KR20220052762A - Novel lactose oxidase from Pseudomonas and method for producing lactobionic acid using the same - Google Patents

Abstract

The present invention provides a method for producing lactobionic acid industrially utilizing a recombinant strain producing lactobionic acid. Furthermore, the present invention identifies the characteristics of a lactose-oxidizing enzyme by isolating the lactose-oxidizing enzyme from a microorganism of the genus Pseudomonas and purifying the same, and is configured such that a recombinant strain producing lactobionic acid is produced by cloning a gene which encodes the lactose-oxidizing enzyme.

Description

Lactose oxidase enzyme derived from microorganisms of the genus Pseudomonas and lactobionic acid production method using the same

The present invention relates to a lactose oxidase enzyme derived from a microorganism of the genus Pseudomonas and a method for producing lactobionic acid using the same.

Lactobionic acid, a type of polyhydroxy bionic acid as a next-generation skin whitening material, is biodegradable and biocompatible in addition to the exfoliating, anti-wrinkle, and skin whitening effects of α-hydroxy acid. , anti-aging, antioxidant, moisturizing, chelating, etc., and has little irritation to the skin, attracting great attention as a next-generation cosmetic material that can replace glycolic acid and lactic acid. Cosmetics containing lactobionic acid It has been commercialized and is now on sale.

Lactobionic acid is a substance formed by β-1,4 bonds of galactose and gluconic acid, and has a molecular weight of 358.3 and is a water-soluble white crystalline compound. Lactobionic acid can be synthesized by the oxidation reaction of the free aldehyde group in lactose, and thus, research on production by chemical, electrochemical, catalytic or biological oxidation is currently being conducted. [Satory et al., Biotechnology letters 19(12) 1205-08, 1997].

Among them, in order to respond to the interest and use of lactobionic acid as a raw material for food and cosmetics, research on biological lactobionic acid production through an environmentally friendly microbial culture method is being intensively conducted. Buckholderia cepacia, and Zymomonas mobilis have been reported. However, the strain with the highest lactobioic acid productivity reported so far is Buckholderia cepacia, which is classified as a pathogenic strain and has a problem in that it cannot be used industrially.

Accordingly, the prior applicant of the present inventors, Republic of Korea Patent Publication No. 10-2018-0047470, promotes the lactobionic acid oxidation reaction from lactose through a culture method optimization study of a non-pathogenic Pseudomonas taetrolens strain, thereby lactobionic acid productivity A method of increasing the .

On the other hand, the problem in the production of lactobionic acid is that there is a difficulty in process development, but the difficulty in securing an excellent strain plays a large role. After confirming the possibility of increasing lactobionic acid production through the development of a culture process of wild-type Pseudomonas tetrorence through previous studies, the development of a recombinant strain promotes the rate of the lactobionic acid conversion reaction to increase the lactobionic acid productivity. It is necessary to study productivity improvement through strain development according to the possibility. In addition, since the activity of the produced enzyme is very different depending on the type of lactobionic acid-producing microorganism, which is currently known, and the environmental conditions, it is necessary to discover a new enzyme with excellent lactobionic acid production ability and to improve the activity of this enzyme. Characteristic studies are required, and there is a great need to develop a strain that has improved lactobionic acid production capacity by overexpressing this enzyme, and research on production technology using it is very important.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are incorporated herein by reference in their entirety to more clearly describe the content of the present invention and the level of the art to which the present invention pertains.

Republic of Korea Patent Publication No. 10-2030776

The present inventors areolate and purify an enzyme having a lactose oxidizing activity from a microorganism of the genus Pseudomonas having the ability to produce lactobionic acid, reveal the characteristics, and lactose oxidase produced by the Pseudomonas strain A gene encoding an easing active enzyme was cloned and expressed successfully in a recombinant strain.

Accordingly, an object of the present invention is to provide a recombinant strain that can be industrially used for lactobionic acid production and a highly efficient lactobionic acid production method using the same.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a recombinant vector for producing a recombinant strain for producing lactobionic acid, comprising a nucleic acid sequence encoding malate dehydrogenase derived from a microorganism of the genus Pseudomonas.

The problem in the production of lactobionic acid is that there is a difficulty in process development, but the difficulty in securing an excellent strain plays a large role. Since the action of the produced enzyme and the oxidizing ability of the substrate are very different depending on the type of microorganism that produces lactobionic acid, which is currently known, a new enzyme with excellent lactobionic acid production capacity has been discovered, and the development of a recombinant strain and it Research on the production technology used is very important.

Accordingly, the present inventors purified the lactose oxidizing enzyme in this strain in order to solve the limitation of lactobionic acid production through culturing of wild-type Pseudomonas microorganisms, and as a result of identifying the characteristics of this enzyme, malate dehydrogenase (malate dehydrogenase) was found to be an enzyme involved in the production of lactobionic acid derived from microorganisms of the genus Pseudomonas.

The Pseudomonas genus microorganisms are Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas moraviensis ( Pseudomonas moraviensis ), Pseudomonas granadensis ( Pseudomonas granadensis ), Pseudomonas Correhensis ( Pseudomonas Koreensis ) and the like, including Pseudomonas Koreensis ( Pseudomonas ) Pseudomonas taetrolens ) is not limited.

In one embodiment, the nucleic acid sequence encoding malate dehydrogenase may include the polynucleotide set forth in SEQ ID NO: 3, wherein the malate dehydrogenase is an amino acid represented by SEQ ID NO: 4 It may include a sequence.

In another embodiment, the malate dehydrogenase may be malate:quinone oxidoreductase (EC 1. 1. 99. 16, GenBank accession number WP_048378186.1).

The malate dehydrogenase derived from the microorganism of the genus Pseudomonas has lactose oxidizing activity, and the malate dehydrogenase has an action temperature of 15° C. to 45° C., and a relative enzyme activity of 60% under the conditions of pH 6.5 to pH 9.0. It may be maintained longer.

The recombinant vector comprising the nucleic acid sequence encoding the malate dehydrogenase may be a plasmid vector, preferably pDSK519.

Another aspect of the present invention is to provide a recombinant strain expressing malate dehydrogenase into which the recombinant vector is introduced.

In one embodiment, the strain may be a Gram-negative bacteria, for example, including but not limited to Terrabacteria, proteobacteria, helical bacteria, sphingobacteria, and supergroups, preferably, the strain is the recombinant vector in E. coli. has been introduced The recombinant E. coli is characterized in that it is possible to produce lactobionic acid from lactose by producing a lactose oxidizing enzyme, unlike wild-type E. coli.

Another aspect of the present invention is to obtain a recombinant vector comprising a nucleic acid sequence encoding a malate dehydrogenase derived from a microorganism of the genus Pseudomonas; And to provide a method for producing a recombinant strain for producing lactobionic acid comprising the step of introducing the recombinant vector into Gram-negative bacteria.

The present invention also provides a method for producing lactobionic acid using the aforementioned recombinant strain and/or a method for producing lactobionic acid using a malate dehydrogenase having lactose oxidizing activity derived from a microorganism of the genus Pseudomonas. do.

The method may include adding a coenzyme PQQ (pyrroloquinoline quinone) to the recombinant strain and/or malate dehydrogenase having lactose oxidizing activity derived from the microorganism of the genus Pseudomonas.

In one embodiment, the lactobionic acid production method comprises the steps of 1) separating and purifying an enzyme involved in the production of lactobionic acid derived from Pseudomonas tetrorence; 2) optimizing enzyme stability and activity for maintaining the activity of the enzyme; 3) constructing a recombinant plasmid and transforming the recombinant E. coli including the plasmid to establish whether it is involved in the production of lactobionic acid producing E. coli and the production of lactobionic acid.

In order to prepare the recombinant microorganism of the present invention, a gene encoding Pseudomonas tetrorence-derived malate dehydrogenase MQO (malate:quinone oxidoreductase, EC 1. 1. 99. 16) was amplified through specific primers through PCR.

Thereafter, a recombinant plasmid was prepared in which the gene was inserted into the expression vector pDSK519 for E. coli using restriction enzymes ( Xba I, BamHI ), which was named pDSK519-MQO.

The recombinant plasmid pDSK519-MQO was transformed into E. coli DH5α to prepare a recombinant E. coli, and the production of lactobionic acid was confirmed with the recombinant strain in a culture medium containing lactose.

In one embodiment of the present invention, in the step of culturing Pseudomonas and E. coli, it contains lactose at a concentration of 1 to 20% by volume, 0.2% yeast extract, 0.5% peptone, and 0.1% beef extract, and 0.5% NaCl can do.

The culturing step may be culturing for 48 to 120 hours at 50 ml to 2.0L of NB medium, 20°C to 37°C.

If necessary, the culture can be cultured according to the lactose concentration, and in a culture medium in the presence of CaCO ₃ as a pH correction material, for example, the lactose concentration in the medium can be varied from 10 g/L to 200 g/L, and , CaCO ₃ It can be carried out with or without addition.

In a preferred embodiment, the culturing of the present invention may be performed by a batch culture method, but is not limited thereto.

In addition, in an embodiment of the present invention, the method for improving productivity through culturing of the Pseudomonas tetrorence strain may be developed as disclosed in Korean Patent Application Laid-Open No. 10-2018-0047470, and Korean Patent Application Laid-Open No. 10-2018- The entire disclosure of 0047470 is incorporated herein by reference.

The present invention isolates and purifies an enzyme protein effective for lactobionic acid conversion of a Pseudomonas tetrorence strain having an existing lactose oxidizing activity, and by clarifying its characteristics, a culture method for improving the activity of this enzyme was confirmed. . In addition, by preparing a recombinant vector and a recombinant strain comprising the gene sequence encoding the enzyme protein of the present invention, it is possible to develop a strain with superior lactobionic acid productivity than the wild-type strain, thereby improving the lactobionic acid conversion rate. Accordingly, the novel recombinant strain can be efficiently used in various industrial fields by improving the production rate in the lactobionic acid mass production process.

1 is an SDS-PAGE electrophoresis picture of lactose oxidizing enzyme (Lane M; Mw standard, Lane 1; Crude enzyme, Lane 2; Ammonium sulfate fraction sample, Lane 3; Hitrap Q chromatography fraction sample, Lane 4; Sephadex G-100 chromatography fraction samples).
2 is a graph showing the optimal temperature and pH of lactose oxidizing enzyme activity expression. A) Relative activity as a function of temperature, B) Thermal stability, C) Relative activity as a function of pH, D) Stability at pH
Figure 3 shows a schematic diagram of a recombinant plasmid for MQO expression in E. coli using the pDSK519 vector.
4 is a result showing whether or not lactobionic acid conversion according to the addition of coenzyme in wild-type E. coli and recombinant E. coli in the presence of lactose.
5 is a graph showing the lactobionic acid production results of wild-type E. coli and recombinant E. coli prepared through the present invention under aerobic fermentation conditions.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples.

Example

Example 1: Purification, Isolation and Molecular Weight of Lactose Oxidizing Enzyme

In order to isolate and purify the enzyme involved in the production of lactobionic acid derived from Pseudomonas tetrorence, an experiment was performed as follows.

Experimental Example 1: Preparation of crude enzyme extract

In order to isolate the enzyme involved in the production of lactobionic acid from the culture medium produced from Pseudomonas tetrolence ( Pseudomonas taetrolens Haynes 1957, KCTC 12501 strain received from the Korea Institute of Bioscience and Biotechnology Biological Resource Center), first, the microorganism is used as a culture medium in Nutrient broth ( NB) (1 g/L beef extract, 2 g/L yeast extract, 5 g/L peptone, 5 g/L NaCl) was used, and 200 g/L lactose For pH correction, 30 g/L CaCO ₃ was added to maintain pH 6.5 or higher during fermentation, and incubated for 1 day at a stirring speed of 200 rpm and 25° C. in a shaker incubator. 1L of this culture solution was centrifuged at 13000rpm, 4℃ for 10 minutes to collect microorganisms, and the collected cell pellet was resuspended in 20mM phosphate buffer (pH 7.2) to make the concentration 10%, and then sonicator (VCX-750, vibra cell, USA) at an amplitude of 30%, 10 sec pulse on, and 30 sec pulse off to break the cell wall of the cell for 20 minutes. After centrifugation at 4° C. and 13000 rpm for 10 minutes to obtain a supernatant, this was referred to as a crude enzyme extract.

Experimental Example 2: Lactose oxidizing enzyme purification

To the crude enzyme solution, 50% by weight to volume ratio ((NH ₄ ) ₂ SO ₄ ) was added at 4° C. and stirred well for 4 hours. The enzyme solution was centrifuged to collect only the solution, and to the solution, an oil having a final concentration of 70% by weight to volume ratio was added at 4° C. and stirred well for 4 hours. After centrifuging the enzyme solution to collect only the precipitate, the precipitate was dissolved in 20 mM potassium phosphate buffer, pH 7.5, and dialyzed overnight in the same buffer. The enzyme solution was centrifuged again to remove insoluble substances, and then purified with a 0.22 μm filter to be used as an enzyme solution.

In this step, the enzyme activity was evaluated by adding 1.350 ml of 0.075 mM 2,6-dichlorophenol indophenol (2,6-dichlorophenol indophenol, DCIP), 5 mM lactose, and 100 mM acetate buffer solution (pH 5.5) containing 4 mM sodium fluoride. 0.15 ml of enzyme solution was reacted for 10 minutes at 30 ° C., and protein concentration was measured by the Bradford method (Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-) dye binding. Anal. Biochem. 1976, 72, 248-254).

Fractionated enzyme samples including lactobionic acid conversion activity were fractionated by dissolving them in a linear gradient of 0-1M NaCl using a HiTrap-Q (GE Healthcare Life Sciences) column as an ion exchange resin, which was a fraction of 4.0 ml. was collected every 2 minutes, and fractions were selected by measuring enzyme activity in each fraction.

In the final purification step, proteins were separated by gel filtration with Sephadex G-100 (Sephadex G-100, 1.5 Х 20 cm, Bio-Rad) equilibrated with 0.1M sodium borate buffer (pH 7.0) based on their molecular weight. do. Table 1 shows the activity and protein concentration of the samples fractionated through each purification step.

active fraction
Total protein (μg)
total activity
(U)
specific activity
(U/μg)
output
(%)
purification drainage Crude enzyme extract 36.2 15.2 0.4 100.0 1.0 50 to 70 % ammonium sulfate 2.5 6.7 2.7 44.1 6.4 HitrapQ ion exchange chromatography 1.1 4.2 3.8 27.6 9.1 Sephadex G-100
gel filtration chromatography 0.3 2.8 9.3 18.4 22.2

Experimental Example 3: Separation of lactose oxidizing enzyme and confirmation of molecular weight

In order to measure the purity and molecular weight of the enzyme purified in the present invention, electrophoresis was performed on 10% SDS-polyacrylamide gel, and the molecular weight was calculated by comparing the movement speed with the protein marker. SDS-PAGE was prepared according to Laemmli's method, and Bio-rad's protein marker (10-250 kDa) was used. As shown in FIG. 1, a single band was shown, and as a result of measuring the molecular weight compared to molecular weight markers composed of standard proteins, the molecular weight of the lactobion production-related enzyme produced by Pseudomonas tetrorence was confirmed to be about 45 kDa. .

After comparing the protein bands by performing Coomassie staining, mass spectrometry was performed on the peptide fraction from which a single protein band was cut. This enzyme was malate:quinone oxidoreductase (MQO, EC 1. 1. 99. 16, GenBank accession number WP_04837818.1) was confirmed.

Example 2: Optimal temperature and pH of lactose oxidizing enzyme

In order to determine the optimum temperature for the enzyme reaction of the lactose oxidizing enzyme confirmed in Example 1, the reaction was carried out in the range of 15 to 65 °C at 5 °C intervals for 10 minutes, and the relative activity of the enzyme was expressed. For thermal stability, the enzyme solution was heat-treated at intervals of 5°C in the range of 15 to 65°C, and the enzyme solution was heat-treated in units of 1 hour, and the residual activity of the enzyme was measured. The optimal pH measurement of this enzyme is 0.1 M acetate buffer (pH 3.5-5.5), 0.1 M phosphate buffer (pH 5.5-8.0), 0.1 M Tris-HCl buffer (pH 8.0-9.0), 0.1 M glycine-NaOH buffer (pH 9.0-12.0) was used. The stability to pH was measured by adding each pH buffer to the enzyme solution and leaving it at room temperature for 1 hour, then the remaining enzyme activity was measured. . Other methods were the same as those of the liquid culture enzyme activity measurement method of Experimental Example <1-2>. The results of the activity changes with respect to the temperature and pH of the enzyme purified in the present invention are shown in FIG. 2 . The activity against temperature was maintained at more than 50% of the maximum activity up to 55 ℃, and the stability to heat decreased rapidly at a temperature of 45 ℃ or higher. The activity to pH was the highest at pH 7.0, and it was confirmed that the relative activity was maintained at 80% of the maximum activity at pH 6.0 to pH 9.0.

Example 3: Effects of metal ions on the activity of lactose oxidizing enzymes and substrate specificity

The activity of the purified enzyme was shown in the specificity (Table 3) according to various metal ions (Table 2) and substrates.

The DCIP activity assay was determined by pre-incubation of the enzyme with 1 mM of various metal ions and reagents for 1 hour. The activity was then measured in the presence of metal ions or reagents. The assayed activity without metal ions and reagents was reported as 100%. The activity of this lactose oxidizing enzyme was inhibited by more than 30% of the maximum activity by ZnCl ₂ , AlCl ₃ , CuCl ₂ , etc., and the activity was improved by FeCl ₂ .

The reaction specificity of this enzyme according to various substrates shows that it reacts with disaccharides such as lactose, cellobiose and maltose, while other compounds do not act as substrates. The values of Michaelis constant (K _m ) and maximum velocity (V _max ) of enzymes involved in lactose production were determined using a sugar substrate at a concentration ranging from 0.2 to 4.5 mM. Values of K _m and V _max were determined from Lineweaver-Burk plots using standard linear regression techniques.

Table 2 below shows the effect of metal ions on lactose oxidizing enzymes.

Inhibitor (1 mM) Relative activity (%) Control 100 CaCl ₂ 72.8±0.8 MgCl ₂ 67.3±1.6 ZnCl ₂ 29.7±2.1 FeCl ₂ 109.4±1.2 AlCl ₃ 30.1±3.8 CuCl ₂ 12.4±2.7 MnCl ₂ 75.2±1.1 NiCl ₂ 65.1±2.4 CoCl ₂ 63.7±0.7 KCl 97.5±0.4 NaCl 95.3±0.9

Table 3 below shows the relative substrate specificity of the lactose oxidizing enzyme.

Substrate Relative activity (%) V _{max (mM/min)} K _m (mM) K _cat (S ^-One ) K _cat /K _m
(S ^-One ·mM ^-One ) Lactose 100 0.05 ± 0.018 0.46 ± 0.17 12.62 27.43 Cellobiose 64.34 0.03 ± 0.014 0.22 ± 0.19 7.57 34.41 Maltose 49.26 0.02 ± 0.007 0.66 ± 0.21 5.05 7.65 Sucrose - ^a - - - - D-glucose - - - - - D-galactose - - - - - D-xylose - - - - - D-mannose - - - - - L-Arabinose - - - - - D-fructose - - - - -

^a : No conversion.

Example 4: Lactobionic acid production from transformed E. coli

Using E. coli ( E. coil ), the purified enzyme was selected as an enzyme involved in the production of lactobionic acid, and an experiment was performed as follows to prepare a recombinant strain for the production of lactobionic acid.

Experimental Example 1: Recombinant plasmid preparation

Enzyme malate:quinone oxidoreductase (MQO, EC 1. 1. 99. 16, GenBank accession number WP_048378186) involved in the production of lactobionic acid from Pseudomonas tetrorence, which has excellent lactobionic acid production ability through lactose (lactose) oxidation. 1) From the Pseudomonas tetrorence genome gene template, forward (SEQ ID NO: 1 SEQ ID NO: MQO_ Xba I_F 5'-TCTAGAAGGAATATGATATGGCGCATAACGAAGCAGTC-3 '), reverse (SEQ ID NO: 2 SEQ ID NO: MQO_ BamH I_R 5'-GGATCCTTACGCGATGCTGG 3') through a polymerase chain reaction using a primer (once at 94°C for 5 minutes; 30 times at 94°C for 30 seconds, at 55°C for 2 minutes for 20 seconds, at 72°C for 30 seconds; once at 72°C for 7 minutes) Xba I as a forward primer The BamHI recognition site was present in the reverse primer to amplify the MQO gene. The amplified gene was isolated through agar gel electrophoresis, and the amplified and isolated DNA fragment was digested with Xba I and BamH I, and then introduced into pDSK519, a conventional plasmid vector for E. coli cleaved with Xba I and BamHI , into a recombinant plasmid was produced.

Experimental Example 2: Preparation of Recombinant Strain

The plasmid pDSK519-MQO prepared in Experimental Example 1 was introduced into recombinant E. coli ( E. coli JM109 Δgcd) by heat shock (42° C.) and transformed. Escherichia coli also possesses the gene for Quinoprotein glucose dehydrogenase (gdh), which is an enzyme involved in the production of lactobionic acid in the presence of the coenzyme PQQ, so the gene was removed by the Quick and Easy Conditional Knockout Kit (Gene Bridges, Heidelberg, Germany) method (Knock- Out) and used as a negative control. The pDSK519 plasmid has a kanamycin resistance gene as a selection marker, colonies into which pDSK519 has been introduced in LB-agar medium containing kanamycin were selected, and the DNA sequencing of the PCR product through colony PCR of the selected colonies (Macrogen, Korea) was confirmed to confirm MQO gene transformation, and recombinant E. coli was plated on an LB (Luria-Bertani) plate (1 % tryptone, 0.5 % yeast extract, 1 % NaCl, 1,5 % agar) in a stationary incubator at 37 ° C. Incubated for 24 hours. Colonies of strains grown on LB plates were inoculated into 200 mL of LB broth containing antibiotics, and OD (600 nm) was 0.6 (measured with a spectrophotometer) at 200 rpm in a stirred incubator at 37 ° C. It was cultured. Through this, a recombinant strain was developed in which the pDSK519-MQO, a recombinant plasmid, was transformed into E. coli.

Example 5: Preparation of MQO-overexpressed recombinant E. coli and confirmation of lactobionic acid production capacity using the same

MQO (malate:quinone oxidoreductase) obtained from wild-type Pseudomonas tetrorence species is a water-soluble glucose dehydrogenase using pyrroloquinolinequinone (PQQ) as a coenzyme. To check whether or not the control group E. coli ( E.coli △ gcd-pDSK) and the new transformed E. coli (E. coli △gcd-pDSK519-MQO) were cultured in a lactose medium containing coenzymes such as PQQ and NAD, the cells were grown and lactobionic acid conversion products were identified.

E. coli ( E. coil △gcd /pDSK519-MQO) strain introduced with MQO gene and control ( E. coil △ gcd /pDSK519) were each added to 2ml of LB medium (Tryptone 10g, Yeast Extract 5g, NaCl 5g) with antibiotic ampicillin. Including 50 μg/ml, incubated for 16 hours at 30°C, and after that, control E. coli and transformed E. coli were lactose 25 g/L, CaCO in 5 ml of LB medium so that the initial OD (600 nm) of each cell was 0.2. ₃ 3 g/L and 20 μM of PQQ or NAD were inoculated and cultured in a stirred incubator at 30° C. and 200 rpm, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the final concentration after 4 hours from the start of the culture. 1 mM was added and cultured for a total of 24 hours. Lactose and lactobionic acid were analyzed using Agilent's high-performance liquid chromatography (HPLC 1260 model) equipped with a Refractive Index Detector (RID). A Coregel ION 300 column was used as the column, and the column temperature was maintained at 70°C. The mobile phase was 0.5mM H ₂ SO ₄ , and the flow was 0.3 ml/min. Under the above conditions, the retention times of lactose and lactobionic acid were confirmed to be 19.2 minutes and 20.4 minutes, respectively, as shown in FIG. 3 .

In E. coli, which overexpressed MQO involved in the production of Pseudomonas tetrorence-derived lactobionic acid in this way, it was confirmed that a lactobionic acid peak not appearing in the control E. coli appeared only under the condition in which the coenzyme PQQ was added. It was confirmed that recombinant E. coli had the ability to convert lactobionic acid by metabolizing lactose by the introduction of the gdh3 gene (FIG. 4).

As a result of comparing the lactobionic acid productivity through lactose 25 g/L fermentation of the wild-type strain and the recombinant strain (Fig. 5), the conversion of lactose metabolism of 23 g/L was completed at 37 hours in the culture condition in which the coenzyme PQQ was added. g/L of lactobionic acid was produced, and it was possible to develop a recombinant E. coli strain capable of metabolizing lactobionic acid with a productivity of 0.62 g/L/h.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Novel lactose oxidase from Pseudomonas and method for producing lactobionic acid using the same <130> P20-124 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> MQO XbaI F <400> 1 tctagaagga atatgatatg gcgcataacg aagcagtc 38 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> MQO BamHI R <400> 2 ggatccttac gcgatgctgg tgttcggttc 30 <210> 3 <211> 1500 <212> DNA <213> Pseudomonas taetrolens <400> 3 atggcgcata acgaagcagt cgacgtagta ctggttggag cgggcatcat gagtgccacc 60 ctcgccgtac tgctcaaaga gctcgacccc ggcattaagc tggaagttgt tgagcagatg 120 gactcaggtg ctgcggagag ttccaacccg tggaacaacg caggcaccgg ccacgccggg 180 ctgtgcgaat tgaactatac gccgcaagcg gctgacggtt cgatcgacat caaaaaggct 240 gtgcatatca atgcacagtt cgaagtctcg aaacagtttt gggcctacct ggcgcaacaa 300 gggaccttcg gctcatgcaa atccttcatc agcccagtgc ctcacctgag ctacgtgcaa 360 ggtgaaaaag gtgtgtcctt cctgaaaaaa cgttttgaag tgctgagcaa gcaccatgcg 420 ttttcagaca tggaatacac cgaagacaag gcaaaaatgg ctgaatggat gccattgatg 480 atgccgggcc gcccggcaga cgaagtcatc tccgccaccc gtgtcatgca cggcaccgac 540 gtcaacttcg gcgccctgac ccagcaactg ctcaagcatt tggccagcgc tcccgacacg 600 cagatcaaat acagcaaaaa agtcactggc ctcaagcgca atggcagcgg ctggacagtg 660 agcatcaaag acaccaacag cggcaacacg cgccacatcg atgccaaatt cgtgttcctc 720 ggtgccggtg gcgcggcctt gccgctgctg caagcctcgg gcattgagga aagcaaaggc 780 tacggcggtt tcccggtcag cggccagtgg ctgcgttgcg acaacccgga agtggtcaag 840 cttcaccagg ccaaggtcta cagccaggcg gccgtaggct ccccaccgat gtcggtaccg 900 catctggaca cccgtgtcgt ggacggcaag aagtccctgt tgtttggacc gtatgccggc 960 ttcacgacca agttcctcaa gcacggttcg ttcatggacc tgccgatgtc gattcgcttg 1020 ggtaatatcg gaccaatgct cgccgtcgcc cgtgacaaca tggatctgac caagtacctg 1080 gtcagtgagg tccgtcaatc gatggagcaa cgcctggact ccctgcgtcg cttctatcca 1140 caggccaagg ccgaagactg gcgtcttgag atcgcgggcc aacgggttca gatcatcaag 1200 aaagacccga aaaaaggcgg cgtgctgcag ttcggtaccg aactggttgc agccaaggac 1260 ggctcattgg ccgccctgct cggcgcctct ccgggcgctt cggtgacggt atcgatcatg 1320 ctcgacctga tcgagcgctg cttcccgcaa aaagcagctg gcgagtgggc gaccaagctc 1380 aaggaaattt tcccggcacg ggaaaaaacc cttgagaccg acgctgagtt gtaccgtgaa 1440 atcagcaccc gcaactccga gttgctggaa ctggtagaac cgaacaccag catcgcgtaa 1500 1500 <210> 4 <211> 499 <212> PRT <213> Pseudomonas taetrolens <400> 4 Met Ala His Asn Glu Ala Val Asp Val Val Leu Val Gly Ala Gly Ile 1 5 10 15 Met Ser Ala Thr Leu Ala Val Leu Leu Lys Glu Leu Asp Pro Gly Ile 20 25 30 Lys Leu Glu Val Val Glu Gln Met Asp Ser Gly Ala Ala Glu Ser Ser 35 40 45 Asn Pro Trp Asn Asn Ala Gly Thr Gly His Ala Gly Leu Cys Glu Leu 50 55 60 Asn Tyr Thr Pro Gln Ala Ala Asp Gly Ser Ile Asp Ile Lys Lys Ala 65 70 75 80 Val His Ile Asn Ala Gln Phe Glu Val Ser Lys Gln Phe Trp Ala Tyr 85 90 95 Leu Ala Gln Gln Gly Thr Phe Gly Ser Cys Lys Ser Phe Ile Ser Pro 100 105 110 Val Pro His Leu Ser Tyr Val Gln Gly Glu Lys Gly Val Ser Phe Leu 115 120 125 Lys Lys Arg Phe Glu Val Leu Ser Lys His His Ala Phe Ser Asp Met 130 135 140 Glu Tyr Thr Glu Asp Lys Ala Lys Met Ala Glu Trp Met Pro Leu Met 145 150 155 160 Met Pro Gly Arg Pro Ala Asp Glu Val Ile Ser Ala Thr Arg Val Met 165 170 175 His Gly Thr Asp Val Asn Phe Gly Ala Leu Thr Gln Gln Leu Leu Lys 180 185 190 His Leu Ala Ser Ala Pro Asp Thr Gln Ile Lys Tyr Ser Lys Lys Val 195 200 205 Thr Gly Leu Lys Arg Asn Gly Ser Gly Trp Thr Val Ser Ile Lys Asp 210 215 220 Thr Asn Ser Gly Asn Thr Arg His Ile Asp Ala Lys Phe Val Phe Leu 225 230 235 240 Gly Ala Gly Gly Ala Ala Leu Pro Leu Leu Gln Ala Ser Gly Ile Glu 245 250 255 Glu Ser Lys Gly Tyr Gly Gly Phe Pro Val Ser Gly Gln Trp Leu Arg 260 265 270 Cys Asp Asn Pro Glu Val Val Lys Leu His Gln Ala Lys Val Tyr Ser 275 280 285 Gln Ala Ala Val Gly Ser Pro Pro Met Ser Val Pro His Leu Asp Thr 290 295 300 Arg Val Val Asp Gly Lys Lys Ser Leu Leu Phe Gly Pro Tyr Ala Gly 305 310 315 320 Phe Thr Thr Lys Phe Leu Lys His Gly Ser Phe Met Asp Leu Pro Met 325 330 335 Ser Ile Arg Leu Gly Asn Ile Gly Pro Met Leu Ala Val Ala Arg Asp 340 345 350 Asn Met Asp Leu Thr Lys Tyr Leu Val Ser Glu Val Arg Gln Ser Met 355 360 365 Glu Gln Arg Leu Asp Ser Leu Arg Arg Phe Tyr Pro Gln Ala Lys Ala 370 375 380 Glu Asp Trp Arg Leu Glu Ile Ala Gly Gln Arg Val Gln Ile Ile Lys 385 390 395 400 Lys Asp Pro Lys Lys Gly Gly Val Leu Gln Phe Gly Thr Glu Leu Val 405 410 415 Ala Ala Lys Asp Gly Ser Leu Ala Ala Leu Leu Gly Ala Ser Pro Gly 420 425 430 Ala Ser Val Thr Val Ser Ile Met Leu Asp Leu Ile Glu Arg Cys Phe 435 440 445 Pro Gln Lys Ala Ala Gly Glu Trp Ala Thr Lys Leu Lys Glu Ile Phe 450 455 460 Pro Ala Arg Glu Lys Thr Leu Glu Thr Asp Ala Glu Leu Tyr Arg Glu 465 470 475 480 Ile Ser Thr Arg Asn Ser Glu Leu Leu Glu Leu Val Glu Pro Asn Thr 485 490 495 Ser Ile Ala

Claims (12)

A recombinant vector for producing a recombinant strain for producing lactobionic acid, comprising a nucleic acid sequence encoding malate dehydrogenase derived from a microorganism of the genus Pseudomonas. The recombinant vector according to claim 1, wherein the malate dehydrogenase has lactose oxidizing activity. The recombinant vector according to claim 1, wherein the maleate dehydrogenase has an action temperature of 15°C to 45°C, and a relative enzyme activity of 60% or more is maintained at a pH of 6.5 to pH 9.0. [Claim 3] The recombinant vector according to claim 1, wherein the nucleic acid sequence is represented by the third sequence in SEQ ID NO: 3. The recombinant vector according to claim 1, wherein the malate dehydrogenase comprises an amino acid sequence represented by SEQ ID NO: 4 in SEQ ID NO:. The method according to claim 1, wherein the malate dehydrogenase is malate:quinone oxidoreductase (EC 1. 1. 99. 16, GenBank accession number WP_048378186.1). Recombinant Vector. A recombinant strain for producing lactobionic acid, transformed with the recombinant vector according to claim 1 . The recombinant strain according to claim 5, wherein the recombinant strain is recombinant E. coli, which, unlike wild-type E. coli, produces a lactose oxidizing enzyme to produce lactobionic acid from lactose. Pseudomonas ( Pseudomonas ) Obtaining a recombinant vector comprising a nucleic acid sequence encoding malate dehydrogenase (malate dehydrogenase) derived from a microorganism; and
introducing the recombinant vector into Gram-negative bacteria
A method for producing a recombinant strain for lactobionic acid production comprising a. A method for producing lactobionic acid using the recombinant strain according to claim 7. 11. The method of claim 10, wherein the method for producing lactobionic acid, characterized in that the addition of the coenzyme PQQ (pyrroloquinoline quinone) to the recombinant strain. A method for producing lactobionic acid using malate dehydrogenase derived from a microorganism of the genus Pseudomonas, wherein the malate dehydrogenase has lactose oxidizing activity. How to produce.

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