The secreted l-arabinose isomerase displays anti-hyperglycemic effects in mice
© Rhimi et al. 2015
Received: 25 August 2015
Accepted: 27 November 2015
Published: 21 December 2015
The l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of d-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts.
The l-arabinose isomerase (l-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP Usp45 ). The l-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei l-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant l-AI with the SP Usp45 . Th l-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the l-AI and galactose, tagatose was produced in vivo and reduced the glycemia index.
We report for the first time the secretion of the intracellular l-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted l-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the l-arabinose isomerase has the ability to produce d-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.
The l-arabinose isomerase (l-AI, EC 126.96.36.199) is an enzyme that mediates the conversion of l-arabinose to l-ribulose in vivo. At industrial scale this enzyme is used for the conversion of d-galactose into d-tagatose, thus it is also referred to as a d-galactose isomerase . The d-tagatose is a d-fructose isomer currently used as a low calorie sweetener . This ketohexose is a rare natural sugar tasting as sucrose and having similar physical properties  although it is not metabolized in humans and consequently has a very low caloric effect [4, 5]. In addition to its sweetener properties, d-tagatose is an anti-hyperglycemic factor and exhibits an efficient anti-biofilm effect. Later, the d-tagatose was considered as a GRAS “Generally Recognized as Safe” sweetener which can be used as a sugar substitute .
As previously reported, the isomerization of d-galactose into d-tagatose is thermodynamically equilibrated allowing the shift of the reaction towards the tagatose production when the temperature is increased [6, 7]. Thus, several thermoactive l-AIs have been isolated from thermophilic microorganisms including Thermotoga, Bacillus and Thermus genera [8–10]. However when performed under alkaline conditions, the isomerization has several drawbacks mainly the production of undesirable sub-products [6, 11]. In order to improve the l-AIs suitability for biotechnological applications, many tools have been used such as: the screening of biodiversity to identify relevant enzymes with interesting properties, protein isolation, molecular modeling and rational design [12–14]. In this context, the thermoactivity, the metallic ions requirement and the catalytic efficiency of several enzymes were probed [15, 16]. Recently, new production procedures of d-tagatose were described. We reported the concomitant bioconversion of d-galactose and d-glucose into d-tagatose and d-fructose, respectively. This original procedure was developed through the co-expression of a d-glucose isomerase and an l-arabinose isomerase in E. coli . Another study  described the hydrolysis of lactose by the β-galactosidase from Pichia pastoris to galactose and glucose and the bioconversion of 30 % of d-galactose into d-tagatose with the addition of the l-AI from Arthrobacter sp. While several studies revealed that numerous efforts have been made to set out a low cost procedure for the industrial production of tagatose, these processes remain costly . This is mainly due to the biocatalyst production costs. Indeed, the l-AIs are intracellular enzymes that require not only the development of profitable procedures for their over-expression but also for their extraction and purification. Compared to the intracellular enzymes, the production of extracellular biocatalysts is an attractive alternative to improve the industrial process profitability.
Although the proteins belonging to the isomerase family are intracellular, in this study we report for the first time an efficient secretion in the extracellular medium of the l-AI from Lactobacillus sakei 23 K by the food grade bacterium Lactococcus lactis. We investigated the properties of the secreted protein and its efficacy to bioconvert galactose into tagatose. We also investigated the functionality of the enzyme, the in vivo isomerization of galactose and the cognate anti-hyperglycemic effect of the produced tagatose in mice model.
Results and discussion
l-AI secretion in L. lactis clpP-htrA strain
l-AI is an intracellular protein, which is an efficient bio-converter of galactose into tagatose. This latter property confers to this enzyme family a strong industrial interest. However, the tagatose production remains to be improved mainly in terms of efficiency and process cost. Many efforts have been focused on the improvement of the biochemical properties of l-AI enzymes and their adaptation to industrial processes. However although the extraction and purification of the intracellular l-AIs increase the cost of production of these biocatalysts, the process steps have hardly been addressed. In this context, we investigated whether an intracellular l-AI could be produced in the extracellular medium. We choose L. lactis as the heterologous host for the expression and secretion test because (1) it is a food grade bacterium, (2) it secretes only one detectable extracellular protein (Usp45) , (3) it possesses only two major proteases, namely ClpP (intracellular) and HtrA (extracellular) (4) it has been used for the successful expression and secretion of several proteins of medical and industrial interest and for in situ delivery .
l-AI specific activity determination in L. lactis NZ9000 harboring the secreted and intracellular enzyme forms
Specific activities (U/mg)
Protein crude extract
21 ± 0.2
10 ± 0.3
13 ± 0.8
Bacterial strains and plasmids used in this work
Escherichia coli BL21 strain harboring the pMR36 plasmid
MG1363 (nisRK genes in chromosome), plasmid free
NZ9000 defective for the htrA gene, plasmid free
NZ9000 defective for the clpP gene, plasmid free
NZ9000 htrA clpP
NZ9000 defective for the htrA and the clpP genes, plasmid free
NZ9000 harbouring the psec:LEISS:araA
NZ9000 clpP harbouring the psec:LEISS:araA
NZ9000 htrA harbouring the psec:LEISS:araA
NZ9000 htrAclpP harbouring the psec:LEISS:araA
Plasmid encoding the l-arabinose isomerase encoding gene (araA) from L. sakei 23 K
Vector derived from pGK12 carrying a chloramphenicol resistance gene and the pnis inducible promoter
Vector derived from pGK12 carrying a chloramphenicol resistance gene and the pnis inducible promoter fused to the signal peptide of the usp45 gene
Vector derived from pGK12 carrying a chloramphenicol resistance gene and the pnis inducible promoter in front of the signal peptide of usp45 fused to a sequence encoding the LEISTCDA polypeptide
pCyt carrying the L. sakei araA gene under the control of the pnis promoter
pSec carrying the L. sakei araA gene fused to the signal peptide of usp45and under the control of the pnis promoter
pSec:LEISS carrying under the control of the pnis promoter, the signal peptide of usp45 associated with the LEISSTCDA encoding fragment and fused to the L. sakei araA gene
Effect of the L. lactis mutant strains on the enzyme secretion efficiency
Specific activity (U/mg)
13 ± 0.8
17 ± 0.4
19 ± 0.3
22 ± 0.6
Altogether, the best production levels of the l-AI in L. lactis were obtained (1) in the pellet using the pCYT:araA (21 ± 0.2 U/mg) and (2) directly in the supernatant with the combination of the L. lactis clpP-htrA deficient strain with the pSEC:LEISS:araA expression plasmid (22 ± 0.3 U/mg).
Functional characterization of the secreted l-AI
To study the biochemical properties of the secreted and the intracellular l-AI we purified this protein from L. lactis and E. coli, respectively. The analysis of the purified protein from E. coli and L. lactis by SDS-PAGE and gel filtration chromatography showed that both purified enzymes have a tetrameric arrangement (Fig. 1). These results evidenced that the secreted l-AI monomer has a functional tetrameric arrangement in the culture supernatant. This observation is also supported by the fact that the secreted L. sakei l-AI and that all l-AIs reported so far, are active as homotetramers or homohexamers [7, 23].
In addition, we studied the activity of the two purified protein preparations as function of the temperature and the pH. Both proteins fractions displayed the same optimal temperature (30–40 °C) and optimal pH (5.0–7.0). The kinetic studies demonstrated that the purified l-AI from L. lactis and E. coli had a catalytic efficiency of 65 ± 0.8 and 64 ± 0.2/mM/min for l-arabinose, respectively. These results established that the secreted protein not only have the same tetrameric arrangement as the protein over-expressed and purified from E. coli, but also the same biochemical and kinetic properties. This underlines again the efficiency of the secretion of this l-AI in L. lactis and the relevance of this mode of production for industrial applications. Such procedure constitutes an original promising tool for the secretion of this protein family in this food-grade host microorganism.
The secreted enzyme efficiently bioconverts d-galactose into d-tagatose
As shown in Fig. 3, the conversion rates were not significantly altered when the pH varied both in case of the induced MRS40 culture and of the purified enzyme. Such phenomenon may be explained by the wide range activity of the L. sakei l-AI .
These results highlighted the ability of the induced MRS40 cells and of the purified secreted l-AI to biocatalyze the d-tagatose production even at neutral and low pH. This underlines again the efficiency of the l-AI secretion as a powerful system for the over-production of an active extracellular l-AI enzyme.
The produced d-tagatose in vivo has an antihyperglycemic effect in mice
Here we report the secretion of the first l-AI from L. sakei 23 K, belonging to the isomerase family, in the food-grade L. lactis microorganism. The secreted l-AI from L. sakei 23 K displays the same biochemical and structural features when compared to the l-AI produced intracellularly in E. coli. Moreover, the L. lactis secreted l-AI efficiently bioconverted the d-galactose into d-tagatose in vivo displaying thus an anti-hyperglycemic effect in mice. In the near future we will focus our efforts on the study of the effect of tagatose concentration on the anti-hyperglycemic response. Furthermore, the optimization and/or isolation of new l-AI having high catalytic efficiency and stability will be of interest for the tagatose production at industrial scale. Such features stress the efficiency of the secretion of l-AI as a profitable way to produce active l-AI for industrial applications.
Bacterial strains, media, plasmids and growth conditions
The bacterial strains and plasmids used in this work are listed in Table 2. E. coli was grown in Luria–Bertani medium at 37 °C . L. lactis was grown in M17 medium supplemented with 1 % glucose at 30 °C without agitation. Plasmids were transformed in L. lactis by electroporation . Plasmids were selected by using antibiotics: 5 µg of chloramphenicol per ml for L. lactis, 10 µg of chloramphenicol per ml for E. coli, and 100 µg of ampicillin per ml for E. coli. Nisin was used at final concentration of 10 ng/ml for L. lactis. 1 mM of IPTG was used with E. coli.
PCR and DNA manipulation
Preparation of plasmid DNA was performed using the Miniprep kit (Promega). DNA digestion with restriction endonucleases and separation of fragments in agarose gel electrophoresis were performed as described by Sambrook et al. . Polymerase chain reactions were carried out in a Gene Amp PCR System 9700 (Applied Biosystems). The amplification reaction mixtures (100 µl) contained Phusion High-Fidelity DNA polymerase buffer, 10 pmol of each primer, 50 ng of DNA template, and 10 units of High-Fidelity DNA polymerase (Fermentas). The cycling parameters were 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s and finally 7 min at 72 °C. PCR products were purified using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Bioscience), following the manufacturer’s instructions.
l-Arabinose isomerase molecular cloning strategy
The pMR36 plasmid was used as a template for the amplification of the gene encoding the l-AI from Lactobacillus sakei 23 K (Table 2). Two primers F-araA: AACTGCAGCATTAAATACAGAAAATTATGAATTTTGG and R-araA: GGACTAGTCCTTATTTAATATTGACGTAAGTCAAATC were used to amplify the araA gene flanked by the SpeI and PstI restriction sites. The resulting PCR fragment was purified and then digested with the latter restriction enzymes. The digested fragment was purified and subsequently ligated to the pCYT, pSEC and pSEC:LEISS vectors (to obtain pCYT:araA, pSEC:araA and pSEC:LEISS:araA, respectively) linearized with SpeI and NsiI restriction enzymes (Table 2). The ligation products were then transformed into either L. lactis NZ9000 or L. lactis clpP-htrA strain . Recombinant clones were analyzed by restriction and generated constructions were confirmed by DNA sequencing using an automated DNA sequencer (MWG Eurofins).
Preparation of crude extracts and protein purification
Recombinant L. lactis were grown until OD600 = 0.6 and induction with 10 ng/ml of nisin (Sigma) was performed during 3 h. The cells were harvested and the supernatant was concentrated using a 100 kDa cut-off membrane. The resulting protein fraction was subjected to an anion exchange chromatography (Mono-Q 5/50GL, GE Healthcare). Purification was achieved by a size exclusion chromatography step (S200 column, Amersham Bioscience) using an ÄKTA purifier system (Amersham Biosciences). The used buffer was 100 mM sodium acetate (pH 5.0) and elution fractions were 0.5 ml.
The harvested L. lactis cells were washed twice with 100 mM sodium acetate buffer (pH 5.0) and disrupted by glass beads (diameter of 212–300 µm, v/v, Sigma). Crude cell extract were recovered by centrifugation (30,000×g, 20 min at 4 °C).
Lactobacillus sakei l-AI produced in E. coli strain (MRS36) was over-expressed and purified as previously reported .
Protein quantification and electrophoresis
Protein concentrations were determined using the Bradford method with bovine serum albumin as standard. The purified enzyme samples were migrated in 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli . Protein bands were visualized by Coomassie brilliant blue R-250 (BioRad) staining.
l-AI activity was established by determining the amount of generated l-ribulose or d-tagatose. Under standard conditions, the reaction mixture contained 0.8 mM Mg2+, 0.8 mM Mn2+, 50 μl of enzyme preparation at a suitable dilution; 5 mM of l-arabinose (or d-galactose) and sodium acetate buffer 100 mM (pH 5.0) to bring the final volume to 1 ml. The reaction mixture was incubated at 35 °C during 1 or 10 min for l-arabinose and d-galactose, respectively, followed by the incubation of the samples at 99 °C during 5 min. The generated l-ribulose (or d-tagatose) was measured by the cysteine carbazole sulfuric-acid method, and the absorbance was measured at 560 nm . The d-tagatose production was also confirmed by high-pressure liquid chromatography (HPLC).
One unit of l-AI activity was defined as the amount of enzyme catalyzing the formation of 1 µmol keto-sugar per min under the above-specified conditions.
Circular dichroism experiments
Circular dichroism (CD) measurements were done with chirascan spectropolarimeter (Applied photophysics). Purified proteins were used at concentration of 1 mg/ml. The CD spectra of enzyme samples in a cuvette (0.1 cm) path length were analyzed in the far-UV region comprised between 200 and 280 nm. Scans were collected at 0.1 nm intervals with a 1 nm bandwidth five times. Each spectrum was corrected by subtracting that of the solution containing the used buffer.
Biochemical and kinetic characterization
The effect of temperature on the activity was determined by incubating the purified enzyme at temperatures ranging from 4 to 55 °C, whereas the pH profile was obtained by measuring the activity at various pH values from 3.0 to 8.5 [3.0–5.0 with sodium acetate buffer, 6.0–7.0 with 2-morpholinoethanesulfonic acid (MES) buffer and 7.5–8.5 with Bicine buffer].
Kinetic properties were studied on the basis of Lineweaver–Burk plots. Assays were done in 100 mM sodium acetate buffer (pH 5.0), 0.8 mM Mg2+, 0.8 mM Mn2+ and 1–800 mM substrate (l-arabinose or d-galactose). Samples were incubated at 35 °C and the amount of keto-sugar generated (l-ribulose or d-tagatose) was determined by the cysteine-carbazole-sulfuric acid method .
In vitro d-galactose bioconversion
Induced MRS40 cells and purified secreted l-AI with a final concentration of 109 CFU/ml and 0.3 mg/ml, respectively, were used to produce d-tagatose. Reactions were carried out under different pH values (pH 5.0, 6.0 and 7.0) by using 10 g/l d-galactose. d-tagatose was determined by cysteine carbazole method .
Animal experiments and glycemia measurement
Male C57BL/6 mice (6–8 weeks old) (Janvier, Le Genest 428 Saint Isle, France or Taconic mice New York, USA) were maintained at the animal care facilities of the National Institute of Agricultural Research (IERP, INRA, Jouy-en-Josas, France) under specific pathogen-free conditions. Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules and approved by the Animal Care Committee COMETHEA (Comité d’Ethique en Expérimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, Jouy en Josas, France). Food intake was stopped 6 h before starting experiments.
L. lactis cellular pellets were harvested by centrifugation (3000g, at 4 °C) and washed three times with sterile PBS. The pellet was suspended in PBS to a final concentration of 109 CFU. Groups of mice (n = 10) received a intragastric administration of either PBS, d-galactose (10 g/l), d-tagatose (10 g/l) or d-galactose (10 g/l) with: (1) MRS40 strain carrying the pSEC:LEISS vector, (2) induced MRS40 strain carrying the pSEC:LEISS:araA vector, (3) non-induced MRS40 strain carrying the pSEC:LEISS:araA vector and (4) the purified enzyme (0.3 mg/ml). The administered volume was 0.2 ml for each condition followed by a glucose challenge (1 g/kg body weight). Glycemia measurements were done by using the Accu-Chek performa system (Roche).
The data reported in this work were plotted using Sigma Plot (Version 9.0). Each value represents the mean for three independent experiments performed in duplicate.
MR participated in the design of the study, experiments and writing the manuscript. LB contributed to the molecular biology assays, animal experiments and writing the manuscript. YH performed molecular biology experiments and enzyme assays. SB participated in the molecular biology and biochemical experiments. NG participates in the animal experiments. AG contributed to the biochemical and the molecular biology experiments. JG performed animal experiments. HM contributed to animal experiments. PL participated in the design of the work and helped in drafting the manuscript. EM contributed in the design, results analysis and writing the manuscript. All authors read and approved the final manuscript.
The French National Institute for Agricultural Research (INRA) supported this work. We thanks Dr Michel JUY and Dr Michel BECCHI from “Institut de Biologie et Chimie des Protéines, Lyon” for their appreciated help in the biophysical experiments and mass spectrometry analysis, respectively.
The authors declare that they have no competing interests.
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