Patent Description:
For the enzymatic hydrolysis of milk and whey products, numerous processes with soluble and immobilized enzyme preparations have been described. If a soluble enzyme preparation is used, lactose is usually hydrolysed batchwise after pasteurization. Hydrolysis overnight at low temperatures is preferred, as this is more practicable than hydrolysis for e. <NUM> hours at <NUM>, where the milk must be processed immediately after hydrolysis.

In the production of UHT (ultra-high temperature processed) milk, enzymes may be added aseptically after heat treatment. However, the requirements for enzyme purity are higher and the enzyme is present in active form in the end product.

Since the enzyme costs are critical for these processes, various immobilization methods with beta-galactosidases have been investigated. In Italy, lactose hydrolysis in milk has long been described with immobilized beta-galactosidase.

In addition to enzymatic reduction of the lactose concentration in milk and whey products, there is also the possibility of selective chromatographic separation of lactose. The world market leader in lactose-free dairy products has been using a combined process, using both enzymatic hydrolysis and selective chromatographic separation of lactose from milk to produce lactose-free milk.

In earlier studies, a new industry-relevant galactosidase has already been found in metagenome studies. A metagenome library of <NUM> million clones was screened for new beta-galactosidases using a three-stage activity-based screening. Six metagenome beta-galactosidases showed improved lactose hydrolysis in milk compared to the commercial enzyme preparation GODO-YNL2 from Kluyveromyces lactis. The best candidate beta-galactosidase M1 (beta-galactosidase 3J33 as described in <CIT>) showed a higher affinity to the substrate lactose and a lower product inhibition to the resulting galactose than the commercial enzyme preparation.

Beta-galactosidases from Paenibacillus thiaminolyticus and Paenibacillus barengoltzii have already been described in this respect. The beta-galactosidase from P. thiaminolyticus had a very low affinity to the substrate lactose. The KM,lactose value was <NUM> ± <NUM>M. However, the determination was carried out at pH <NUM> and at a temperature of <NUM>. For the beta-galactosidase PbGal2A from P. barengoltzii, the KM,lactose value of <NUM> ± <NUM>M was significantly higher.

Thus, although a considerable number of studies have been published investigating beta-galactosidases from different sources for lactose hydrolysis, only a small number of enzyme preparations, mainly obtained from the yeasts K. lactis and K. fragilis, are applied for industrial applications. However, the industrially used enzymes possess serious disadvantages.

The beta-galactosidases utilised in the food industry are significantly inhibited by the hydrolysis-generated product D-galactose whereas the substrate affinity to the substrate lactose is not favourable (i.e., the KM-value for lactose is too high (><NUM>M)). As a result, the enzymatic hydrolysis of lactose in food requires a correspondingly large amount of enzymes (cost factor) and the time required for effective lactose hydrolysis is high (time factor). The latter entails a high risk of contamination of the process.

In addition to their hydrolytic activity, beta-Galactosidases also can have a transgalactosylation activity. High transgalactosylation activity is essential to production of galacto-oligosaccharides (GOS) which have several health benefits and are recognized as prebiotics. Thus, the production of GOS is of great interest.

In this context, <CIT> describes a method of producing milk products containing GOS at low temperature using beta-galactosidase enzymes having transgalactosylation activity. Further, Uniprot database entry A0A098MAF9 shows the amino acid sequence of Paenibacillus wynnii beta-galactosidase.

In summary, the technical problem underlying the present invention is the provision of beta-galactosidases for use in the production of lactose-depleted and lactose-free and GOS-enriched dairy products which are characterized by low product inhibition, high substrate affinity and high enzyme activity.

The solution to the above technical problem is achieved by the embodiments characterized in the claims.

In particular, in a first aspect, the present invention relates to the use of an enzyme having beta-galactosidase and transgalactosylation activity in the production of (i) lactose-depleted or lactose-free, and (ii) galacto-oligosaccharide-enriched dairy products, wherein said enzyme is derived from Paenibacillus wynnii, and wherein said enzyme comprises (a) the amino acid sequence of SEQ ID NO: <NUM>; or (b) an amino acid sequence having at least <NUM>% sequence identity to SEQ ID NO: <NUM> and having beta-galactosidase and transgalactosylation activity.

As used herein, the term "enzyme having beta-galactosidase activity" relates to a glycoside hydrolase enzyme that catalyses the hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides through the breaking of a glycosidic bond.

The enzyme having beta-galactosidase and transgalactosylation enzyme activity used in the present invention is either (i) the P. wynnii beta-galactosidase having the amino acid sequence of SEQ ID NO: <NUM>, the advantageous use of which in the production of lactose-depleted or lactose-free dairy products, as well as in the production of GOS-enriched dairy products, has been discovered in the present invention, or (ii), in the case of an enzyme having at least <NUM>% sequence identity to SEQ ID NO: <NUM> and having beta-galactosidase and transgalactosylation activity, a respective beta-galactosidase derived therefrom.

The term "lactose-depleted dairy product" as used herein relates to a dairy product whose lactose content has been reduced with respect to its original lactose content. In specific embodiments, the lactose content has been reduced by at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%.

Furthermore, the term "lactose-free dairy product" as used herein relates to a dairy product no longer containing any lactose, or containing lactose to a maximum of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% (w/v).

The terms "galacto-oligosaccharide-enriched dairy product" or "GOS-enriched dairy product" as used herein relate to a dairy product whose GOS content has been increased with respect to its original GOS content. In specific embodiments, a relative GOS yield of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, up to e.g. <NUM>%, can be achieved. Further, in specific embodiments, the GOS content in GOS-enriched dairy products according to the present invention is increased by a factor of at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or more. Specific GOS contents in GOS-enriched dairy products according to the present invention are for example at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, at least <NUM>/L, or more.

According to the present invention, the production of GOS-enriched dairy products, i.e., the increase of the GOS content in the dairy products, is achieved by way of the generation of GOS by the enzyme having beta-galactosidase activity.

In this context, the terms "galacto-oligosaccharide" and "GOS" as used herein relates to oligosaccharides that are produced from lactose by a beta-galactosidase, having <NUM> to <NUM> saccharide units, with the terminal saccharide being glucose and the remaining saccharide units being galactose. The linkages within the GOS molecule are beta-(<NUM>→n) linkages, whereas n can be <NUM>, <NUM>, <NUM> and <NUM>. GOS display both structural and functional similarities with human breast milk oligosaccharides and, due to these similarities, are used as prebiotic in baby food.

The term "dairy products" as used herein is not particularly limited and includes a type of food produced from or containing the milk of mammals, preferably cattle, water buffaloes, goats, sheep, camels, and humans. In preferred embodiments, the dairy product is milk, preferably bovine milk.

The term "enzyme comprising an amino acid sequence having at least <NUM>% sequence identity to SEQ ID NO: <NUM> and having beta-galactosidase and transgalactosylation activity" relates to polypeptides that can comprise any number of amino acid substitutions, additions, or deletions with respect to the amino acid sequence of SEQ ID NO: <NUM>, provided that the resulting polypeptides fulfil the requirement of having at least <NUM>% sequence identity to SEQ ID NO: <NUM> and retain biological activity of a beta-galactosidase and transgalactosylase. In this context, the term "retains the biological activity of a beta-galactosidase" as used herein relates to polypeptides that have at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, preferably at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, <NUM>%, or more than <NUM>% of the activity of the P. wynnii beta-galactosidase having the amino acid sequence of SEQ ID NO: <NUM>, as determined in a standard beta-galactosidase activity assay known in the art.

While the number of amino acid substitutions, additions, or deletions is generally only limited by the above proviso concerning the sequence identity and biological activity of the resulting polypeptide, it is preferable that the resulting polypeptide has at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% sequence identity to the amino acid sequence of SEQ ID NO: <NUM>.

Means for determining the sequence identity of an amino acid sequence to a reference sequence are known in the art.

In a second aspect, the present invention relates to a method for the production of a (i) lactose-depleted or lactose-free, and (ii) galacto-oligosaccharide-enriched dairy product, comprising the step of incubating a dairy product with an enzyme having beta-galactosidase and transgalactosylation activity as defined for the uses of the present invention under conditions and for a duration of time suitable to hydrolyse lactose present in said dairy product and to generate galacto-oligosaccharides in said dairy product.

In this second aspect, all relevant definitions and limitations indicated above for the uses of the present invention apply in an analogous manner. In particular, the dairy products and the enzyme having beta-galactosidase and transgalactosylation activity are as defined above.

Means for reducing the lactose content in dairy products by way of using an enzyme having beta-galactosidase activity, as well as respective reaction conditions and incubation durations are not particularly limited and are known in the art. In specific embodiments, the methods of the present invention are performed at a temperature of <NUM> to <NUM> and for a duration of <NUM> to <NUM> hours.

Similarly, means for increasing the GOS content, i.e., means for generating GOS in dairy products by way of using an enzyme having beta-galactosidase and transgalactosylation activity, as well as respective reaction conditions and incubation durations are not particularly limited and are known in the art. In specific embodiments, the methods of the present invention are performed at a temperature of <NUM> to <NUM> and for a duration of <NUM> to <NUM> hours.

In preferred embodiments, the lactose content in the lactose-depleted or lactose-free dairy product is at most <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% (w/v).

In the present invention, a beta-galactosidase derived from P. wynnii was produced recombinantly and characterized in detail. Surprisingly and advantageously, this beta-galactosidase is characterized by (i) a high affinity to its substrate lactose, (ii) a lack of product inhibition by galactose, (iii) a high enzyme activity, and (iv) an activity of generating GOS.

Specifically, by cultivating a recombinant strain, an active beta-galactosidase could be provided. The beta-galactosidase showed a high affinity to the substrate lactose, with a KM,lactose-value of <NUM> ± <NUM>M. The influence of galactose was investigated in Novo buffer (i.e., a buffer which has a similar ion concentration to milk) at <NUM>. No inhibition of beta-galactosidase up to a concentration of <NUM>M could be detected. During the hydrolysis of lactose in milk with an enzyme activity of <NUM> nkatlactose, <NUM>/mLmilk, <NUM> % of the lactose had been hydrolysed after <NUM> (<NUM>/L). Already after <NUM> no more lactose could be detected. During hydrolysis using the industrial enzyme Opti-Lactase (with the same enzyme activity), the lactose content was <NUM> times higher (<NUM>/L) after <NUM>. After <NUM> a lactose content of <NUM>/L could still be detected. During hydrolysis, the synthesis of galacto-oligosaccharides (GOS) was qualitatively investigated. While Opti-Lactase produced hardly any GOS and only low-molecular saccharides, beta-galactosidase from P. wynnii synthesized higher-molecular GOS.

For the hydrolysis of lactose in milk to be effective, the affinity of the enzyme to the substrate lactose must be high, i.e., the KM value must be low. The beta-galactosidase from P. wynnii used in the present invention possesses a lower KM value than the beta-galactosidases known in the art and used as technical enzyme preparations (cf. Table <NUM>). In this context, the beta-galactosidase designated as "Metagenome M1" in the following table, as well as elsewhere herein, denotes metagenome beta-galactosidase 3J33 as described in <CIT>.

In addition the beta-galactosidase from P. wynnii used in the present invention is not inhibited by the hydrolysis-generated product galactose which leads to a more effective hydrolysis of lactose (cf. Table <NUM>).

During lactose hydrolysis in milk (scale of ~<NUM>), which was carried out at <NUM> to <NUM> using beta-galactosidase from P. wynnii according to the present invention and Opti-Lactase (K. lactis) over a period of <NUM>, lactose was degraded significantly faster using beta-galactosidase from P. After <NUM>, <NUM> % had been hydrolysed. This corresponded to a concentration of <NUM>/L. After <NUM> no more lactose could be detected by HPLC (detection minimum <NUM>/L). In comparison to this, using Opti-Lactase, after <NUM> a ten times higher lactose concentration was present (<NUM>/L). After <NUM> a lactose concentration of <NUM>. <NUM>/L could be determined (cf. the Examples, below).

The present invention discloses the following amino acid sequences:.

The present invention will be further illustrated by the following example without being limited thereto.

The construction of the expression vector for the beta-galactosidase from Paenibacillus wynnii was based on the genomic DNA sequence of the beta-galactosidase from Paenibacillus wynnii DSM18334 (European Molecular Biology Laboratory: EMBL coding: KGE19535. <NUM>; SEQ ID NO: <NUM>) available at the EMBL, European Bioinformatics Institute. One nucleotide was exchanged for cloning, but this did not result in an amino acid exchange. In general, standard molecular biology methods according to Sambrook and Russell (<NPL>) were used. Cloning in pET-20b (+) was followed by transformation in E. coli XL-<NUM> blue cells to introduce a C-terminal His6-tag in frame, resulting in the construct pET-20b-P. For the expression of the P. wynnii beta-galactosidase, the construct was transformed in E. coli BL21(DE3) cells.

coli BL21(DE3)-pET20b-P. -beta-gal was cultivated in 2YTAmp medium (<NUM> L-<NUM> tryptone, <NUM> L-<NUM> yeast extract, <NUM> L-<NUM> NaCl containing <NUM> µg mL-<NUM> ampicillin concentration) with <NUM> % glucose (w/v) in a bioreactor with <NUM> operating volume. Before inoculating the bioreactor different precultures were prepared. A single colony of E. coli BL21(DE3)-pET20b-P. -beta-gal was picked from a LB agar plate and transferred into a prepared test tube containing <NUM> 2YTAmp medium with <NUM> % (w/v) glucose and incubated on an orbital shaker at <NUM> rpm for <NUM> (preculture I). Preculture I was transferred after <NUM> into a baffled flask containing <NUM> 2YTAmp medium and <NUM> % (w/v) glucose (preculture II). Preculture II was incubated on an orbital shaker at <NUM> rpm for <NUM> before it was added to a baffled flask containing 2YTAmp medium with <NUM> % (w/v) glucose (preculture III). After incubation for <NUM> on an orbital shaker (<NUM> rpm) preculture III was added sterilely to <NUM> 2YTAmp medium with <NUM> % (w/v) glucose that was prepared in a bioreactor. The following parameters were chosen for the cultivation: <NUM> rpm, air gassing vvm = <NUM>, and pH <NUM>. The pH was adjusted using <NUM> M NaOH and <NUM> M H<NUM>PO<NUM>. The cells were cultivated at <NUM> up to an OD<NUM> of <NUM>. For the expression of the recombinant protein the cells were cooled down to <NUM> and induced with <NUM>M isopropyl-beta-D-<NUM>-thiogalactopyranoside (IPTG). After further <NUM> cultivation at <NUM> the cells were harvested using a continuous working centrifuge (CEPA Rapid centrifuge Type GLE, flow rate <NUM> min-<NUM>, <NUM>,<NUM>, <NUM>). The harvested cells were washed twice with saline and centrifuged for <NUM> at <NUM>,<NUM> and <NUM>.

<NUM>M potassium phosphate buffer with <NUM>M MgCl<NUM> (pH <NUM>) was added to the cells to prepare a <NUM> % (w/v) cell suspension. The cells were disrupted on ice using a sonificator (cycle <NUM>, amplitude <NUM> %). Cell disruption was carried out in ten cycles of one-minute pulsed intervals followed by one-minute breaks. After disruption the cell suspension was centrifuged for <NUM> at <NUM> g and <NUM>. The cell pellet was discarded and the enzyme in the supernatant was used for further experiments.

The recombinant P. wynnii beta-galactosidase with a hexa histidine-tag was purified by nickel affinity chromatography (Biofox <NUM> IDALow XK16, <NUM>, Knauer, Berlin). The disrupted cells were centrifuged for <NUM> at <NUM> and <NUM>,<NUM>. The supernatant was centrifuged again for <NUM> at <NUM> and <NUM>. Afterwards, the supernatant was filtrated (<NUM> µm) and <NUM> of the crude extract were injected into an ÄKTA FPLC system (GE Healthcare, Freiburg). The column was previously equilibrated with <NUM> CV binding buffer. The conditions for the purification of P. wynnii beta-galactosidase are listed in Table <NUM>. The eluted proteins were detected by an UV detector at λ = <NUM> and fractionated in <NUM> fractions. The fractions in which an UV signal was detected were pooled. Size exclusion chromatography columns (PD10 columns, GE Healthcare, Freiburg) were used to change the buffer to <NUM>M potassium phosphate buffer (pH <NUM> containing <NUM>M magnesium chloride).

Binding buffer: <NUM>M potassium phosphate buffer containing <NUM>M NaCl Elution buffer: <NUM>M potassium phosphate buffer containing <NUM>M NaCl and <NUM> imidazole.

The beta-galactosidase activity was determined using either the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside (oNPGal) or the natural substrate of the enzyme lactose.

The enzyme activity of beta-galactosidase was determined in <NUM> scale using oNPGal as substrate at <NUM>. For the assays a final concentration of <NUM>M oNPGal dissolved in <NUM>M potassium phosphate buffer (pH <NUM> containing <NUM>M magnesium chloride) was used. Substrate and enzyme solution were preheated separately. The reaction was performed in a temperature-controlled cuvette using a spectrophotometer by adding enzyme solution to oNPGal solution. The increase of absorbance at <NUM> as result of o-nitrophenol release was measured for <NUM>. The activity was calculated from the slope of the straight line.

One nanokatal is defined as the amount of enzyme that catalyses the release of <NUM> nmol of o-nitrophenol from oNPGal per second. The amount of the released o-nitrophenol was determined using a calibration curve (range: <NUM>-<NUM>M o-nitrophenol).

The beta-galactosidase activity was determined using lactose as a substrate. Beta-galactosidase hydrolyses the beta-<NUM>,<NUM>-glycosidic bond in lactose and release D-glucose and D-galactose.

For the substrate solution <NUM>M lactose was dissolved in Novo buffer (the composition of which is depicted in Table <NUM>). The beta-galactosidase activity was determined in a thermo shaker in <NUM> reaction tubes at <NUM>. The enzyme mix and the lactose solution were preincubated at <NUM> for <NUM>. To start the enzymatic reaction <NUM> µL of the enzyme mix were added to <NUM> µL lactose solution. The reaction was carried out for <NUM> at <NUM>. The reaction was stopped by adding <NUM> µL <NUM> M HClO<NUM> to the enzyme-substrate-mix. The mix was kept on ice for <NUM> before it was centrifuged at <NUM><NUM> rcf and <NUM> for <NUM>. <NUM> µL of the supernatant were transferred to <NUM> µL <NUM> M KOH to neutralise the solution. The neutralisation step was checked using pH paper and the pH was adjusted if necessary. The solution was kept on ice for another <NUM> before it was centrifuged at <NUM><NUM> rcf at <NUM> for <NUM>. All assays were performed in triplicate.

The glucose concentration was determined for the calculation of the beta-galactosidase activity with lactose as a substrate. The glucose concentration was determined using an enzymatic assay. The commercial kit hexokinase/glucose-<NUM>-phosphate dehydrogenase (Megazyme, Wicklow, Ireland) was used. The D-glucose is phosphorylated to glucose-<NUM>-phosophate by the enzyme hexokinase using ATP as cosubstrate. In a coupled reaction glucose-<NUM>-phosphate is oxidized to gluconate-<NUM>-phosphate by the enzyme glucose-<NUM>-phosphate dehydrogenase while NADP+ is reduced to NADPH which was quantified photometrically at <NUM>. The amount of the formed NADPH equals the amount of glucose in the solution. The determination of the glucose concentration was performed using a robotic liquid handling system (MultiPROBE II EX, Packard Bioscience, Meriden, CT, USA). The hexokinase/glucose-<NUM>-phosphate dehydrogenase suspension was diluted <NUM>:<NUM> with H<NUM>Odd before use. TRA buffer which contained NADP+ and ATP was prepared according to Table <NUM>.

For the glucose determination <NUM> µL TRA buffer were prepared in a <NUM> well plate and <NUM> µL of the sample solution was added. After <NUM> the absorption at λ = <NUM> was measured in a plate reader (A1). Subsequently, <NUM> µL of the diluted hexokinase/glucose-<NUM>-phosphate dehydrogenase were added to each well. The plate was shaken in the plate reader and incubated at room temperature. After <NUM> the absorption at λ = <NUM> was measured again (A2). With the absorption difference ΔA = A2 - A1 the glucose concentration was calculated using a calibration curve (range; <NUM>-<NUM>/L glucose).

The kinetic parameters were determined using the natural substrate lactose. The reaction matrices and conditions were chosen in order to simulate conditions in milk systems. The chosen reaction matrices were Novo buffer (cf. Table <NUM>, above) and milk diluted with Novo buffer. The reaction temperature was <NUM> and the pH value was <NUM>.

For the determination of the KM values of P. wynnii beta-galactosidase lactose concentrations ranged from <NUM>M to <NUM>M. In order to obtain different lactose concentrations in milk, milk was diluted with Novo buffer. It was used a beta-galactosidase activity of <NUM> nkatLactose,<NUM>/mL for the determination in milk and <NUM> nkatLactose,<NUM>/mL in buffer. The enzyme activities were measured under initial reaction velocity conditions of the enzyme. The reaction times for the initial velocity of the enzymes were determined in previous experiments. The kinetic parameter KM was analysed using the Enzyme Kinetics Module in SigmaPlot <NUM> (Systat Software, Inc. , San Jose, USA).

The KM values determined are summarized in Table <NUM>. In the case of the studies in Novo buffer the enzyme activity decreased again with increasing lactose concentration. For this reason, in addition to the Michaelis Menten evaluation, the evaluation also included an evaluation taking into account a substrate inhibition.

The influence of galactose was investigated with P. wynnii beta-galactosidase and the commercial beta-galactosidase Opti-Lactase (Optiferm GmbH, Oy-Mittelberg (D)). The determination of the enzyme activity in Novo buffer was conducted with a lactose concentration of <NUM>M and <NUM>M respectively. To this solutions <NUM> or <NUM>M galactose were added. The determination of the enzyme activity was performed as described above.

<FIG> shows the relative enzyme activity of the two beta-galactosidases. First, <NUM> and <NUM>M galactose respectively were added to <NUM>M lactose (A). The enzyme activity of the technical enzyme preparation decreased with increasing galactose concentration. An activity of <NUM> % was determined. In contrast, galactose did not have an inhibitory effect on the activity of the beta-galactosidase from P. In another experiment <NUM>M galactose was added to <NUM>M lactose (B). Here, the same effect as in (A) could be observed. Furthermore, the comparison of enzyme activity at <NUM> and <NUM> showed that with lower lactose concentrations the activity of Opti-Lactase decreased significantly. The determined enzyme activity decreased from <NUM> ± <NUM> mkatLactose,<NUM>/L to <NUM> ± <NUM> mkatLactose,<NUM>/L. The enzyme activity of the beta-galactosidase from P. wynnii did not change significantly (<NUM> ± <NUM>µkatLactose,<NUM>/L).

The hydrolysis of lactose in milk was performed both with the beta-galactosidase from P. wynnii and with the technical enzyme preparation Opti-Lactase (K. The hydrolysis was conducted in a <NUM> scale at approx. <NUM> to <NUM>. For the conversion an enzyme activity of <NUM> nkatLactose, <NUM>/mL milk was used. Samples were taken over a period of <NUM>. Therefore <NUM>µL samples were taken and stopped with perchloric acid as described above and the lactose content was determined by HPLC (cf. Example <NUM>, below). GOS synthesis was investigated by HPLC and thin-layer chromatography (cf. Example <NUM>, below).

<FIG> shows the course of the lactose hydrolysis in milk over time. The beta-galactosidase from P. wynnii exhibited a better performance in lactose hydrolysis from the beginning. After <NUM> hours, <NUM>% of the lactose had already been hydrolysed with P. wynnii beta-galactosidase compared to <NUM>% with Opti-Lactase. After <NUM> <NUM> % lactose was hydrolysed which corresponds to a residual milk lactose content of <NUM>/L with P. wynnii beta-galactosidase and after <NUM> no more lactose could be detected by HPLC (detection limit <NUM>/L). In comparison to P. wynnii beta-galactosidase, with Opti-Lactase lactose amounts of <NUM>/L after <NUM>, <NUM>/L after <NUM>, and <NUM>/L even after <NUM> could be determined.

An Agilent Series <NUM> HPLC system, equipped with a Shodex HILICpakVG-<NUM>4E column (<NUM> × <NUM>, <NUM> µm, Shodex) and a low-temperature evaporative light scattering detector (ELSD Sedex 85LT, Sedere, Alfort ville Cedex, France) at <NUM> and <NUM> bar was used to determine the concentration of lactose. The column was eluted with <NUM> % (v/v) acetonitrile, <NUM>% (v/v) methanol, and <NUM>% (v/v) double-distilled water at a flow rate of <NUM>/min at <NUM>.

Thin layer chromatography was used in order to visualize the products (lactose, glucose, galactose and galacto-oligosaccharides (GOS)) generated from milk lactose by enzymatic conversion with the two beta-galactosidases (cf. Example <NUM>, above). Aliquots of <NUM> µL of the sample prepared were spotted on TLC plates (Silica gel 60F254, Merck, Whitehouse Station, NJ), and developed in a solvent system of <NUM>-butanol/<NUM>-propanol/water (<NUM>:<NUM>:<NUM>). The plates were developed by a colouring agent (<NUM>M N-<NUM>-naphthyl ethylenediamine dihydrochloride and <NUM> % sulfuric acid in methanol) at <NUM> until the spots became visible.

<FIG> shows the HPLC analysis of the samples from lactose hydrolysis by means of Opti-Lactase and <FIG> the analysis using beta-galactosidase from P. In addition to the decrease in lactose and the increase in galactose or glucose, the formation of GOS over time can be observed. It was obvious that the GOS synthesis during the hydrolysis of lactose with Opti-Lactase occurred much later than during the hydrolysis with P. wynnii beta-galactosidase. While during the conversion of lactose by Opti-Lactase only a minimal GOS formation could be detected after <NUM>, the conversion by P. wynnii beta-galactosidase showed the formation of GOS after <NUM> already. The highest yield of GOS was approximately <NUM> for the conversion with Opti-Lactase and <NUM> with P. wynnii beta-galactosidase. However, the yield of Opti-Lactase was qualitatively lower than that of P. wynnii beta-galactosidase. In addition, different GOS were formed. While Opti-Lactase synthesized rather low molecular weight GOS, P. wynnii beta-galactosidase generated also higher molecular weight GOS.

The metagenome beta-galactosidase was produced and purified using nickel affinity chromatography as described above (cf. Example <NUM>). After purification, the specific activity of M1 beta-galactosidase was <NUM> nkatoNPGal,<NUM> mg-<NUM>.

The further investigations with M1 focused mainly on a comparison of the technical applicability for the hydrolysis of lactose in milk. Therefore, the influence of galactose and glucose on the enzyme activity was investigated, as well as the hydrolysis of lactose in milk which was performed under the same conditions as the hydrolysis with P. wynnii beta-galactosidase of the present invention. In addition, the stability of the two enzymes was investigated.

The influence of galactose was also investigated with the metagenome M1 beta-galactosidase as described before. The determination of the enzyme activity in Novo buffer was conducted with a lactose concentration of <NUM>M and <NUM>M respectively. To this solutions <NUM> or <NUM>M galactose were added.

<FIG> shows the relative enzyme activity of the beta-galactosidase M1. In both cases, the enzyme activity of the beta-galactosidase M1 did not change significantly. This means galactose did also not have an inhibitory effect on the M1 beta-galactosidase activity.

In table <NUM>, the comparison of the results of the investigations of beta-galactosidase M1 and P. wynnii is depicted. With M1, no influence of galactose on activity was observed, with P. wynnii beta-galactosidase galactose a slightly activating effect on enzyme activity could be shown.

In addition to galactose, the effect of released glucose on the enzyme activity of the two beta-galactosidases was also of interest. Due to the experimental setup (analysis), it was not possible to measure the influence of glucose with the natural substrate lactose. Therefore, oNPGal was used for the determination of beta-galactosidase activity in the presence of glucose as known in the art. In addition, the influence of galactose and the influence of an equimolar concentration of both glucose and galactose was tested.

The enzyme activity of P. wynnii beta-galactosidase increased with increasing galactose concentration (<NUM>M: <NUM> % activity) as seen in <FIG>. The presence of glucose showed a much stronger activating effect on the enzyme activity of P. wynnii beta-galactosidase (at <NUM>M glucose: <NUM>% activity) compared to beta-galactosidase M1 (cf. Table <NUM>). Only a slight improving effect on the enzyme activity (at MIX-<NUM>: <NUM>% activity) was observed for the equimolar mixture of glucose and galactose, compared to the exclusive addition of glucose.

In summary, P. wynnii beta-galactosidase showed an activating effect in the presence of both glucose and galactose, while M1 only in the presence of glucose also showed an activating effect, which is less turned out. In the presence of galactose, M1 does not show inhibition but is not activated either.

The hydrolysis of lactose in milk was performed both with the beta-galactosidase M1 and the P. wynnii beta-galactosidase. The hydrolysis was conducted as described previously in a <NUM> scale at approx. <NUM> to <NUM>. For the conversion, an enzyme activity of <NUM> nkatLactose, <NUM> mL-<NUM> milk was used. Samples were taken over a period of <NUM>.

In <FIG>, the results of lactose hydrolysis with both the P. wynnii beta-galactosidase and M1 were presented for comparison. Both enzymes showed a similar course of lactose in the first <NUM> hours. However, less lactose was hydrolysed by M1 after <NUM> hours. After <NUM>, <NUM> L-<NUM> lactose was still detected, whereas with P. wynnii beta-galactosidase no lactose could be detected after <NUM>. One reason for the better conversion of P. wynnii beta-galactosidase over time could be related to the higher activation of glucose and galactose released by the lactose hydrolysis. Another reason could be a lower stability of M1.

The storage stability of the two beta-galactosidases M1 and P. wynnii were investigated in NOVO buffer at <NUM>. Already after <NUM> days, only a residual activity of <NUM>% could be determined with M1, while with P. wynnii beta-galactosidase no loss of activity should be determined. After <NUM> days a residual activity of <NUM>% could be determined with P. wynnii beta-galactosidase.

To determine the turnover number, the specific activity of the enzymes on lactose at <NUM> was determined. The following formula was used to determine the turnover number: <MAT>.

In Table <NUM>, the calculated turnover numbers of the two beta-galactosidases are depicted.

The turnover number of P. wynnii beta-galactosidase is twice as high compared to the turnover number of M1. This shows also the higher efficiency of the P. wynnii beta-galactosidase.

In conclusion, it could be shown that the P. wynnii beta-galactosidase of the present invention could be activated in the presence of glucose and galactose while the metagenome M1 beta-galactosidase was not inhibited by galactose but only activated by glucose, wherein the activation was lower than with the P. wynnii beta-galactosidase. wynnii beta-galactosidase of the present invention showed a total lactose conversion in milk after <NUM> while M1 had not completely hydrolysed the lactose after <NUM> (<NUM> % conversion) under the same conditions. It could also be shown that the P. wynnii beta-galactosidase is more stable than M1.

Altogether P. wynnii beta-galactosidase showed more favorable characteristics of the technical applicability for the hydrolysis of lactose in milk than M1.

In further studies it could also be shown that the beta-galactosidase from P. wynnii according to the present invention is able to produce galacto-oligosaccharides (GOS). In this comparative study, the experimental Enzyme B (Opti-B) which is distributed by Optiferm GmbH, Germany, was used. This enzyme is originated from B. Compared to a commercially available enzyme, the GOS yield is similar and the remaining lactose content is lower. As further enzyme preparation, Saphera <NUM> was used which is an industrial beta-galactosidase preparation from Novozymes A/S. The beta-galactosidase is originated from Bifidobacterium bifidum. The preparation is used in batch and aseptic application for the hydrolysis of lactose in dairy products.

With these and the following (cf. Example <NUM>, infra) experiments, it could be shown that P. wynnii beta-galactosidase of the present invention shows both a good performance of lactose hydrolysis in milk and at the same time a good transgalactosylation potential for the formation of GOS.

Since it was shown in preliminary experiments that GOS were formed only with P. wynnii beta-galactosidase and Opti-B and not with Saphera <NUM>, the bioconversions were carried out only with the first two. Pharma-lactose is highly purified lactose (<NUM> % lactose) and was used for the bioconversions to exclude as far as possible the components of lactose influencing the performance of the beta-galactosidases.

For the determination of the transgalactosylation activity of the P. wynnii beta-galactosidase, pharma-lactose (<NUM> L-<NUM>) was dissolved in potassium phosphate buffer (concentrations varying from <NUM> to <NUM>M) containing <NUM>M MgCl<NUM> (pH <NUM>) at <NUM>. After cooling down, the solution (V: <NUM>) was transferred to a small reaction vessel (<NUM>) which was tempered to <NUM>. To start the reaction, the beta-galactosidase (<NUM> Ulactose,<NUM> mL-<NUM>) was added to the solution. On several time points, samples (<NUM> µL) were taken and heated to <NUM> for <NUM> to inactivate the beta-galactosidase. The samples were stored at -<NUM> and used for further determination of the sugar concentrations (cf. For the determination of the residual activity, samples (<NUM> µL) were taken after <NUM>, <NUM>, <NUM> and <NUM> and the buffer was exchanged using PD10 columns to remove the sugars. Afterwards, the residual beta-galactosidase activity was measured by the oNPGal assay.

For a possible further application of P. wynnii beta-galactosidase, the production of galacto-oligosaccharides was done without buffer salts. Therefore, pharma-lactose (<NUM> L-<NUM>) was dissolved in H<NUM>Odd. Also, <NUM> Ulactose,<NUM> mL-<NUM> were applied. The remaining procedure was done as described above.

To compare the transgalactosylation activity of P. wynnii beta-galactosidase, the bioconversions were also done with the enzyme preparation Opti-B (Optiferm GmbH).

Before using HPLC, <NUM> µL of the sample was added into a <NUM> reaction tube and was mixed with <NUM> µL fructose (<NUM> L-<NUM>) as internal standard. Afterwards, <NUM> µL of the mixture was transferred into a HPLC vial. If necessary, the samples were diluted with H<NUM>Odd before mixing with the internal standard.

Sugars were separated for quantification using high pressure liquid chromatography (HPLC). Therefore, two consecutive carbohydrate Ca<NUM>+-HPLC-columns (<NUM> x <NUM>; CS, Langerwehe, Germany) were used. Molecule separation was based on size and the different interactions of the hydroxy groups of the sugar molecules with the Ca<NUM>+-ions of the columns.

The columns were attached to an Agilent <NUM> Series HPLC system (Agilent Technologies, Santa Clara, USA) equipped with an evaporative light scattering detector (ELSD Sedex 85LT; Sedere, Olivet, France) which was operated at <NUM> and <NUM> bar for detection. The columns were tempered to <NUM> using the Croco-Cil HPLC column oven (SCO Seitz Chromatographie Produkte GmbH, Weiterstadt, Germany). The system was operated with a pressure of <NUM> bar. An automated sampler applied <NUM> µL sample on the columns. Elution was carried out with a constant flow rate of <NUM> min-<NUM> of H<NUM>Odd for <NUM>.

The parameters of the used method are listed in the following Table <NUM>.

The area of the peaks corresponded to respective analyte amounts, defined by calibration. For the preparation of the calibration curves, lactose (<NUM> - <NUM> L-<NUM>), glucose (<NUM> - <NUM> L-<NUM>) and galactose (<NUM> - <NUM> L-<NUM>) were dissolved in H<NUM>Odd. The limit of detection (LOD) of lactose, glucose and galactose are <NUM> L-<NUM>, <NUM> L-<NUM> and <NUM> L-<NUM>, respectively. For each sample, an individual correction factor was calculated and multiplied with the peak area of lactose, glucose and galactose, respectively. The correction factor was calculated by dividing the mean value of all fructose peaks (internal standard) of a measurement series by the peak of fructose in the corresponding sample.

After determination of the sugar concentrations using HPLC, two different parameters can be calculated.

The lactose conversion describes how much lactose was processed by the beta-galactosidase during the bioconversion and is calculated with following equation: <MAT> With:.

The GOS yield describes the GOS concentration as a percentage of initial lactose concentration. <MAT> With:.

The GOS production with P. wynnii beta-galactosidase was done under different process conditions to investigate the influence of the different parameters on GOS yield and lactose conversion. Therefore, the salt and the enzyme concentration were varied, respectively. The results of the bioconversions are shown in Table <NUM>.

The highest lactose conversion (<NUM> %) and theoretical GOS yield (<NUM> %) was determined when pharma-lactose was dissolved in <NUM>M potassium phosphate + <NUM>M MgCl<NUM> (pH <NUM>) and <NUM> Ulactose mL-<NUM> was applied. Dissolving the pharma-lactose in H<NUM>Odd resulted in a lower lactose conversion as well as in a lower GOS yield, although nearly the same residual activity than in <NUM>M buffer was measured after <NUM>. Therefore, it is assumed that potassium and phosphate ions as well as MgCl<NUM> may influence the conformation and activity of the beta-galactosidase.

The results of the bioconversions with Opti-B are summarized in Table <NUM>. Since the preparation Opti-B was developed to produce GOS, it was used in this study for comparison of the transgalactosylation activity. Thus, two bioconversions in buffered and unbuffered system were conducted.

When pharma-lactose was dissolved in H<NUM>Odd, more lactose was converted during the process while a minor GOS yield was reached. This may be due to a higher water availability in the salt-free system and thus the beta-galactosidase kinetic was shifted towards the hydrolysis reaction.

In this experiment, all three enzyme preparations, the P. wynnii beta-galactosidase of the present invention, Opti-B and Saphera <NUM> were used.

The hydrolysis of lactose in milk was done in a small Schott bottle (V: <NUM>). Therefore, <NUM> milk (H-milk, "Schwarzwaldmilch", <NUM> % fat) was incubated on a magnetic stirrer at <NUM> - <NUM>. To prevent microbial growth, <NUM> % (w/v) sodium azide was added. To start the reaction <NUM> nkatoNPGal,<NUM> mL-<NUM> of the respective beta-galactosidase was added. At several time points samples (<NUM> µL) were taken and heated for <NUM> at <NUM> to inactivate the enzyme. Afterwards, the samples were stored at -<NUM> until analysis. The lactose concentration was measured after sample preparation by Carrez Clarification using HPLC (see above). For qualitative analysis, a thin-layer chromatography was done as described before.

To compare the performance of the P. wynnii beta-galactosidase of the present invention, this experiment was also done with the industrial enzyme preparations Opti-B (Optiferm GmbH) and Saphera <NUM> (Novozymes A/S).

To remove non-soluble proteins and fats, a carrez precipitation of the milk samples was done before the samples were analyzed via HPLC. Carrez I and Carrez II were prepared as known in the art. The sample (<NUM> µL) was mixed with <NUM> µL carrez I, <NUM> µL carrez II, <NUM> µL NaOH (<NUM> M) and <NUM> µL H<NUM>Odd. Then, the mixture was centrifuged (<NUM>, <NUM> rpm, <NUM>) and the supernatant was filtered using Minisart SRP4 syringe filter (<NUM> µm).

Using BgaPW and Saphera <NUM>, the lactose was under <NUM> L-<NUM> (LOD) after <NUM> and <NUM>, respectively. Whereas with Opti-B, still <NUM> L-<NUM> lactose was detected after <NUM>.

For qualitative analysis of the hydrolysis of lactose in milk and the GOS production during this process, a thin-layer chromatography was done. In <FIG>, the thin layer chromatography of the approaches with the three beta-galactosidases using <NUM> nkatoNPGal,<NUM> mL-<NUM>milk are shown. On the lanes <NUM>-<NUM> the standard solutions of glucose and galactose and a mix of all sugars were applied.

The decrease of the lactose concentration was observed on the thin-layer chromatography of the hydrolysis with Opti-B. However, lactose was still detected after <NUM>. The production of GOS as well as the further degradation of GOS was also observed. A very similar chromatogram was seen with P. wynnii beta-galactosidase, but lactose was hydrolyzed completely after <NUM>. Furthermore, different GOS pattern were observed with the TLC of P. wynnii beta-galactosidase and Opti-B. With Opti-B, a main GOS spot was observed whereas with P. wynnii beta-galactosidase, different GOS spots were seen. Using Saphera <NUM> for lactose hydrolysis resulted in a completely lactose conversion after <NUM> without production of GOS. So, all thin-layer chromatographies confirm the HPLC analysis.

Claim 1:
Use of an enzyme having beta-galactosidase activity in the production of
(i) lactose-depleted or lactose-free, and
(ii) galacto-oligosaccharide-enriched
dairy products, wherein said enzyme is derived from Paenibacillus wynnii, and wherein said enzyme comprises
(a) the amino acid sequence of SEQ ID NO: <NUM>; or
(b) an amino acid sequence having at least <NUM>% sequence identity to SEQ ID NO: <NUM> and having beta-galactosidase and transgalactosylation activity.