The focus of this work was to employ B. coagulans MA-13 as source of enzymes to improve the digestibility and the nutritional value of food containing oligosaccharides indigestible by the human gut as well as to set up an eco-friendly production of prebiotics from diary food waste. A combination of -omic technologies based on mass spectrometry with conventional biochemical approaches has been employed to exploit the applicative potential of B. coagulans MA-13 in these biotechnological contexts.
Screening and identification of the glycosyl hydrolases activities of Bacillus coagulans MA-13
Annotation of glycosyl hydrolases
Whilst a full functional annotation of B. coagulans MA-13 genome is under way (manuscript in preparation), herein we show the annotation of the GH(s) repertoire, using dbCAN2 meta server [22] (Table 1). Seventeen enzymes have been identified, among which some families (GH3, GH15, GH32, GH36, GH42, GH70, GH73) are represented by a single member, whereas all the others include diverse glycosyl hydrolases. A set of GH(s) connected to starch degradation has been identified and includes GH13 and GH65 representatives. This finding is in line with the fact that starch is the major carbohydrate present in beans and B. coagulans MA-13 was isolated from a canned beans manufacturing process [5, 26]. Three GHs members belonging to families 18 and 73 are related to the sporulation pathways of B. coagulans. Few Carbohydrate-Binding Modules (CBMs) were found in GH13 and GH18 members. The presence of a sucrose-6-phosphate hydrolase (GH32) mirrors the ability of B. coagulans MA-13 to grow on an inexpensive sucrose-rich carbon source (molasses) [6]. Finally, GH36 and GH42 members have been identified and interestingly lactic bacteria producing both α- and β-galactosidases are of interest for the food industry [10].
Table 1
Predicted GH in the genome of B. coagulans MA-13. The automated CAZyme annotation has been carried out using dbCAN2 metaserver, integrated with HMMER, DIAMOND and Hotprep databases [22]. The presence of signal peptides in the proteins are reported as: Y = 100%, N = 0%. Abbreviations: Glycosyl hydrolases (GH) and carbohydrate-binding modules (CBM).
NCBI Reference Sequence
|
GH family
|
Signal Peptide
|
RAST
annotation
|
WP_195850490.1
|
GH3
|
N
|
β-glycosyl hydrolase
|
WP_019720988.1
|
CBM34 + GH13_20
|
N
|
Neopullulanase (EC 3.2.1.135)
|
WP_133536160.1
|
GH13_31
|
N
|
Oligo-1,6-glucosidase (EC 3.2.1.10)
|
WP_195850265.1
|
GH13_31
|
N
|
Oligo-1,6-glucosidase (EC 3.2.1.10)
|
WP_133536961.1
|
GH13_5
|
N
|
Glucan 1,4-α-maltohexaosidase (EC 3.2.1.98)
|
WP_195850265.1
|
CBM48 + GH13_9
|
N
|
1,4-α-glucan (glycogen) branching enzyme (EC 2.4.1.18)
|
WP_061575462.1
|
GH0
|
N
|
Phosphorylase b kinase regulatory subunit β
|
WP_133537568.1
|
CBM50 + GH18
|
N
|
Spore cortex-lytic enzyme, N-acetylglucosaminidase SleL
|
WP_133536804.1
|
CBM50 + GH18
|
N
|
spore peptidoglycan hydrolase (N-acetylglucosaminidase) (EC 3.2.1.-)
|
WP_133537667.1
|
GH32
|
N
|
Sucrose-6-phosphate hydrolase (EC 3.2.1.26)
|
WP_133537615.1
|
GH36
|
N
|
α-galactosidase (EC 3.2.1.22)
|
WP_133536219.1
|
GH42
|
N
|
β-galactosidase (EC 3.2.1.23)
|
WP_133536548.1
|
GH65
|
N
|
α,α-trehalose phosphorylase (2.4.1.64)
|
WP_133536158.1
|
GH65
|
N
|
Maltose phosphorylase (EC 2.4.1.8)
|
WP_133536578.1
|
GH65
|
N
|
Maltose phosphorylase (EC 2.4.1.8)
|
WP_133537168.1
|
GH70
|
Y (1–34)
|
hypothetical protein
|
WP_195850162.1
|
GH73
|
N
|
endo-β-N-acetylglucosaminidase (EC 3.2.1.96)
|
Screening of the intracellular and extracellular GH activities
To discover GH enzymatic activities, intracellular cell extracts and secretome of B. coagulans MA-13 were tested on a panel of artificial substrates. Cells were cultivated in LB rich medium with the purpose of detecting a baseline of activities under standard growing conditions. Cultures were collected at exponential growth phase (0.5–0.6 OD600nm) and 0.8 µg of total intracellular and extracellular protein preparations were assayed over the following substrates: PNP-β-glu, ONP-β-glu, PNP-α-glu, PNP-β-xyl, PNP-α-man, PNP-β-man, PNP-β-fuc, PNP-α-fuc, PNP-α-rha, ONP-β-gal, PNP-β-gal, PNP-α-gal, PNP-α-ara.
Intracellular enzymatic activities were revealed only on a subset of substrates, i.e. ONP-β-gal, PNP-β-gal, PNP-α-gal, PNP-α-glu, PNP-α-ara (Fig. 1). By comparing this result with the annotated list of B. coagulans MA-13 GHs, the hydrolytic activity towards PNP-α-glu could be traced back to representative(s) of GH13 family (subfamily 31), whereas the correlation with the activity on PNP-α-ara is not obvious (Table 1). The hydrolysis of β- (ONP-β-gal, PNP-β-gal) and α-galactosidic (PNP-α-gal) linkages might be related to GH42 and GH36 members, respectively (Fig. 1). Indeed, B. coagulans MA-13 genome bears two genes, i.e. locus tag: E2E33_010705 (WP_133536219.1) and locus tag: E2E33_000265 (WP_133537615.1), encoding for a GH42 and for a GH36, respectively (Table 1). As shown in Fig. 1, the specific activity recorded on ONP-β-gal and PNP-α-gal was significantly higher compared to the other substrates tested.
The presence of secreted GHs was verified by testing supernatants on the same substrates and the only relevant activity was detected on PNP-α-gal (Fig. 1). All together, these results indicate that enzymes hydrolyzing β- and α-galactosidic linkages are constitutively over-expressed under standard growing conditions. These enzymes catalyze the hydrolysis of terminally joined galactosidic residues in simple galactose-containing oligosaccharides as well as in complex polysaccharides and have the potential to improve the digestibility of some RFO-containing food and of milk-based products [14, 15].
Identification of the hydrolytic activities through mass spectrometry
To identify the enzymes involved in the hydrolysis of β-galactosidic linkages, cell extracts were analysed through zymography. Active-bands on ONP-β-gal and PNP-β-gal resided in the same upper gel region (not shown). These bands were excised, the proteins were in-gel trypsinized and the peptides were extracted and analysed by LC-ESI-MS/MS. Proteins were identified by using MASCOT search engine to explore the B. coagulans MA-13 protein database and compared to sequences present in a complete annotated database (UniProt) by using BLAST Search Form. The best alignments (minimum E- value) were obtained with proteins from B. coagulans strain 36D1. As expected, α-galactosidase (Uniprot code: G2TQE8) was identified both in intra- and extracellular protein extract together with other unrelated co-migrating proteins. The putative GH42 (Uniprot code: G2TH90) was identified as the only enzyme potentially responsible for the hydrolytic activity on PNP-β-gal, since the other co-migrating proteins/enzymes clearly belonged to unrelated metabolisms (Additional file 1). The only exception might be represented by a GH36 member (Uniprot code: G2TQE8), that, based on CAZy classification, was however not predicted to be active on PNP-β-gal substrate. Then, the presence of this enzyme was likely due to similar migration properties of GH42 in the zymography gel. By a first inspection of proteins identified in correspondence of bands active on ONP-β-gal with at least 2 peptides, no enzymes linked to the hydrolysis of β-1-4 linkages were found. Decreasing the detection threshold up to 1 peptide, β-galactosidase (Uniprot code: G2TQE8) was detected (Additional file 1). Overall, results obtained from enzymatic screening and mass spectrometry analysis indicated the presence of a single enzyme (GH42, accession number: MBF8418755) involved in the hydrolysis of β-linkage. The enzyme specific activity associated to ONP-β-gal and PNP-β-gal was particularly high (Fig. 1), thus suggesting that either the enzyme was over-expressed under basal growth conditions or its specific activity was significantly high. To assess the culture conditions suitable to further increase the expression levels of β-galactosidase, we resolved to analyze the induction profile of this enzyme using a selective medium. By adding 0.1% lactose into a minimal medium (0.1% yeast), a significant increase (∼30-fold) of the β-galactosidase activity was observed (Additional file 2, Supplementary Fig. 1). This result is in line with the fact that most β-galactosidases play a major role in lactose metabolism and this substrate is the best carbon source for inducing the maximum production of β-galactosidase in Gram + and Gram- bacteria [27, 28].
Furthermore, both cell extracts and secretome of B. coagulans MA-13 cells grown in LB medium were tested on PNP-α-gal, since hydrolytic activity on α-linkages was detected inside and outside the cells (Fig. 1). The activity bands of intra- and extra-cellular proteins displayed the same electrophoretic mobility, lying within the 130–180 KDa gel region. As shown by mass spectrometry analysis, the GH 36 enzyme (Uniprot code: G2TQE8) was found in both samples (Additional file 1) suggesting that this enzyme might exert its hydrolytic activity on intracellular and extracellular α-1-6 galactans. The list of intracellular proteins identified through mass spectrometry analysis included also another GH enzyme (namely, an Arabinogalactan endo-β-1,4-galactanase), however, this latter was not found in the annotated B. coagulans MA-13 genome (Additional file 1 and Table 1). The remaining co-migrating proteins identified in the extracellular and intracellular samples were related to other metabolic pathways (Additional file 1).
Analysis of the protein sequence did not highlight any typical signal peptide (Tat or Sec system) at the N-terminus of α-galactosidase through dbCAN database (Table 1), thus raising questions on how this protein is actually secreted and why this enzyme has a dual cellular localization. A reasonable explanation is that B. coagulans MA-13 exploits a leader-less secretion system, namely ESAT-6 Secretion System (ESS) which has been discovered in Firmicutes and Actinobacteria [29–31]. In this system, proteins lacking a canonical signal peptide can be secreted through the combined action of two molecular components, namely EsxA and EsxB. The relative genes are both present in the B. coagulans MA-13 genome (data not shown) and are arranged in a cluster, likewise for other bacteria [29–31]. Moreover, many of the proteins secreted through ESS share some distinguishing and conserved features that include a WXG amino acid motif in the central region of the protein. Interestingly, this motif has been identified in the middle of the sequence (W368 and G370) of the α-galactosidase (Accession number: MBF8416840, 730 aa) as well in the enolase (Uniprot code: G2TP79) which was found extracellularly along with the α-galactosidase (Additional file 1).
To further confirm the presence of this enzyme in the supernatant, B. coagulans MA-13 cells were grown in a minimal medium supplemented with galactomannans (locust bean gum). These are insoluble polymers that cannot be translocated inside cells and bear α-1,6-linkages, thus being natural potential substrates of α-galactosidases. Enzymatic assays carried out on the supernatants using PNP-α-gal, revealed that α-galactosidase was induced (about 4-fold) in the presence of galactomannans compared to the control cells cultivated only in yeast (Fig. 2A). Moreover, the analysis of cell extract indicated that the levels of intracellular and extracellular enzymatic activities were comparable upon the cultivation of cells in the presence of locust bean gum. Conversely, the distribution of α-galactosidase was strongly biased toward its intracellular localization when yeast was used as the only carbon source (Fig. 2A), thus suggesting that the presence of galactose-containing polymer such as locust bean gum in the medium plays a role in the secretion of α-galactosidase. All together, these results, along with the lack of a mannanase gene in B. coagulans MA-13 genome (Table 1), strongly supports the hypothesis that this microorganism can rely solely on the activity of an external α-galactosidase to metabolize these galactomannans. By assaying the supernatants of locust bean gum grown cells through zymography, the α-galactosidase activity was promptly revealed (Fig. 2B) and a similar result was obtained by using other complex carbon sources derived from food waste (not shown). The identification by mass spectrometry of the enzymes responsible of this in-gel activity was hindered by a strong contamination of polymers probably deriving from substrates used for enzymatic assay. However, the electrophoretic mobility of this band (within the 130–180 KDa gel region) was identical to that identified as GH36 (Uniprot code: G2TQE8, Additional file 1) thus indicating that the enzymatic activity revealed by zymography, can be ascribed to the same protein. Besides our experimental evidences, the extracellular localization of the α-galactosidase has been previously described for another closely related B. coagulans strain [18, 32] which was found to be able to grow on galactose-containing polymers (melibiose, raffinose, and stachyose) as well as for other soil microorganisms [33] and for Bacillus megaterium [34]. It is known that galactomannans are present in seeds of beans and in general RFOs (raffinose, stachyose, and verbascose) that contain α 1-6-linked galactose units, are particularly abundant in these legumes [9]. Since B. coagulans MA-13 was isolated from manufactured canned beans, the α-galactosidase might be a key enzyme for the host metabolism along with the β-galactosidase. Indeed, manufacturing bean wastes represent a lactose-free environment, however, other genes encoding GH42 enzymes from prokaryotes are unlikely to encounter lactose, suggesting that the substrate for these enzymes in their natural environment, might be represented also by more complex oligo- and polysaccharides [35].
The experimental evidence of the considerable induction of β-galactosidase expression upon addition of lactose to the growth medium prompted us to analyse the effect of this inexpensive substrate also on the production of α-galactosidase. Indeed, previous studies have described the induction of this enzyme on galactose-containing oligosaccharides or galactose [36]. Then, the enzymatic activity on PNP-α-gal was also measured upon lactose supplementation to the medium and a 2-fold induction was observed (Additional file 2, Supplementary Fig. 1). However, it is not clear whether the true inducer of B. coagulans MA-13 α-galactosidase is lactose or galactose; indeed, the latter might be produced at high intracellular concentration as hydrolysis product of the over-expressed β-galactosidase in the presence of lactose. From an applicative point of view, setting up growth conditions suitable for the expression of both β-and α-galactosidases is of great interest and only few studies have described the production of both enzymes by the same strain [11, 12, 36].
A thoroughly biochemical characterization of a closely related recombinant α-galactosidase from B. coagulans ATCC 7050 (identity percentage 97.4%) has been recently published [18]. Therefore, we focused on the study of the β-galactosidase enzyme, since there is no evidence about the ability of β-galactosidases from other B. coagulans strains to produce GOS upon transglycosylation reactions.
Sequence analysis, cloning and expression of BcGalB
The gene (E2E33_010705) encoding for the putative β-galactosidase (herein named as BcGalB), has been identified within a cluster of genes encoding for a lacI family regulator, an hypothetical Major Facilitator Superfamily Transporter related to multi-drug resistance mechanisms and other small hypothetical proteins. This genetic arrangement is also present in B. coagulans ATCC 7050. Then, the gene is not included in an operon encoding also for a lactose-permease and a transacetylase, likewise the well-known E. coli lac-operon. Specifically, the hypothetical galactose-lactose permease encoding sequence is quite distant (≃7,000 nt) from BcGalB gene, thus suggesting that its expression might not be subjected to the same regulative circuit of lac operon, consisting in the concomitant over-expression of the permease upon exposure of cells to lactose. Accordingly, we did not observe repression of BcGalB in lactose-free medium as described for E. coli; rather, the constitutive expression of the enzyme was quite high under standard growth conditions and the induction fold observed in the presence of lactose was significant but quite low if compared to other systems (Fig. 1) [37].
There is no report about any transcriptional cross-regulation which might account for the genetic proximity between β-galactosidase gene and choline-operon. The only functional connection has been found in a β-galactosidase from Streptococcus mitis, which bears a Choline Binding Domain (CBD) at its C-terminus. However, this β-galactosidase uses CBD domain as an attachment anchor to molecular components (such as lipo-teichoic acids) to bind to cell-wall. Instead, BcGalB has an intracellular localization and therefore this genetic juxtaposition remains murky (Fig. 3) [38]. BcGalB bears three typical domains of the GH42 family as suggested by CD-Search and other reports [39, 40]. E2E33_010705 was amplified by PCR from the genomic DNA of B. coagulans MA-13 and expressed in E. coli Rosetta™(DE3) pLysS cells as soluble, intracellular histidine-tagged protein (C-terminus). The overexpression system and purification method applied were quite efficient, since the enzyme was purified to homogeneity by His-trap affinity chromatography, with an overall yield of about 10 mg for 1 liter of culture with an yield of 82% (Additional file 3, Supplementary Table 1). As revealed by SDS-PAGE analysis (Additional file 2, Supplementary Fig. 2), BcGalB displayed a single band with a molecular mass of ~ 75 kDa. This concurred with the molecular mass of BcGalB deduced from the nucleotide sequence of the E2E33_010705 gene and the identity of the protein was verified by mass spectrometry (data not shown). The recombinant protein was analyzed by size-exclusion chromatography coupled with a triple-angle light scattering QELS. This analysis revealed that BcGalB is a hexamer in solution (not shown). Since seven cysteines are present on BcGalB sequence, the enzyme was analyzed on SDS-PAGE in the presence of β-mercaptoethanol as a reducing agent (Additional file 2, Supplementary Fig. 2). BcGalB was present only in monomeric form under this condition, thus pointing to the role of at least some of the cysteines in the oligomerization state. It is worth noting that β-galactosidases can be found in diverse oligomeric forms, such as dimeric (halophilic Haloferax alicantei [41]), trimeric (thermophilic Geobacillus stearothermophilus [42]), tetrameric (acidophilic archaeon Sulfolobus solfataricus [43]) and hexameric (hyperthermophilic Thermotoga maritima [44]) arrangements. This latter structure is uncommon among thermophilic GH42 members, whereas some GH2 β-galactosidases exhibit this supramolecular organization. At the best of our knowledge, the correlation between the hexameric structure and biochemical features of β-galactosidases is not obvious although a general correlation between oligomeric states and thermal stability has been proposed for thermophilic enzymes [23, 45].
Characterization and stability properties of BcGalB
The influence of pH and temperature on the enzymatic activity was evaluated using ONP-β-gal as a substrate. Upon testing the enzyme in the interval 4.0–10.0, the optimal pH was set at 5.0 (Fig. 4A). Interestingly, BcGalB retained 70% of its activity from 5.0 to 7.0 whilst a sharp decrease was observed at pH 4.0 (Fig. 4A). Despite this drop at acidic pH values, it is worth noting that the enzyme exhibited a relevant stability at different pH values, ranging from acidic to alkaline ones.
As shown in Fig. 4B, in the pH range from 5.0 to 7.0 the enzyme retained more than 70% of its activity up to 24 hours (Additional file 2, Supplementary Fig. 3). The exploitation of β-galactosidases in the dairy industry is related to the optimal pH for hydrolysis [46]. Lactose is a hygroscopic sugar exhibiting low solubility that might cause crystallization as well as technological issues for certain products in the dairy industry. The solubility and sweetness can be increased by the lactose hydrolysis into the two glucose and galactose units [47]. In this context, BcGalB might be successfully employed in slightly acid and/or sweet whey hydrolysis, because of its activity and stability in a wide range of pH values.
From the industrial point of view, the enzyme should be stable both at low (preventing the proliferation of microorganisms and nutrients in milk) and at high temperatures (pasteurization) [47]. The dependence of BcGalB from temperature was studied and the maximal activity was found at 60 °C (Fig. 4C) that is quite similar to that of β-galactosidases from other B. coagulans strains [19, 39, 48]. Moreover, BcGalB exhibited high stability at a temperature of 50 °C given that approximately 60% of its initial activity was retained after incubation up to 24 hours (Fig. 4D). Moreover, the half-life at its optimal temperature was 4 hours (Fig. 4D). The loss of activity at 60 °C is counterbalanced by the high specificity activity of BcGalB (i.e. about 4300 U/mg, Additional file 3, Supplementary Table 1) meaning that the catalytic performance of the enzyme is still consistent for an efficient hydrolysis at high temperature by employing small quantities of protein. Interestingly, the thermophilic nature and thermal stability of BcGalB can be exploited in the production of lactose-free dairy products by coupling the thermization to the hydrolysis of lactose preventing microbial contamination, decreasing viscosities of the substrate solution and reducing the cost of the whole process [47].
Finally, enzymes employed in the preparation of lactose-free products are positively selected for their relatively high activity at neutral pH and stability at low temperature [47]. In this regard, BcGalB might be a good candidate not only because of its high specific activity at neutral pH but also for its stability at 4 °C up to several months (not shown).
Effects of metal ions and monosaccharides on BcGalB activity
It is well known that ions affect the catalytic performance of β-galactosidases. For instance, the activity of yeast enzymes isolated from Kluyveromyces lactis and K. fragilis depends on the presence of Mn2+ or Na+, and Mn2+, Mg2+, K+, respectively [49]. Moreover, some metal ions such as Ca2+, Mg2+ and Mn2+ can act as cofactors for β-galactosidases and their presence might significantly enhance their activities. Finally, it has been reported that Ca2+ and heavy metals inhibit the enzyme activity of several β-galactosidases. For examination of the metal ion requirements, BcGalB was assayed in the presence of 1 mM mono- and divalent ions after dialysis of the enzyme in 10 mM EDTA. Results from this study were overall in agreement with former analyses conducted on other B. coagulans β-galactosidases (Additional file 2, Supplementary Fig. 4) [19, 39, 48]. Worth mentioning is the negligible effect of Ca2+ on the activity up to 2 mM of BcGalB thus allowing its use in dairy-industries processes since Ca2+ is one of the prime elements in milk. Cu2+ is the only ion affecting the enzyme activity (60% reduction), as reported for other β-galactosidases. Indeed, some metal ions, such as Fe3+ and Cu2+, could inactivate the enzyme by inducing structural changes upon interaction with the protein [50, 51].
In order to foresee the employment of BcGalB in the manufacturing of lactose-free products, the effect of galactose and glucose on enzyme activity was also studied. The inhibitory effect exerted by the lactose hydrolysis products on BcGalB activity seems different from previous studies since glucose affected the BcGalB enzymatic activity more than galactose (Fig. 5). Moreover, since lactose hydrolysis produces equimolar amounts of the two sugar units, we resolved to investigate the combined influence of galactose and glucose. A stronger decrease of the enzymatic activity was observed especially at high concentration of the sugars although the effect is not additive. Furthermore, xylose and arabinose were included in these experiments since the former is an acceptor of transgalactosylation reactions whereas the latter is one of the substrates of BcGalB although the specific activity towards PNP-α-ara is lower than on ONP-β-gal (see below). These two monosaccharides had a minor effect on the enzymatic activity compared to galactose and glucose, since BcGalB retained at least 66% of the activity at the highest concentrations tested (Fig. 5). Finally, as part of the general biochemical characterization of BcGalB, the effect of surfactants (SDS and Tween 20), reducing (DTT and β-mercaptoethanol) and chaotropic (urea and guanidine chloride) agents, was studied. The enzyme activity significantly decreased only in the presence of SDS whereas it retained at least 65% of the relative activity when tested with all the other agents (Additional file 3, Supplementary Table 2).
Catalytic properties of BcGalB
The hydrolytic activity of BcGalB was tested on different ortho- or para-nitrophenyl synthetic glycosides as well as on natural polysaccharide substrates and specificity of the enzyme was determined by carrying out individual reactions with each of the compounds as indicated in Material and Methods section. As shown in Table 2, the highest specific activity was recorded on ONP-β-gal, whereas the enzyme performed less efficiently on para-substituted substrates. As shown in Fig. 1 analysis of the intracellular cell extract revealed the presence of enzyme(s) able to hydrolyze PNP-α-ara. Interestingly, a lower but still significant activity of BcGalB was found on PNP-α-ara suggesting that the enzyme is endowed with an ancillary activity on a different substrate. Then, the observed enzymatic activity in the cell extract on PNP-α-ara can be traced back, at least in part, to BcGalB (Fig. 1 and Table 2). This accessory activity is surprising, since it has never been described for other thermophilic GH 42 β-galactosidases and it will be a matter of further investigation [52]. Some β-galactosidases can support the growth of environmental microorganisms from hot springs, soils and hypersaline sites where lactose is not present; rather, plant biomasses are preferential carbon and energy sources. Since B. coagulans MA-13 was isolated from beans processing waste, it is conceivable that BcGalB can be involved also in the hydrolysis of arabino-derived oligosaccharides in vivo.
Lactose, which is the natural substrate for most β-galactosidases, is translocated inside cells through specific lactose-transporters [53]. Therefore, the hydrolytic performance of BcGalB on this substrate was also studied and the specific activity was found to be 1283 U/mg, which is a quite high value compared to β-galactosidases from other B.coagulans strains [19, 39, 48].
Table 2 Substrate specificity of BcGalB. The highest activity of BcGalB is toward ONP-β-gal, whilst the enzyme is not active on the arylic compounds PNP-β-xyl and PNP-β-glu. These latter two substrates are shown since they have been used in transglycosylation reactions (see below). Abbreviation: not detected (N.D.).
Substrate
|
Specific activity (U/mg)
|
ONP-β-gal
PNP-β-gal
PNP-α-ara
PNP-β-xyl
PNP-β-glu
D-lactose
|
4373.4 ± 77.6
795.9 ± 3.9
328.5 ± 11.6
N.D.
N.D.
1283.0 ± 24.7
|
The kinetic parameters of BcGalB were evaluated using both the preferred artificial substrate and lactose under standard reaction conditions (Table 3). Results of this analysis highlighted that BcGalB showed the highest affinity towards ONP-β-gal (KM = 0.72 mM) and interestingly this value is among the lowest determined so far among mesophilic and thermophilic β-galactosidases [14, 40, 54]. Moreover, even among closely related β-galactosidases from other B. coagulans strains, BcGalB displays the highest affinity towards this substrate [19, 39, 48].
Table 3
Kinetic parameters of BcGalB. KM, kcat and kcat/KM values towards the natural and artificial substrates are reported. Standard deviations were lower than 2% of the calculated values.
Substrate
|
KM (mM)
|
kcat (s− 1)
|
kcat/KM (mM− 1 s− 1)
|
ONP-β-gal
D-lactose
|
0.723
136.2
|
5466.7
1603.7
|
756.2
11.8
|
Interestingly, the enzymatic activity on lactose was not affected by Ca2+ and even a slight increase (114%) was recorded (data not shown). The KM was found to be higher than for the artificial substrate; however, previous studies have revealed that most GH42 β-galactosidases prefer to hydrolyze chromogenic substrates while showing weaker lactose hydrolysis activity. Although GH2 β-galactosidases perform better than GH42 representatives on lactose hydrolysis, BcGalB exhibits a significant specific activity toward lactose [19, 39, 48, 55]. Accordingly, B. coagulans MA-13 is able to grow on lactose by over-producing BcGalB (Additional file 2, Supplementary Fig. 1), whereas several prokaryotes possessing a GH42 gene are unable to utilize this substrate [35]. This indicates that BcGalB can sustain the host metabolism through hydrolysis of either lactose or more complex oligosaccharides.
Transgalactosylation activity of BcGalB
To study whether BcGalB was endowed with transgalactosylation activity, the artificial substrate ONP-β-gal was tested in auto condensation reactions. TLC analysis revealed the synthesis of transgalactosylation products already after 10 minutes of reaction (Fig. 6A, lane S 10) and additional signals were clearly visible after 20 minutes (Fig. 6A, lane S 20), demonstrating that in the early stages of the reaction the donor was promptly consumed in favor of synthesis of transgalactosylation products (lower red circles). Most importantly, these compounds were not hydrolyzed by BcGalB up to 18 hours (Fig. 6A, lane S ON) although the addition of fresh BcGalB to the transgalactosylation mixture, caused their complete hydrolysis (data not shown). Then, the persistence of the transgalactosylation products up to 18 hours can be due to the combined effect of partial inactivation of BcGalB occurring after 4 hours at 60 °C (Fig. 4D) and of the inhibitory effect on the enzymatic activity due to D-galactose accumulation (Fig. 5).
BcGalB is also able to catalyze the synthesis of hetero-oligosaccharides when ONP-β-gal was used as a donor whereas PNP-β-glu and PNP-β-xyl were the acceptors. Since none of these latter two glycosides were substrates of BcGalB (Table 2), the activity of the enzyme could be followed under standard conditions. TLC analysis showed the synthesis of transgalactosylation products whose migration properties were apparently similar to those found in homo-condensation reactions (Fig. 6B and C). However, in the reaction with ONP-β-glu and PNP-β-xyl additional signals (highlighted in blue, Fig. 6B and in green Fig. 6C) that can be traced back to the formation of hetero-oligosaccharides, were detected.
The transgalactosylation products were analysed by ESI-MS (Table 4) after carrying out all the reaction for 18 hours. In all spectra, the galactose as the product of the hydrolytic activity of the β-gal enzyme was detected, as well as the presence of the substrate(s) (ONP-β-gal, ONP-β-glu, PNP-β-xyl). From the mass analysis of the sample mixture reported in Fig. 6A (ONP-β-Gal was the substrate) three different products were present, consisting in the addition from 1 up to 3 Gal units to ONP-β-Gal (Fig. 6A, Table 4), as shown by TLC separation (lower red circles). The ESI-MS analysis of sample containing ONP-β-Gal and PNP-β-Glu as donor and acceptors, respectively showed the presence of two transgalactosylation products (Fig. 6B, blue circles) counting a mass increasing of 1 or 2 esose (Gal/Glu) in respect to the initial isobaric substrates indicating the synthesis of di- and trisaccharides (Fig. 6B, Table 4). These reaction products could be due to both homo- and hetero-condensation of glucose and galactose molecules; unfortunately, they were not distinguishable by ESI-MS analysis, having the same molecular weight (Table 4). In the sample reported in Fig. 6C (containing ONP-β-Gal/PNP-β-Xyl as donor and acceptor, respectively) 4 different products were detected by ESI-MS: 2 molecules deriving from homo-condensation reaction, presenting 1 or 2 Gal molecules added to the ONP-β-Gal, and 2 molecules coming from hetero-condensation reactions, containing 1 xylose and 1 or 2 Gal units (Fig. 6C, Table 4).
Since from a biotechnological perspective, the ONP-β-gal is useless as a donor in industrial processes, the natural, plentiful and inexpensive substrate lactose was used as the glycosyl donor and acceptor in the synthesis of glycoconjugates. A high initial lactose concentration of 160 mM was chosen to enhance GOS synthesis over hydrolysis. TLC analysis revealed the appearance of hydrolysis products as well as of several GOS signals already after 10 minutes of incubation (Fig. 6D, lanes SD0 to SD ON, S10 and S ON). This result indicates that BcGalB is able to produce GOS at the expenses of lactose hydrolysis in a short time range. As the reaction proceeded, lactose was consumed, glucose and galactose were formed upon lactose hydrolysis, but we did not observe a concurrent increase of GOS amounts at least as judged by the intensity of the spots (Fig. 6D, lanes S10-S ON). This indicates that the two reactions reached a dynamic equilibrium in which GOS production reached a plateau before lactose was completely hydrolyzed. As judged by ESI-MS analysis, a transgalactosylation product consisting probably of a lactose molecule increased of a galactose unit (m/z value of 527.2 Table 4, Fig. 6D) was detected (S ON) along with the signal corresponding to the substrate D-lactose.
Table 4
Transgalactosylation products identified by ESI-MS. All components were detected as adducts with Na+. The observed and theoretical molecular weights are reported. The ESI-MS analysis can not distinguish between the epimer Gal and Glu, which are reported as alternatives in the interpretation of MS spectra obtained with the couple ONP-β-Gal/PNP-β-Glu: ONP-β-Gal as acceptor and donor, respectively.
Acceptor:Donor
|
Transgalactosylation products
|
MNa+Theoretical (Da)
|
MNa+observed (Da)
|
(ONP-β-Gal: ONP-β-Gal)
|
1. (ONP-β-Gal + Gal) Na+
|
486.408
|
486.142
|
2. (ONP-β-Gal + 2 Gal) Na+
|
648.565
|
648.205
|
3. (ONP-β-Gal + 3Gal) Na+
|
810.722
|
810.277
|
(ONP-β-Gal/PNP-β-Glu: ONP-β-Gal)
|
1. (ONP-β-Gal/PNP-β-Glu + Gal/Glu) Na+
|
486.408
|
486.150
|
2. (ONP-β-Gal/PNP-β-Glu + 2Gal/2Glu) Na+
|
648.565
|
648.224
|
(PNP-β-Xyl:ONP-β-Gal)
|
1. (ONP-β-Gal + Gal) Na+
|
486.408
|
486.159
|
2. (ONP-β-Gal + 2Gal) Na+
|
648.565
|
648.195
|
3. (PNP-β-Xyl + Gal) Na+
|
456.382
|
456.146
|
4. (PNP-β-Xyl + Gal + Gal) Na+
|
618.539
|
618.211
|
(D-Lactose:D-Lactose)
|
1. (Lactose + Gal) Na+
|
527.477
|
527.202
|
All together these data indicate that the thermophilic BcGalB is effective in the production of GOS from lactose. Moreover, lactose solubility in water is rather low in comparison to other carbohydrates; then, reaching lactose concentration high enough to favor transgalactosylation reactions is a difficult task. Since lactose solubility increases exponentially with temperature, GOS synthesis can benefit from carrying out reactions with thermostable enzymes and thermophilic microorganisms.