catalyze the isomerization reaction at mesophilic and lower temperatures,21 there has been no publication regarding lactulose production using mesophilic CEs so far. It was believed that the CE property of the isomerization reaction was exclusively performed by the enzymes from the thermophilic microorganisms. The aim of the current study was to evaluate whether the other well-known CEs from mesophilic microorganisms, such as RaCE, and CE from Bacteroides f ragilis (Bf CE) and from Flavobacterium johnsoniae (FjCE), are also able to catalyze the isomerization reaction, leading to production of lactulose from lactose. The complete epi- and isomerization reaction progress of lactose with RaCE was investigated for further insights. Revealing also that mesophilic CEs are able to catalyze the isomerization reaction, all CEs will most likely follow the same reaction mechanisms just with different kinetic performances regarding the epimerization and the isomerization reactions.
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MATERIALS AND METHODS Materials, chemicals, and enzymes. All chemicals were of analytical grade or higher and purchased from Sigma-Aldrich (St. Louis, MO), Carl Roth (Karlsruhe, Germany), or Merck (Darmstadt, Germany). The T4 DNA ligase was supplied by Fisher Scientific (Schwerte, Germany), and restriction enzymes and DNA polymerase were acquired from New England Biolabs (Ipswich, MA). Microorganisms, plasmids, and cloning. The strains Escherichia coli XL-1 Blue and E. coli BL21 (DE3) were used for the cloning and expression of the proteins. For the expression of FjCE, the construct pET-20bFJCE was built as reported previously.22 The expression vectors for RaCE and the CE from Dyadobacter fermentans (Df CE) were built analogously to Krewinkel et al.,22 resulting in the constructs pET-20bDFCE and pET-20bRACE. Therefore, the genomic DNA sequences of the CEs from Ruminococcus albus DSM 20455 (EMBL coding: BAF81108.1) and Dyadobacter fermentans DSM 18053 (EMBL coding: ACT93198.1) were used. The construction of the expression vectors for Bf CE and the CE from Rhodothermus marinus (RmCE) was based on the genomic DNA sequence of the CEs from Bacteroides f ragilis DSM 2151 (European Molecular Biology Laboratory: EMBL coding: BAH23773.1) and Rhodothermus marinus DSM 4252 (EMBL coding: BAK61777.1), available at the EMBL, European Bioinformatics Institute.23 The codon usage was optimized for expression in E. coli using Gene Designer (DNA2.0 Inc., Menlo Park, CA). The genes were purchased as synthetic genes from Life Technologies (Carlsbad, CA). In general, standard molecular biology methods according to Sambrook and Russell were used.24 Cloning in pET-20b (+) was followed by transformation in E. coli XL-1 Blue cells to introduce a C-terminal His6-tag in frame, resulting in the constructs pET-20bBFCE and pET-20bRMCE. The identification, construction, and cloning of the expression vectors for the expression of the CEs from Butyrivibrio sp. AE2015 (B15CE) and Firmicutes bacterium CAG:534 (FbCE) was based on the amino acid sequence of the B
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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vessel (working volume: 750 μL) at 10 ± 1 °C below the optimal temperature of the respective CE over the course of 8 days. Sugar analysis of the reaction mixture was carried out via HPLC (see above). Additional conversions with 10 μkatepi/mL CE were carried out under similar conditions in order to obtain reaction mixtures with suitable lactulose concentration for thinlayer chromatography (TLC) analysis (see below). Enzymatic batch conversions were conducted with an initial enzyme activity of 20 μkatepi/mL in order to observe the full reaction progress of the epi- and isomerization of lactose by RaCE. The conversions were carried out in 10 mM PIPES buffer, pH 7.5, containing 100 mM NaCl and 150 mM lactose as a substrate. The conversions were conducted in a 1.5 mL reaction vessel (working volume: 750 μL) at 20 °C. The experiments were carried out in duplicate in two separate reaction vessels. A third vessel without the addition of the enzyme was used as a negative control. Samples for sugar quantification via HPLC (see above) were taken over the course of 14 days. Thin layer chromatography. Thin layer chromatography was used in order to visualize the products (epilactose and lactulose) generated from lactose by enzymatic conversion with the CEs. Reactions were performed as stated above. The reaction was terminated by adding 150 μL of a mix of 80% (v/ v) acetonitrile and 20% (v/v) methanol to 50 μL of reaction mixture. The samples were incubated for 15 min on ice following a centrifugation step (20,000g, 5 min, 4 °C). Subsequently, 160 μL of the supernatant were mixed with 40 μL of double-distilled water. Aliquots of 1 μL of the sample prepared were spotted on TLC plates (Silica gel 60F254, Merck, Whitehouse Station, NJ), and developed in a solvent system of 2-propanol/acetic acid/5 g/L aqueous boric acid (60:3:10, v/ v). The plates were developed by a coloring agent (2 M phosphoric acid, 190 mM aniline, 100 mM diphenylamine in ethanol) at 100 °C until the spots became visible. Statistical analysis. Unless otherwise stated, all enzymatic assays and analytic measurements were performed at least in duplicate. Statistical analyses, calculation, and visualization were carried out using Excel 2007 (Microsoft Corporation, Redmond, WA). Sequence analysis. The neighbor-joining phylogenetic tree (with distance corrections) of the nine cellobiose 2epimerases was generated by Phylogeny.f r.31 Sequences were aligned with MUSCLE (v3.8.31) configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions (i.e., containing gaps and/or poorly aligned) were removed with Gblocks (v0.91b). The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT). The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.131) and 4 gammadistributed rate categories to account for rate heterogeneity across sites. The γ shape parameter was estimated directly from the data (γ = 2.058). Reliability for the internal branch was assessed using the aLRT test (SH-Like). Graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v198.3). The sequence alignment of multiple CE proteins was carried out using Clustal Omega web services,32 and the percent identity matrix results of the CE alignment were created by Clustal2.1.
albumin as a standard. A sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis was performed for examination of the quality of the CE expression and purification.28 Therefore, a precast 4−15% (w/v) Mini-PROTEAN TGX gradient gel was used (Bio-Rad, München, Germany). The protein bands were stained with Coomassie Brilliant Blue R-250, according to the method of Fairbanks et al. (1971).29 The protein molecular mass standard (2−212 kDa) was purchased from New England Biolabs (Ipswich, MA). The molecular weight of the respective CE was estimated with the protein identification and analysis tools using the ExPASy Server by Gasteiger et al.30 Determination of CE activity. The determination of CE activity, unless otherwise stated, was carried out at 10 °C below the respective optimum (TOpt.-10 °C) of the enzyme for a suitable amount of time in 10 mM PIPES buffer with the respective optimal pH (pHOpt.), containing 100 mM NaCl and 150 mM substrate. The enzymes used in this study and their respective TOpt., and pHOpt are listed in Table 1. The reaction Table 1. Temperature (TOpt.) and pH (pHOpt.) Values for the Optimal Epimerization Activity of B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE, and RmCE enzyme
TOpt. [°C]
pHOpt. [−]
source
B15CE Bf CE Df CE FbCE FjCE RaCE RmCE
60 45 50 45 35 30 80
7.0 7.5 7.7 7.5 8.4 7.5 6.3
25 35 33 25 33 34 38
was carried out with suitably diluted enzyme solution in a total volume of 200 μL. The enzymatic reaction was stopped by adding 600 μL of 80% (v/v) acetonitrile, 20% (v/v) methanol, containing 100 mM urea as an internal standard for subsequent high performance liquid chromatography (HPLC) analysis (see below). The stopped samples were incubated for 15 min on ice followed by a centrifugation step (20,000g, 5 min, 4 °C) to remove precipitated protein. The samples were analyzed subsequently by HPLC to determine the concentration of the reaction products. One katal (kat) of activity was defined as the amount of enzyme required to produce 1 mol of product per s. HPLC method for sugar analysis. An Agilent Series 1100 HPLC system, equipped with a Shodex HILICpak VG-50 4E column (4.6 × 250 mm, 5 μm, Shodex) and a low-temperature evaporative light scattering detector (ELSD Sedex 85LT, Sedere, Alfort ville Cedex, France) at 50 °C and 3 bar was used to determine the concentrations of lactose, epilactose, and lactulose. The column was eluted with 77.5% (v/v) acetonitrile, 15% (v/v) methanol, and 7.5% (v/v) double-distilled water at a flow rate of 1 mL/min at 40 °C. After mixing 400 μL of the supernatant (see above) with 100 μL of double-distilled water, 5 μL of the sample prepared was injected into the HPLC system. Retention times were 5.0 min for urea, 11.9 min for lactulose, 13.3 min for epilactose, and 15.1 min for lactose. Enzymatic sugar conversions. Enzymatic conversions with an initial epimerization activity of 1 μkat/mL (μkatepi/mL) CE were carried out for the demonstration of lactulose production from lactose by all CEs. The reactions were performed in 10 mM PIPES buffer with the respective optimal pH, containing 100 mM NaCl and 150 mM lactose as a substrate. The conversions were conducted in a 1.5 mL reaction C
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Neighbor-joining phylogenetic tree with distance corrections (a) and percent identity matrix (b) of CsCE, DtCE and the seven CEs: B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE used in this study. Phylogenetic tree analysis was created by Phylogeny.f r and percent identity matrix by Clustal2.1 based on the amino acid sequence of the respective CE (Genbank accession number; GB ID). The scale bar indicates the genetic distance, representing the conservation between nine cellobiose 2-epimerase homologues.
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RESULTS In this work seven mainly mesophilic CEs were recombinantly produced using E. coli BL21 (DE3) as an expression host. After purification the CEs were investigated upon their ability to catalyze a C2-epimerization and -isomerization reaction on lactose. Furthermore, the complete enzymatic conversion of lactose with the mesophilic enzyme RaCE was conducted in order to display the progression of lactulose formation over the time course of 2 weeks. Selection, production and purification of seven CEs. An assortment of seven CEs was selected in order to cover the optimal temperature and pH range of known CEs.8,25 Simultaneously, the selection was aimed to contain several well investigated CEs to increase the strength of evidence and to attempt an extrapolation of the results obtained onto the whole class of CEs. The seven CEs chosen were B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE with the highest epimerization activities between 30 and 80 °C and between pH 6.3 and 8.4, respectively (see Table 1). An amino acid sequence-based alignment was carried out using Clustal Omega32 in order to give an overview of the homology of the seven CEs investigated in this study and how they are related to CsCE and DtCE, the most widely investigated CEs known to catalyze the isomerization reaction (Figure 1). The neighbor-joining phylogenetic tree with distance corrections is shown in Figure 1a. While the upper five CEs (DtCE, RaCE, CsCE, FbCE and B15CE) are from bacteria of the phylum Firmicutes, the remaining four CEs (RmCE, Df CE, FjCE, and Bf CE) belong to bacteria of the phylum Bacteroidetes. The identity matrix of the relations between the CEs is given in Figure 1b. The seven CEs chosen show moderate amino acid sequence homologies with CsCE ranging between 34.7 and
52.9%. Among the seven CEs chosen in this study, FbCE and B15CE were found to be the most similar to CsCE, with amino acid sequence homologies of 52.9% and 52.0%, respectively. Df CE (34.7%) showed the least similarity to CsCE. Among B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE, homologies ranged from 34.8 to 63.6%. All seven CEs were expressed with the respective pET20bCE construct in E. coli BL21 (DE3). The purified CE preparations showed the most strongly pronounced band at about 42 kDa (see Figure 2). This is in good agreement with the theoretically calculated masses ranging from 42 kDa for B15CE to 47.4 kDa for RmCE. The purified CE preparations were used for all further investigations. Lactulose and epilactose generation by all CEs. In order to test for the isomerization activity of all seven CEs in the current study, enzymatic conversions of lactose were carried out with an elevated initial epimerization activity of 1 μkatepi/ mL. Reactions were carried out at 10 °C below the temperature of optimal epimerization activity of the respective enzyme to minimize the risk of thermal inactivation. The conversions with Bf CE, FbCE and FjCE produced less than 3% of total sugar (%ts) lactulose. Therefore, in order to ensure visibility of the produced lactulose on the TLC plates additional conversions with these three CEs were carried out over the course of 8 days with an initial enzyme activity of 10 μkatepi/mL. The formation of lactulose as a reaction product of the enzymatic conversion of lactose with B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE is shown in Figure 3a with HPLC and in Figure 3b with TLC analysis. As expected, epilactose was detected for all reactions in addition to lactulose. Neither epilactose nor lactulose could be detected in the negative control (N) in Figure 3b. D
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Lactulose production from 150 mM lactose in 10 mM PIPES, 0.1 M NaCl over the course of 8 days with 1 μkatepi/mL of B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE at TOpt.-10 °C, pHOpt.
Figure 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (4−15%, w/v gel, Coomassie stained, 5 μg protein per lane) showing the CE preparations purified by His-Trap affinity chromatography (Biofox IDAlow, 6.4 mL). Lane 1: Protein molecular mass marker (2−212 kDa, NEB); lane 2: B15CE; lane 3: Bf CE; lane 4: Df CE; lane 5: FbCE; lane 6: FjCE; lane 7: RaCE; lane 8: RmCE. The arrow indicates the expected size of the CEs purified.
with the highest lactulose formation, produced 19%ts of lactulose after 8 days. No trend was observable concerning a correlation between the phyla of the original organism, optimal pH or temperature of the respective CE and the amount of lactulose generated after 8 days. Apart from the lactulose produced, the ratio of lactose and epilactose to one another was observed to be at a constant level of about 70% lactose to 30% epilactose for all conversions (data not shown). Specific enzyme activities for epi- and isomerization of lactose. The specific epimerization and isomerization activity of the CEs using 150 mM lactose as a substrate is shown in Table 2. While the specific epimerization activity ranged from about 430 to 5200 nkat/mg, the specific isomerization activity ranged from about 9 to 1300 pkat/mg.
Lactulose formation during 8 days. The progression of lactulose generation over the course of 8 days with 1 μkatepi/ mL CE at 10 °C below the optimal epimerization temperature in 10 mM PIPES buffer at optimal pH, containing 100 mM NaCl and using 150 mM lactose as a substrate, is shown in Figure 4. The seven CEs can be divided into three groups based on their lactulose production. Bf CE, FbCE and FjCE comprise the group with the lowest lactulose production of less than 3%ts after 8 days. The second group comprising moderate lactulose production levels (6−9%ts after 8 days) consists of B15CE, Df CE, and RaCE. RmCE, as the sole CE in the third group
Figure 3. HPLC chromatograms (a) and TLC plate (b) for the sugar analysis of epilactose and lactulose formation from lactose by all CEs. The peaks and spots are urea as internal standard (IS), lactulose (1), epilactose (2) and lactose (3). Mixtures of sugars (10 mM for HPLC, 40 mM for TLC) were used as a reference (I). Negative control (150 mM lactose, 70 °C, 24 h) (N). Conversion of 150 mM lactose in 10 mM PIPES, 0.1 M NaCl, pHOpt. at TOpt.-10 °C with 1 μkatepi/mL B15CE for 8 days (II), 10 μkatepi/mL Bf CE for 7 days (III), 1 μkatepi/mL Df CE for 8 days (IV), 10 μkatepi/mL FbCE for 7 days (V), 10 μkatepi/mL FjCE for 7 days (VI), 1 μkatepi/mL RaCE for 8 days (VII) and 1 μkatepi/mL RmCE for 2 days (VIII). E
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Specific enzyme activity (EAspec.) of all tested CE catalyzing the epimerization and isomerization of 150 mM lactose as a substrate in 10 mM PIPES, pHOpt., containing 100 mM NaCl at TOpt.-10 °C enzyme
EAspec. epimerization [nkat/mg]
EAspec. isomerization [pkat/mg]
B15CE Bf CE Df CE FbCE FjCE RaCE RmCE
431 ± 17 1280 ± 33 782 ± 26 982 ± 12 809 ± 11 647 ± 21 5190 ± 73
23.1 ± 0.8 8.88 ± 0.5 36.7 ± 1.4 13.7 ± 0.4 8.69 ± 0.1 16.5 ± 0.7 1300 ± 37
Figure 5. Time course study of the enzymatic conversion of 150 mM lactose by 20 μkatepi/mL RaCE in 10 mM PIPES, pH 7.5, containing 100 mM NaCl at 20 °C over 14 days.
Therefore, all CEs tested exhibit isomerization activity, although the activity values are between 4,000 (RmCE) and 140,000 (Bf CE) times lower for the specific isomerization reaction of lactose compared to the respective epimerization reaction. Although RmCE displayed both, the highest epimerization activity (5188 ± 73 nkat/mg) and the highest isomerization activity (1300 ± 37 pkat/mg), no correlation for the other CEs between epimerization activity and isomerization activity could be observed. In addition, no correlation between the isomerization activity and the phyla of the originating organism, the optimal temperature or the optimal pH for epimerization activity was observed. Surprisingly, the amino acid sequence homology of the seven CEs to CsCE did not correlate with the specific isomerization activity of the respective CE either. While RmCE displayed the highest isomerization activity, it has the second lowest homology to CsCE at 38.8%. On the other hand, FbCE, which displayed the highest homology to CsCE (52.9%), had the fourth lowest isomerization activity (13.7 ± 0.4 pkat/mg). Complete enzymatic conversion of lactose with RaCE. In the current study, it was shown for the first time that B15CE, Bf CE, Df CE, FbCE, FjCE, RaCE and RmCE, were able to catalyze both, the epimerization and the isomerization of lactose. An enzymatic batch conversion was conducted in order to investigate whether the reaction progress using a mesophilic CE proceeds in a similar pattern to the reaction progress catalyzed by the thermophilic CsCE. Therefore, the conversion was carried out with an initial activity of 20 μkatepi/mL RaCE at 20 °C in 10 mM PIPES at pH 7.5, containing 100 mM NaCl using 150 mM lactose as the substrate. The progression of the lactose, epilactose and lactulose proportions are shown in Figure 5. After an initial increase of the epimerization product epilactose up to a content of about 30%ts after 5 min, the epilactose content decreases over the course of the remaining reaction time. By contrast, the lactulose content steadily increases over the whole course of the reaction. As a result, the lactose content first decreases quickly to a content of about 70%ts after 5 min and decreases more slowly after the maximum of epilactose content was produced. The lactose/epilactose ratio, regardless of lactulose, remained at a constant level of about 70% to 30%, respectively, throughout the course of the conversion (see Supporting Information Figure S1). The final sugar content reached after 21 days of enzymatic conversion were 56.8 ± 1.1%ts lactulose, 13.3 ± 0.3%ts epilactose and 29.9 ± 0.7%ts lactose.
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DISCUSSION Cellobiose 2-epimerases are a widely investigated enzyme class and have been discovered in a variety of microorganisms.8,33 All CEs are reported to catalyze a C2-epimerization on β-1,4glycosidic bound mannose and glucose in mannobiose, cellobiose and lactose, respectively. However, so far, only four CEs, namely CsCE, CoCE, DtCE and StCE have been published to catalyze an additional isomerization reaction on the same moiety leading to a transformation into β-1,4glycosidic-bound fructose.11−14 The current study demonstrates for the first time that CEs described previously, such as RaCE and Bf CE, are able to catalyze the isomerization reaction as well. RaCE was the first CE described and has, since then, been widely investigated.17,34−37 Additionally, Bf CE and RmCE have been the subject of several studies.15,35,38,39 Among the four exclusively thermophilic CEs that have been published to catalyze the isomerization reaction, CsCE is the most extensively investigated one.21,40−42 The seven CEs of this work were mainly mesophilic and were chosen in order to cover a wide range of optimal temperatures and pH values. The moderate homology range of 34.7 to 63.6% among the seven CEs of this study and CsCE as well as DtCE (Figure 1) was in agreement with a recent publication using a polymerase chain reaction-based approach to identify novel CEs from environmental samples.18 In that study, 71 ce-like genes with amino acid sequence homologies of 41−80% to known CEs were identified.8,18 To the best of our knowledge, this is the first published data to show the isomerization reaction and therefore the lactulose production from lactose by CEs other than CsCE, CoCE, DtCE and StCE.11−14 Since previous studies reported of an increase in isomerization activity when an increase in thermostability was achieved until now it was unclear whether the thermostability of CsCE, CoCE, DtCE and StCE was imperative to their isomerization activity.40,41 Among all tested CEs in this study, the one with the highest temperature optimum (RmCE; TOpt. 80 °C) showed the highest isomerization activity. However, in this work it was shown for the first time that the isomerization reaction was catalyzed by mesophilic CEs (Bf CE, FbCE, FjCE and RaCE), too. Although it has to be mentioned that the four mesophilic CEs with temperature optima between 30 and 45 °C showed lower isomerization activities compared to B15CE (TOpt. 60 °C) and Df CE (TOpt. 50 °C), respectively. Therefore, F
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Figure 6. Clustal Omega sequence alignment of CEs used in this study in comparison with CsCE and DtCE. The GenBank accession numbers are given after the respective CE. The highly conserved residues of the catalytic center are highlighted with green bars. The indicated catalytic His residue corresponds to His259 in Ruminococcus albus CE (RaCE).9 The red bars show the position of amino acid residue exchanges in CsCE, causing a CsCE mutant with high isomerization activity.40
the hypothesis that all CEs might be capable of catalyzing both the epimerization and the isomerization reaction to a certain extent seems admissible. The specific isomerization activity of
the CEs in this study ranged between 9 and 1300 pkat/mg and were therefore more than 138 times lower than previously reported for CsCE and more than 4000 lower than the G
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Figure 7. Reaction scheme diagram of the proposed isomerization and epimerization reaction of cellobiose 2-epimerases.
respective epimerization activity.41 The results indicate that, as previously reported for CsCE,21 the isomerization reaction is the slower reaction of CEs and, in most cases, seems to be so slow that it has not been detected until now. Due to the extraordinary high specific enzyme activity of CsCE for the isomerization reaction, the new found isomerization reaction of the other CEs will not challenge CsCE as the most likely and most efficient CE to be adapted for industrial lactulose production. However, the newly found isomerization reaction for mesophilic CE may deepen the understanding of the enzyme class of CE and their catalytic behavior. Surprisingly, no correlation was observed between the specific isomerization activity and the epimerization activity of the CEs tested in this study. In addition, the phyla of the originating organism, the optimal pH as well as the amino acid sequence homology do not allow a reliable prediction about the extent of isomerization activity of a certain CE. A recent publication reported on a CsCE mutant where 5 amino acid residues (R5M; A12S; I52V; F231L; K328I) were substituted by random mutagenesis that catalyzed an enhanced conversion of lactose resulting solely in the production of lactulose.41 All but one (I52V) of the particular mutations was found outside the supposed active site. R5M, A12S and K328I are located in different α-helices at the enzyme surface far away from the putative catalytic center, presumably influencing isomerization activity and thermal stability by long-range structural effects. Also at the protein surface, but in a β-strand region close to the active site entrance cleft, the amino acid mutation F231L is located. Interestingly, the sequence alignment of nine CE proteins revealed that the phenylalanine at position 231 is conserved in all of them (Figure 6). Since phenylalanine is a bulky amino acid residue, its replacement by leucine might favor substrate access and increase lactose affinity for enhanced side-reaction activity of lactulose formation. The mutation of a hydrophobic isoleucine residue at position 52 of CsCE to the smaller valine residue was proposed to influence the flexibility of the active site pocket and therefore contributes to the orientation of the reaction.38 However, DtCE, Df CE, and FjCE
already possess a valine residue at this position, while RaCE, FbCE, B15CE, RmCE and Bf CE have an isoleucine at the respective position similar to the wild type CsCE (Figure 6). Therefore, it remains unclear which structural effects influence the epimerization and isomerization capability of the enzyme directly. Nevertheless, it can be assumed, based on the data presented in the current study, that CEs generally have the ability to catalyze both the epimerization and the isomerization on β-1,4-glycosidic-bound mannose and glucose in oligosaccharides, although in varying extents. This is in accordance with a recently published review including structure−function relationship of CE where the author supposes that the His residue corresponding to His259 of RmCE might transfer H2 to C1 of the cis-enediol intermediate in other CEs to catalyze the isomerization reaction of the reducing end D-glucose or Dmannose residue (Figure 7).8 Since this His residue is highly conserved throughout all known CEs (Figure 6; His259) the assumption is confirmed that at least all CEs have the ability to catalyze the isomerization activity. The experimental evidence for that is given in this study, showing a highly diverse extent of isomerization activity. This is presumably caused by amino acid residues outside the active center, which are influencing the flexibility of substrate entrance and affinity as well as thermal stability of the respective CE. However, structural information on CEs with high isomerization activity is not sufficient to conclusively determine the mechanism of lactulose formation catalyzed by CEs. Further biochemical and structural analysis is required for a better understanding of the structure−function relationship of CEs. Regarding the physiological function, for some CEs the isomerization activity might help to improve the usage of environmental β-mannan by converting mannobiose into 4-Oβ-D-mannopyranosyl-D-glucopyranose and 4-O-β-D-mannopyranosyl-D-fructofuranose. After phosphorylation α-D-mannosyl 1-phosphate, D-glucose8,10 and D-fructose would be available to enter at different points into the catabolic pathways of the bacterium and therefore improving the substrate usage efficiency. However, due to the more than 4000 times higher H
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ACKNOWLEDGMENTS Many thanks go to Susanne Herr (Institute of Food Science and Biotechnology) for her help regarding the gene cloning and Wolfgang Claaßen (Institute of Food Science and Biotechnology) for his help regarding the cultivations.
epimerization activity of CEs it seems unlikely that the isomerization reaction of CEs undertakes more than the role of a side activity in the physiological function of the enzyme. In order to investigate the full reaction progress using a mesophilic CE, a batch conversion with 20 μkatepi/mL RaCE and 150 mM lactose as a substrate was conducted. The conversion led to the production of about 57%ts lactulose and 13%ts epilactose with a remaining 30%ts lactose after 21 days, which is similar to the final sugar contents reported earlier.8,43 The reaction progress of the epimerization and isomerization with CsCE and DtCE using lactose as a substrate has been reported previously.11,12 In the study, 150 U/mL CsCE (which corresponds to about 2.5 μkatepi/mL) was used for an enzymatic batch conversion with 700 g/L (corresponding to about 2 M) lactose as a substrate. The authors reported a lactulose production of 180 g/L, which corresponds to about 526 mM or 25.7%ts after a reaction time of 30 min.12 A comparable lactulose production (26.5%ts) was achieved after 45 h in the current study using RaCE with an 8-fold higher initial activity (20 μkatepi/mL). However, the pattern of the reaction behaved in a similar manner with an initial increase of the epilactose content followed by a steady decrease. In contrast, the lactulose content steadily increased. Moreover, it was observed that the interrelated sugar ratios of lactose toward epilactose remained at a constant level of about 70% lactose to 30% epilactose. The same behavior of the interrelated sugar ratios has been observed recently for enzymatic conversions with CsCE and DtCE in a buffered system using lactose, epilactose and lactulose as a substrate.43 In addition, this lactose/epilactose ratio is also in good accordance with earlier reports about the epimerization yield of RaCE and other CEs.8 In conclusion, seven CEs from various, mostly mesophilic microorganisms were investigated regarding their ability to catalyze an isomerization reaction in addition to their characteristic C2-epimerization reaction. The CEs were applied in high amounts and for long reaction times. All CEs displayed isomerization activity. Moreover, RaCE displayed a reaction progress for the epimerization and isomerization of lactose similar to CsCE, resulting in similar final sugar proportions. The results presented indicate that all CEs are most likely able to catalyze both the C2-epimerization and the isomerization of their substrates.
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REFERENCES
(1) Aït-Aissa, A.; Aïder, M. Lactulose: Production and use in functional food, medical and pharmaceutical applications. Practical and critical review. Int. J. Food Sci. Technol. 2014, 49, 1245−1253. (2) Seki, N.; Hamano, H.; Iiyama, Y.; Asano, Y.; Kokubo, S.; Yamauchi, K.; Tamura, Y.; Uenishi, K.; Kudou, H. Effect of lactulose on calcium and magnesium absorption: a study using stable isotopes in adult men. J. Nutr. Sci. Vitaminol. 2007, 53 (1), 5−12. (3) Seki, N.; Saito, H. Lactose as a source for lactulose and other functional lactose derivatives. Int. Dairy J. 2012, 22 (2), 110−115. (4) Schuster-Wolff-Bühring, R.; Fischer, L.; Hinrichs, J. Production and physiological action of the disaccharide lactulose. Int. Dairy J. 2010, 20 (11), 731−741. (5) Aider, M.; Halleux, D. De. Isomerization of lactose and lactulose production: review. Trends Food Sci. Technol. 2007, 18 (7), 356−364. (6) Mayer, J.; Kranz, B.; Fischer, L. Continuous production of lactulose by immobilized thermostable β-glycosidase from Pyrococcus f uriosus. J. Biotechnol. 2010, 145 (4), 387−393. (7) Guerrero, C.; Vera, C.; Plou, F.; Illanes, A. Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases. J. Mol. Catal. B: Enzym. 2011, 72 (3−4), 206−212. (8) Saburi, W. Functions, structures, and applications of cellobiose 2epimerase and glycoside hydrolase family 130 mannoside phosphorylases. Biosci., Biotechnol., Biochem. 2016, 80 (7), 1294−1305. (9) Fujiwara, T.; Saburi, W.; Inoue, S.; Mori, H.; Matsui, H.; Tanaka, I.; Yao, M. Crystal structure of Ruminococcus albus cellobiose 2epimerase: structural insights into epimerization of unmodified sugar. FEBS Lett. 2013, 587 (7), 840−846. (10) Senoura, T.; Ito, S.; Taguchi, H.; Higa, M.; Hamada, S.; Matsui, H.; Ozawa, T.; Jin, S.; Watanabe, J.; Wasaki, J.; Ito, S. New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase. Biochem. Biophys. Res. Commun. 2011, 408 (4), 701− 706. (11) Kim, J.-E.; Kim, Y.-S.; Kang, L.-W.; Oh, D.-K. Characterization of a recombinant cellobiose 2-epimerase from Dictyoglomus turgidum that epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides. Biotechnol. Lett. 2012, 34 (11), 2061−2068. (12) Kim, Y.-S.; Oh, D.-K. Lactulose production from lactose as a single substrate by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Bioresour. Technol. 2012, 104, 668−672. (13) Park, C.-S.; Kim, J.-E.; Lee, S.-H.; Kim, Y.-S.; Kang, L.-W.; Oh, D.-K. Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2epimerase. Biotechnol. Lett. 2013, 35 (11), 1873−1880. (14) Chen, Q.; Levin, R.; Zhang, W.; Zhang, T.; Jiang, B.; Stressler, T.; Fischer, L.; Mu, W. Characterization of a novel cellobiose 2epimerase from thermophilic Caldicellulosiruptor obsidiansis for lactulose production. J. Sci. Food Agric. 2017, DOI: 10.1002/jsfa.8148. (15) Fujiwara, T.; Saburi, W.; Matsui, H.; Mori, H.; Yao, M. Structural insights into the epimerization of β-1,4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzyme epimerizing non-anomeric hydroxyl groups of unmodified sugars. J. Biol. Chem. 2014, 289 (6), 3405−3415. (16) Itoh, T.; Mikami, B.; Hashimoto, W.; Murata, K. Crystal structure of YihS in complex with D-mannose: Structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase. J. Mol. Biol. 2008, 377 (5), 1443−1459. (17) Tyler, T. R.; Leatherwood, J. M. Epimerization of disaccharides by enzyme preparations from Ruminococcus albus. Arch. Biochem. Biophys. 1967, 119, 363−367.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05599. Details on the reaction progress of RaCE; Figure S1, interrelated sugar ratios of lactose and epilactose (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +49-711-459-22576; Fax: +49-711-459-24267; E-mail: t.
[email protected]. ORCID
Wanmeng Mu: 0000-0001-6597-527X Timo Stressler: 0000-0001-9392-4031 Notes
Ethical Statement. This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare no competing financial interest. I
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry (18) Wasaki, J.; Taguchi, H.; Senoura, T.; Akasaka, H.; Watanabe, J.; Kawaguchi, K.; Komata, Y.; Hanashiro, K.; Ito, S. Identification and distribution of cellobiose 2-epimerase genes by a PCR-based metagenomic approach. Appl. Microbiol. Biotechnol. 2015, 99 (10), 4287−4295. (19) Krewinkel, M.; Kaiser, J.; Merz, M.; Rentschler, E.; Kuschel, B.; Hinrichs, J.; Fischer, L. Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose. J. Dairy Sci. 2015, 98 (6), 3665−3678. (20) Chen, Q.; Zhang, W.; Zhang, T.; Jiang, B.; Mu, W. Characterization of an epilactose-producing cellobiose 2-epimerase from Thermoanaerobacterium saccharolyticum. J. Mol. Catal. B: Enzym. 2015, 116, 39−44. (21) Rentschler, E.; Schuh, K.; Krewinkel, M.; Baur, C.; Claaßen, W.; Meyer, S.; Kuschel, B.; Stressler, T.; Fischer, L. Enzymatic production of lactulose and epilactose in milk. J. Dairy Sci. 2015, 98 (10), 6767− 6775. (22) Krewinkel, M.; Gosch, M.; Rentschler, E.; Fischer, L. Epilactose production by 2 cellobiose 2-epimerases in natural milk. J. Dairy Sci. 2014, 97 (1), 155−161. (23) Kulikova, T.; Akhtar, R.; Aldebert, P.; Althorpe, N.; Andersson, M.; Baldwin, A.; Bates, K.; Bhattacharyya, S.; Bower, L.; Browne, P.; Castro, M.; Cochrane, G.; Duggan, K.; Eberhadt, R.; Faruque, N.; Hoad, G.; Kanz, C.; Lee, C.; Leinonen, R.; Lin, Q.; Lombard, V.; Lopez, R.; Lorenc, D.; Mc William, H.; Mukherjee, G.; Nardone, F.; Pastor, M. P. G.; Plaister, S.; Sobhany, S.; Stoehr, P.; Vaughan, R.; Wu, D.; Zhu, W.; Apweiler, R. EMBL Nucleotide Sequence Database in 2006. Nucleic Acids Res. 2007, 35 (SUPPL. 1), 16−20. (24) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory Manual; Cold Spring Harbour Laboratory Press: New York, NY, 2001; Vol. 1. (25) Krewinkel, M. Discovery, production and characterization of epimerases and lipases with potential relevance in milk processing; Dissertation, University of Hohenheim: 2015. (26) Rentschler, E.; Schwarz, T.; Stressler, T.; Fischer, L. Development and validation of a screening system for a β-galactosidase with increased specific activity produced by directed evolution. Eur. Food Res. Technol. 2016, 242, 2129−2138. (27) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (28) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5259), 680−685. (29) Fairbanks, G.; Steck, T. L.; Wallachl, D. F. H. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 1971, 10 (13), 2606−2617. (30) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel, R. D.; Balroch, A. Protein identification and analysis tools on the ExPASy Server. In Theproteomics protocols handbook; Walker, J. M., Ed.; Humana Press Inc.: Totowa, NJ, 2005; pp 571− 607. (31) Dereeper, A.; Guignon, V.; Blanc, G.; Audic, S.; Buffet, S.; Chevenet, F.; Dufayard, J.-F.; Guindon, S.; Lefort, V.; Lescot, M.; Claverie, J.-M.; Gascuel, O. Nucleic Acids Res. 2008, 36 (April), 465− 469. (32) McWilliam, H.; Li, W.; Uludag, M.; Squizzato, S.; Park, Y. M.; Buso, N.; Cowley, A. P.; Lopez, R. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 2013, 41 (Web Server issue), 597− 600. (33) Ojima, T.; Saburi, W.; Yamamoto, T.; Mori, H.; Matsui, H. Identification and characterization of cellobiose 2-epimerases from various aerobes. Biosci., Biotechnol., Biochem. 2013, 77 (1), 189−193. (34) Ito, S.; Taguchi, H.; Hamada, S.; Kawauchi, S.; Ito, H.; Senoura, T.; Watanabe, J.; Nishimukai, M.; Ito, S.; Matsui, H. Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme. Appl. Microbiol. Biotechnol. 2008, 79 (3), 433−441.
(35) Senoura, T.; Taguchi, H.; Ito, S.; Hamada, S.; Matsui, H.; Fukiya, S.; Yokota, A.; Watanabe, J.; Wasaki, J.; Ito, S. Identification of the cellobiose 2-epimerase gene in the genome of Bacteroides f ragilis NCTC 9343. Biosci., Biotechnol., Biochem. 2009, 73 (2), 400−406. (36) Kawahara, R.; Saburi, W.; Odaka, R.; Taguchi, H.; Ito, S.; Mori, H.; Matsui, H. Metabolic mechanism of mannan in a ruminal bacterium, Ruminococcus albus, involving two mannoside phosphorylases and cellobiose 2-epimerase: discovery of a new carbohydrate phosphorylase, β-1,4-mannooligosaccharide phosphorylase. J. Biol. Chem. 2012, 287 (50), 42389−42399. (37) Saburi, W.; Yamamoto, T.; Taguchi, H.; Hamada, S.; Matsui, H. Practical preparation of epilactose produced with cellobiose 2epimerase from Ruminococcus albus NE1. Biosci., Biotechnol., Biochem. 2010, 74 (8), 1736−1737. (38) Ojima, T.; Saburi, W.; Sato, H.; Yamamoto, T.; Mori, H.; Matsui, H. Biochemical characterization of a thermophilic cellobiose 2epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785. Biosci., Biotechnol., Biochem. 2011, 75 (11), 2162−2168. (39) Sato, H.; Saburi, W.; Ojima, T.; Taguchi, H.; Mori, H.; Matsui, H. Immobilization of a thermostable cellobiose 2-epimerase from Rhodothermus marinus JCM9785 and continuous production of epilactose. Biosci., Biotechnol., Biochem. 2012, 76 (8), 1584−1587. (40) Shen, Q.; Zhang, Y.; Yang, R.; Hua, X.; Zhang, W.; Zhao, W. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis. J. Mol. Catal. B: Enzym. 2015, 120, 158−164. (41) Shen, Q.; Zhang, Y.; Yang, R.; Pan, S.; Dong, J.; Fan, Y.; Han, L. Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Food Chem. 2016, 207, 60−67. (42) Wang, M.; Yang, R.; Hua, X.; Shen, Q.; Zhang, W.; Zhao, W. Lactulose production from lactose by recombinant cellobiose 2epimerase in permeabilised Escherichia coli cells. Int. J. Food Sci. Technol. 2015, 50 (7), 1625−1631. (43) Kuschel, B.; Claaßen, W.; Mu, W.; Jiang, B.; Stressler, T.; Fischer, L. Reaction investigation of lactulose-producing cellobiose 2epimerases under operational relevant conditions. J. Mol. Catal. B: Enzym. 2016, DOI: 10.1016/j.molcatb.2016.11.022.
J
DOI: 10.1021/acs.jafc.6b05599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
