CN115322976B

CN115322976B - Glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase and encoding gene and application thereof

Info

Publication number: CN115322976B
Application number: CN202210677494.1A
Authority: CN
Inventors: 郑兆娟; 黄礼荣; 刘鹏; 欧阳嘉; 李鑫
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2024-01-30
Anticipated expiration: 2042-06-15
Also published as: CN115322976A

Abstract

The invention discloses a glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase and a coding gene and application thereof. The glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase provided by the invention can be expressed by a host cell to produce the enzyme, and the enzyme has a wide substrate spectrum and can oxidize the anomeric carbon hydroxyl groups of monosaccharides and disaccharides into carboxyl groups to generate corresponding sugar acids. The enzyme and the host expressing the enzyme can be used as a biocatalyst for preparing a series of sugar acids with high added value, the yield of target products is high, byproducts are avoided, the conversion time is short, and the preparation method is simple, mild in condition and environment-friendly, and has an industrialization prospect.

Description

Glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase and encoding gene and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to a glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase and a coding gene and application thereof.

Background

Both the aldose and the reducing disaccharide in the monosaccharide contain free anomeric carbon, the hydroxyl group on the anomeric carbon can be oxidized into carboxyl group by the catalyst to generate the corresponding sugar acid. For example, the products of oxidation of the anomeric carbon hydroxyl groups of glucose, galactose and lactose are gluconic acid, galactonic acid and lactobionic acid, respectively. The sugar acids have important application value. For example, gluconic acid and galactonic acid can be used as food acidity regulators by selectively replacing citric acid, and can be used for developing related products such as sweeteners, drug intermediates, dispersants and the like; the lactobionic acid can be used for moisturizers in cosmetics, additives in food industry, drug carriers in medicine industry and the like, and has extremely wide application fields. The preparation method of the sugar acid generally comprises a chemical method and a biological method, and the chemical method is mainly adopted at present. The biological method generally uses enzyme or microorganism cells containing specific enzyme as a catalyst, has mild reaction conditions and is environment-friendly, and the related defects of the chemical method can be greatly avoided. However, the reports of related enzymes are few at present, and most enzymes have the defects of low substrate concentration, long conversion time, difficult cofactor regeneration, low conversion rate and the like.

There are a large number of proteins annotated as the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase (glucose/quick/shikimate family membrane-bound PQQ-dependent dehydrogenase) in the NCBI database, and the specific function of these proteins has not been verified, and very few literature reports on this class of enzymes have been reported.

Fully excavates gene resources, screens out enzymes with wide substrate spectrum and strong catalytic capability for sugar acid production, and is key to realizing industrialization of biological sugar acid preparation process.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention provides a glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase, and a coding gene and application thereof, and provides a new enzyme source for a biological sugar acid preparation process.

In order to solve the technical problems, the invention discloses a glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase which is a protein with an amino acid sequence shown as SEQ ID No.2 or a protein which has at least 80% homology with SEQ ID No.2 and has the protein function shown as SEQ ID No. 2.

The invention further provides a nucleotide sequence for encoding the PQQ-dependent membrane-bound dehydrogenase of the glucose quinic acid shikimate family.

Preferably, the nucleotide sequence encoding the amino acid sequence shown as SEQ ID No.2 is shown as SEQ ID No. 1.

There is provided a genetically engineered expression vector comprising the polynucleotide described above. Methods well known to those skilled in the art can be used to construct the recombinant expression vector. These methods include recombinant DNA techniques, DNA transformation techniques, and the like. The DNA encoding the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase may be operably linked to multiple cloning sites in a vector to direct mRNA synthesis to express the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase, or for homologous recombination. In a preferred embodiment of the present invention, pBBRMCS-2 is used as the expression vector.

In another aspect, the invention provides a genetically engineered expression strain comprising a recombinant expression vector as described above or a polynucleotide as described above integrated into the genome.

The original strain of the genetically engineered expression strain is pseudomonas putida or genetically modified pseudomonas putida, wherein the genetically modified pseudomonas putida is obtained by deleting a glucose dehydrogenase encoding gene gcd on the genome of the pseudomonas putida. The Pseudomonas putida is preferably KT2440 (ATCC No. 47054).

In one embodiment, the gene encoding the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase may be amplified first, then ligated with a plasmid, and the resulting recombinant plasmid is electrotransformed into competent cells of pseudomonas putida, thereby obtaining a genetically engineered expression strain. Meanwhile, the host pseudomonas putida can be genetically modified, and the glucose dehydrogenase encoding gene gcd is deleted, so that the genetically engineered expression strain is obtained.

In another embodiment, the gene encoding the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase may be integrated into the Pseudomonas putida genome, and a Pseudomonas putida engineering strain may be constructed as a genetically engineered expression strain instead of the glucose dehydrogenase encoding gene gcd therein.

The invention further provides the PQQ-dependent membrane-bound dehydrogenase of the glucose quinic acid shikimate family, or a nucleotide sequence for encoding the dehydrogenase, or application of the genetically engineered expression strain in preparing corresponding sugar acids by oxidizing the anomeric carbon hydroxyl groups of monosaccharides and disaccharides, wherein the monosaccharides are aldoses, and the disaccharides are disaccharides with reducibility.

When the method is used, monosaccharide and disaccharide are used as substrates, a genetic engineering expression strain or the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase is used as a catalyst, and an acid neutralizer is added at the same time to perform a conversion reaction to obtain the corresponding sugar acid compound.

Preferably, the acid neutralizer is preferably calcium carbonate or sodium hydroxide.

Specifically, the conditions of the conversion reaction are: the reaction temperature is 25-40 ℃, the reaction pH is 5.5-7.5, and the reaction system contains metal ion Mg ²⁺ Or Fe (Fe) ³⁺ Or Ca ²⁺ 0.1 to 2.0mM. The temperature is preferably 30-35 ℃, the pH is preferably 6.0-6.5, and the metal ion is preferably Mg ²⁺ The Mg is ²⁺ The concentration is preferably 0.5 to 1.0mM.

The beneficial effects are that: the glucose quinic acid shikimic acid family PQQ-dependent membrane-bound dehydrogenase substrate has wide spectrum, strong catalytic capability, low similarity with other enzymes with activities of oxidized monosaccharide and disaccharide anomeric carbon hydroxyl reported in the current literature, obvious difference and more gene resources for constructing genetic engineering bacteria with high-efficiency sugar acid production capability by using genetic engineering technology. The genetic engineering bacteria and the biological conversion process of the genetic engineering bacteria on the series of monosaccharides and disaccharides can realize the efficient preparation of a series of high-added-value sugar acids, the yield of target products is high, byproducts are avoided, the conversion time is short, and the preparation method is simple, mild in condition and environment-friendly, and has good industrialization prospect.

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

In the following examples, the sugar acid yield was calculated as follows:

example 1 PCR amplification of the glucose quinic acid shikimate family PQQ-dependent Membrane-associated dehydrogenase encoding Gene and construction of genetically engineered bacteria.

The PQQ-dependent membrane-bound dehydrogenase encoding gene sieve of the glucose quinic acid shikimate family is selected from Pseudomonas fragi (Pseudomonas fragi) NL20W, and the strain is preserved in China center for type culture Collection with a preservation date of 2021 and a preservation number of CCTCC NO: M2021245.

Genomic DNA of Pseudomonas fragi NL20W was extracted using the Promega genome extraction kit. The target gene is obtained by PCR amplification from the genome DNA by using a synthesized primer, the nucleotide sequence of the target gene is shown as SEQ ID NO.1, and the coding amino acid sequence of the target gene is shown as SEQ ID NO. 2. By Blast comparison, the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase protein sequence has very low similarity with the enzyme protein sequence which has the capacity of preparing corresponding sugar acid by oxidizing monosaccharide and disaccharide anomeric carbon hydroxyl and has been reported. Specifically, the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase in the present invention has a similarity of 67.99% to glucose dehydrogenase from pseudomonas putida KT2440 (ATCC No. 47054). Glucose dehydrogenase (gcd) from Pseudomonas putida KT2440 (ATCC No. 47054) is capable of oxidizing xylose and galactose, but is very weak in oxidizing lactose (see comparative example 1) and is incapable of oxidizing arabinose (see comparative example 2). The substrate spectrum of the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase is different from that of the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase, and various substrates such as xylose, galactose, lactose and the like, arabinose and the like can be oxidized.

According to conventional molecular biology methods, use is made ofUni Seamless Cloning and Assembly Kit (from all)Gold of formula (la) the target gene obtained by PCR amplification was cloned into pBBR1MCS-2 plasmid (available from Addgene). The specific operation is as follows:

(1) Designing two pairs of primers for amplifying target genes and plasmids respectively;

(2) The primers for amplifying the target gene are as follows:

an upstream primer: 5'-TTACTCAGCCAGTTTGAACG-3'

A downstream primer: 5'-ATGAGCACTGAAGGTGCTTT-3'

(3) The primers for amplifying the plasmid are:

an upstream primer: 5'-AAAGCACCTTCAGTGCTCATAGCTGTTTCCTGTGTGAAAT-3'

A downstream primer: 5'-CGTTCAAACTGGCTGAGTAAGCGTTAATATTTTGTTAAAA-3'

(4) The PCR product was purified using a purification kit and then followed by the following stepsUni Seamless Cloning and Assembly Kit (Beijing full gold Biotechnology Co., ltd.) manual to link the fragment of interest to the plasmid.

(5) The resulting recombinant plasmid was electrotransformed into competent cells of Pseudomonas putida KT2440 (ATCC No. 47054). Among them, competent preparation method and electrotransformation method are described in patent CN113073072a. After electrotransformation, transferring the bacterial liquid in the electrorotating cup into a centrifuge tube, and culturing for 1h on a shaking table at 30 ℃ for cell resuscitation. After resuscitating, the cells are screened on an LB solid plate containing kanamycin resistance, grown colonies are picked and inoculated into an LB liquid culture medium containing kanamycin resistance, the culture is carried out at 30 ℃ until the medium phase of logarithm, and the thalli are collected and stored in a low-temperature refrigerator for standby. The strain is the genetically engineered expression strain in the invention.

Example 2: the genetically engineered expression strain prepared in example 1 was used as a biocatalyst to oxidize lactose.

Taking one-loop bacteria from an agar slant culture medium (components: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, kanamycin 50mg/L and agar powder 18 g/L) for preserving pseudomonas putida genetic engineering expression strains, inoculating the one-loop bacteria into a liquid culture medium (components: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L and kanamycin 50 mg/L), and culturing for 12 hours at 30 ℃ and 200rpm to activate strains; and (3) inoculating the activated bacterial liquid into the same culture medium according to the volume percentage of 1%, performing expansion culture, performing centrifugal collection on bacterial cells after culturing for 10 hours at 30 ℃ and 200rpm, washing the bacterial cells for 2 times by using physiological saline, and performing centrifugal separation to obtain the microbial whole cells, namely the biocatalyst.

The cells were resuspended in phosphate buffer (200 mM, pH 7.0) and mixed with lactose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 8g dry weight/L, lactose was 50g/L, and calcium carbonate was added to give a concentration of 7.3g/L. The reaction was carried out at 30℃and pH 7.0 at 200rpm, and the concentration of lactose and lactobionic acid was measured by intermediate sampling. And calculating the lactobionic acid yield according to a formula. The data show that after 24 hours of reaction, the lactobionic acid yield was 100%.

Example 3: lactose was oxidized using the genetically engineered expression strain of example 1 as a biocatalyst.

The Pseudomonas putida genetically engineered expression strain in example 1 was used as a biocatalyst. The biocatalyst cell preparation method was the same as in example 2.

The cells were resuspended in phosphate buffer (200 mM, pH 6.0) and mixed with lactose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 8g dry weight/L, lactose was 50g/L, magnesium chloride was 0.5mM, and calcium carbonate was added to give a concentration of 7.3g/L. The reaction was carried out at 30℃and pH 6.0 at 200rpm, and the concentration of lactose and lactobionic acid was measured by intermediate sampling. And calculating the lactobionic acid yield according to a formula. The data show that after 10 hours of reaction, the lactobionic acid yield was 100%.

Example 4: the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase coding gene is integrated into the pseudomonas putida KT2440 (ATCC No. 47054) genome to replace the glucose dehydrogenase coding gene gcd, so that a pseudomonas putida engineering strain is constructed, and the pseudomonas putida engineering strain is used as a biocatalyst to oxidize lactose.

Genomic DNA of Pseudomonas fragi NL20W and Pseudomonas putida KT2440 (ATCC No. 47054) were extracted, respectively, using the Promega genome extraction kit. The upstream homology arm to gcd was amplified from Pseudomonas putida KT2440 (ATCC No. 47054) genomic DNA using the synthesized primers up-f and up-r, and the downstream homology arm to gcd was amplified from Pseudomonas putida KT2440 (ATCC No. 47054) genomic DNA using the synthesized primers down-f and down-r. The synthetic primers target-f and target-r are used for PCR amplification from Pseudomonas fragi NL20W genome DNA to obtain the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase coding gene. The vector fragment was amplified using the synthesized primers pK18-f and pK18-r with plasmid pK18mobSacB as template.

Wherein, the related primer sequences are as follows:

up-f：5’-ttcgagctcggtaccggcgaaacctggcga-3’

up-r：5’-accttcagtgctcatcgtaggttctccgtcagg-3’

down-f：5’-aaactggctgagtaagcgacaccgctcccg-3’

down-r：5’-tctagaggatccccgtccttgaggaagaactcgcg-3’

target-f：5’-ctgacggagaacctacgatgagcactgaaggtgc-3’

target-r：5’-ctgcgggagcggtgtcgcttactcagccagtttgaac-3’

pK18-f：5’-tcctcaaggacggggatcctctagagtcg-3’

pK18-r：5’-ggtttcgccggtaccgagctcgaattcgt-3’

the PCR product was purified using a purification kit and then followed by the following stepsUni Seamless Cloning and Assembly Kit an instruction manual the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase encoding gene was ligated to upstream and downstream homology arms to obtain linear fragments. The obtained linear fragment was used as a template, and the fragment was amplified in large amounts using the primers up-f and down-r. Thereafter, according to->-Uni Seamless Cloningand Assemble Kit instruction manual to join the desired fragment and the vector fragment obtained as described above to obtain a recombinant plasmid.

The resulting recombinant plasmid was electrotransformed into competent cells of Pseudomonas putida KT2440 (ATCC No. 47054). Among them, the competent preparation method and the electrotransformation method are described in example 1. Colonies on the plates were picked and inoculated into LB liquid medium containing kanamycin for cultivation at 30℃to mid-log phase. And (3) performing bacterial liquid PCR verification by using the primers up-f and down-r to obtain the bacterial strain capable of amplifying the long fragment and the short fragment simultaneously, namely the correct single exchange target bacteria. The correct single-exchange target bacteria were inoculated into LB liquid medium and cultured overnight at 30 ℃. The culture solution is transferred to an LB liquid culture medium containing 15% of sucrose for culture to mid-log phase, and transferred to an LB liquid culture medium containing 15% of sucrose for culture to mid-log phase. Properly diluting the bacterial liquid (generally 10 ^-6 Or 10 ^-7 ) Spread on LB solid plates containing 15% sucrose, and incubated at 30℃until single colonies appear. The growing single bacteria are selected, bacterial liquid PCR verification is carried out by using primers up-f and down-r, and the strain which can only amplify short fragments is obtained as the correct double-exchange target bacteria, and the original glucose dehydrogenase coding gene of the strain is completely replaced by the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase coding gene of the invention on the genome of the strain.

The Pseudomonas putida engineering strain constructed in the embodiment is used as a biocatalyst.

The biocatalyst cell preparation was the same as in example 2, except that the medium did not contain kanamycin.

The cells were resuspended in phosphate buffer (200 mM, pH 6.0) and mixed with lactose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 8g dry weight/L, lactose was 50g/L, magnesium chloride was 0.5mM, and calcium carbonate was added to give a concentration of 7.3g/L. The reaction was carried out at 35℃and pH 6.0 at 200rpm, and the concentration of lactose and lactobionic acid was measured by intermediate sampling. And calculating the lactobionic acid yield according to a formula. The data show that after 15 hours of reaction, the lactobionic acid yield was 100%.

Example 5: the genetically engineered expression strain of example 1 was used as a biocatalyst to oxidize arabinose.

The Pseudomonas putida genetically engineered expression strain in example 1 was used as a biocatalyst.

The biocatalyst cell preparation method was the same as in example 2.

The cells were resuspended in phosphate buffer (200 mM, pH 6.5) and mixed with arabinose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 4g dry weight/L, the concentration of arabinose was 50g/L, the concentration of magnesium chloride was 1.0mM, and calcium carbonate was added to give a concentration of 15g/L. The reaction was carried out at 30℃and pH 6.5 at 200rpm, and the concentration change of arabinose and arabinose concentration was detected by intermediate sampling. And calculating the yield of the arabinonic acid according to a formula. The data show that after 4 hours of reaction, the yield of arabinonic acid is 100%.

Example 6: deleting the glucose dehydrogenase encoding gene gcd to construct genetically modified pseudomonas putida.

Pseudomonas putida KT2440 (ATCC No. 47054) has the ability to oxidize xylose and galactose, and the glucose dehydrogenase encoding gene gcd on its genome is deleted, at which time the strain no longer has the ability to oxidize xylose and galactose (see comparative examples 3 and 4). The strain is taken as a host, and the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase in the invention is expressed therein, and the oxidizing ability of the genetically engineered expression strain to xylose and galactose is derived from the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase in the invention.

For deletion of the gene gcd encoding glucose dehydrogenase, see patent CN 113186232a for specific technical embodiments.

Example 7: a genetically engineered expression strain expressing the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase was constructed using the genetically engineered Pseudomonas putida of example 6 as a host.

Technical embodiment is the same as in example 1, but the competent cells of step (5) in example 1 are replaced with genetically engineered pseudomonas putida deleting gcd in example 6.

Example 8: lactose oxidation Using the genetically engineered expression Strain of example 7 as a biocatalyst

The Pseudomonas putida genetically engineered expression strain in example 7 was used as a biocatalyst. The biocatalyst cell preparation method was the same as in example 2.

The cells were resuspended in phosphate buffer (200 mM, pH 6.0) and mixed with lactose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 8g dry weight/L, lactose was 50g/L, magnesium chloride was 1.0mM, and calcium carbonate was added to give a concentration of 7.3g/L. The reaction was carried out at 30℃and pH 6.0 at 200 rpm. Intermediate sampling measures lactose and the concentration change of lactose. And calculating the lactobionic acid yield according to a formula. The data show that after 11 hours of reaction, the lactobionic acid yield was 100%.

Example 9: the genetically engineered expression strain of example 7 was used as a biocatalyst to oxidize galactose.

The cells were resuspended in phosphate buffer (200 mM, pH 6.0) and mixed with galactose, and the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 4g dry weight/L, galactose was 50g/L, magnesium chloride was 1.0mM, and calcium carbonate was added to give a concentration of 13g/L. The reaction was carried out at 30℃and pH 6.0 at 200 rpm. Intermediate sampling measures the concentration changes of galactose and galactose. And calculating the yield of the galactonic acid according to a formula. The data show that after 2.5 hours of reaction, the yield of galactonic acid is 100%.

Example 10: the genetically engineered expression strain of example 7 was used as a biocatalyst to oxidize xylose.

The cells were resuspended in phosphate buffer (200 mM, pH 6.0) and mixed with xylose, the concentration of each substance in the reaction system was adjusted so that the concentration of the biocatalyst was 4g dry weight/L, xylose was 15g/L, magnesium chloride was 1.0mM, and calcium carbonate was added to give a concentration of 5g/L. The reaction was carried out at 30℃and pH 6.0 at 200 rpm. Intermediate sampling detects changes in xylose and xylose concentration. And calculating the yield of the xylonic acid according to a formula. The data show that after 4 hours of reaction, the xylitol yield is 100%.

Comparative example 1: lactose was oxidized with Pseudomonas putida KT2440 (ATCC No. 47054) as a biocatalyst.

Pseudomonas putida KT2440 (ATCC No. 47054) is used as a biocatalyst. The biocatalyst cell preparation was the same as in example 2, except that the medium did not contain kanamycin. Biocatalytic conditions were the same as in example 2.

And calculating the lactobionic acid yield according to a formula. The data show that after 24 hours of reaction, the lactobionic acid yield was 26%.

Comparative example 2: pseudomonas putida KT2440 (ATCC No. 47054) was used as a biocatalyst for oxidation of arabinose.

Pseudomonas putida KT2440 (ATCC No. 47054) is used as a biocatalyst. The biocatalyst cell preparation was the same as in example 2, except that the medium did not contain kanamycin.

Biocatalytic conditions were the same as in example 5.

The data show that arabinonic acid was not detectable after 24 hours of reaction.

Comparative example 3: galactose was oxidized with the gcd deleted pseudomonas putida constructed in example 6 as a biocatalyst.

The genetically engineered pseudomonas putida with gcd deleted in example 6 was used as a biocatalyst.

Biocatalytic conditions were the same as in example 9.

The data show that after 24 hours of reaction, no galactonic acid could be detected.

Comparative example 4: xylose was oxidized with the gcd deleted Pseudomonas putida constructed in example 6 as biocatalyst

Biocatalytic conditions were the same as in example 10.

The data show that no xylitol was detected after 24 hours of reaction.

Comparative example 5: lactose was oxidized with the gcd deleted Pseudomonas putida constructed in example 6 as biocatalyst

Biocatalytic conditions were the same as in example 8.

The data show that no lactobionic acid was detected after 24 hours of reaction.

In conclusion, the genetically engineered bacterium and the biological conversion process of the genetically engineered bacterium on the series of monosaccharides and disaccharides can be used for efficiently preparing a series of high-added-value sugar acids, the yield of target products is high, byproducts are avoided, the conversion time is short, and the preparation method is simple, mild in condition and environment-friendly, and has a good industrialization prospect.

Sequence listing

<110> university of Nanjing forestry

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Met Ser Thr Glu Gly Ala Phe Ser Arg Ser Arg Leu Leu Pro Ser Leu

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Leu Gly Ile Leu Leu Leu Leu Met Gly Leu Ala Met Leu Ala Gly Gly

20 25 30

Ile Lys Leu Val Thr Leu Gly Gly Ser Trp Tyr Tyr Leu Leu Ala Gly

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Ile Gly Phe Gly Leu Ser Gly Ala Leu Leu Ile Ala Gly Arg Arg Ala

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Ala Leu Ala Leu Tyr Ala Leu Thr Leu Phe Ala Ser Thr Val Trp Ala

65 70 75 80

Leu Met Glu Val Gly Leu Asp Trp Trp Gln Leu Val Pro Arg Leu Ala

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Met Trp Phe Ala Ile Gly Ile Val Leu Leu Leu Pro Trp Phe Arg Arg

100 105 110

Pro Val Leu Arg Gly Gln Ser Ala Pro Leu Ala Thr Gly Ala Leu Ser

115 120 125

Val Ala Val Val Leu Ala Gly Ala Ala Ala Leu Ala Ser Gln Phe Thr

130 135 140

Ser Pro Gly Glu Ile Lys Gly Gln Leu Asp Arg Asp Ala Val Pro Gly

145 150 155 160

Met Thr Asn Ala Ala Pro Ala Met Pro Asp Gly Asp Trp Gln Ser Tyr

165 170 175

Gly Arg Thr Ala Phe Gly Asp Arg Tyr Ser Pro Leu Lys Glu Ile Thr

180 185 190

Pro Glu Asn Ala His Lys Leu Val Pro Ala Trp Thr Phe Arg Thr Gly

195 200 205

Asp Met Pro Gly Glu Gly Asp Pro Gly Glu Thr Thr Ala Glu Asn Thr

210 215 220

Pro Leu Lys Val Asn Gly Met Leu Tyr Val Cys Thr Pro His Ser Gln

225 230 235 240

Val Ile Ala Leu Asp Pro Asp Thr Gly Lys Glu Ile Trp Arg Tyr Asp

245 250 255

Pro Lys Ile Ser Thr Gln Asn Ala Glu Asn Phe Lys Gly Trp Ala His

260 265 270

Met Thr Cys Arg Gly Val Thr Tyr His Asp Glu Asn Ala Tyr Ala Lys

275 280 285

Ala Ser Thr Glu Gln Ser Ala Ala Glu Pro Ala Ala Ala Thr Ser Ser

290 295 300

Asn Ser Cys Pro Arg Arg Leu Phe Leu Pro Thr Ala Asp Thr Arg Leu

305 310 315 320

Ile Ala Leu Asn Ala Asp Thr Gly Lys Pro Cys Glu Asp Phe Gly Asp

325 330 335

His Gly Ser Val Asp Leu Arg His Asn Ile Gly Ser Phe Ala Pro Gly

340 345 350

Gly Tyr Tyr Ser Thr Ser Pro Pro Ala Val Thr Lys Asp Leu Val Val

355 360 365

Ile Gly Gly His Val Thr Asp Asn Ile Ser Asn Asp Glu Pro Ser Gly

370 375 380

Val Ile Arg Ala Tyr Asp Val Arg Thr Gly Lys Leu Val Trp Asn Trp

385 390 395 400

Asp Ser Gly Asn Pro Glu Lys Thr Thr Pro Ile Ala Glu Gly Glu Thr

405 410 415

Tyr Thr Arg Asn Ser Pro Asn Met Trp Ser Met Phe Ala Val Asp Glu

420 425 430

Asp Leu Gly Met Leu Tyr Leu Pro Met Gly Asn Gln Thr Pro Asp Gln

435 440 445

Phe Gly Gly Asp Arg Thr Glu Asp Ser Glu Arg Tyr Ala Ala Gly Ile

450 455 460

Thr Ala Leu Asp Ile Asn Thr Gly Lys Val Arg Trp Tyr Arg Pro Leu

465 470 475 480

Thr His His Asp Leu Trp Asp Met Asp Val Gly Gly Gln Pro Thr Leu

485 490 495

Met Asp Leu Lys Thr Ala Asp Gly Val Lys Pro Ala Leu Leu Ala Ser

500 505 510

Thr Lys Gln Gly Ser Ile Tyr Val Met Asp Arg Arg Thr Gly Glu Ala

515 520 525

Ile Val Pro Ile Thr Glu Ile Pro Ala Pro Gly Gly Ala Val Glu Gly

530 535 540

Asp His Thr Ala Pro Thr Gln Pro Arg Ser Asp Leu Asn Met Ile Pro

545 550 555 560

Pro Val Leu Thr Glu Arg Asp Met Trp Gly Val Thr Pro Phe Asp Gln

565 570 575

Met Leu Cys Arg Ile Asn Phe Lys Ser Leu Arg Tyr Asp Gly Met Tyr

580 585 590

Thr Pro Pro Ser Leu Gln Gly Ser Ile Val Tyr Pro Gly Asn Phe Gly

595 600 605

Val Phe Asp Trp Gly Gly Ile Ser Val Asp Pro Val Arg Gln Ile Ala

610 615 620

Phe Leu Asn Pro Ser Tyr Met Ala Phe Thr Ser Lys Leu Val Pro Gln

625 630 635 640

Ala Asp Val Ala Ala Met Gly Pro Arg Lys Gly Glu Thr Ser Gly Val

645 650 655

Gln Pro Asn Lys Gly Ala Pro Tyr Gly Val Ile Leu Glu Pro Leu Leu

660 665 670

Ser Pro Leu Gly Leu Pro Cys Gln Ala Pro Ala Trp Gly Tyr Val Ala

675 680 685

Ala Val Asp Leu Thr Asn Asn Glu Val Ile Trp Lys His Lys Asn Gly

690 695 700

Thr Val Arg Asp Ser Ser Pro Val Pro Ile Pro Leu Ser Met Gly Val

705 710 715 720

Pro Ser Leu Gly Gly Thr Phe Thr Thr Ala Gly Gly Val Ala Phe Leu

725 730 735

Ser Gly Thr Leu Asp Gln Tyr Leu Arg Ala Tyr Asp Val Ser Asn Gly

740 745 750

Lys Val Leu Trp Glu Gly Arg Leu Pro Ala Gly Gly Gln Thr Thr Pro

755 760 765

Met Thr Tyr Thr Gly Lys Asp Gly Thr Gln Tyr Val Leu Val Met Ala

770 775 780

Gly Gly His Gly Gly Leu Gly Thr Lys Lys Gly Asp Tyr Val Met Ala

785 790 795 800

Phe Lys Leu Ala Glu

805

<210> 3

<211> 20

<212> DNA

<213> an upstream primer for amplifying a target Gene (Artificial Sequence)

<400> 3

ttactcagcc agtttgaacg 20

<210> 4

<211> 20

<212> DNA

<213> amplification of the primer for downstream of the target Gene (Artificial Sequence)

<400> 4

atgagcactg aaggtgcttt 20

<210> 5

<211> 40

<212> DNA

<213> amplification of the upstream primer of the plasmid (Artificial Sequence)

<400> 5

aaagcacctt cagtgctcat agctgtttcc tgtgtgaaat 40

<210> 6

<211> 40

<212> DNA

<213> amplification of the downstream primer of the plasmid (Artificial Sequence)

<400> 6

cgttcaaact ggctgagtaa gcgttaatat tttgttaaaa 40

<210> 7

<211> 30

<212> DNA

<213> up-f(Artificial Sequence)

<400> 7

ttcgagctcg gtaccggcga aacctggcga 30

<210> 8

<211> 33

<212> DNA

<213> up-r(Artificial Sequence)

<400> 8

accttcagtg ctcatcgtag gttctccgtc agg 33

<210> 9

<211> 30

<212> DNA

<213> down-f(Artificial Sequence)

<400> 9

aaactggctg agtaagcgac accgctcccg 30

<210> 10

<211> 35

<212> DNA

<213> down-r(Artificial Sequence)

<400> 10

tctagaggat ccccgtcctt gaggaagaac tcgcg 35

<210> 11

<211> 34

<212> DNA

<213> target-f(Artificial Sequence)

<400> 11

ctgacggaga acctacgatg agcactgaag gtgc 34

<210> 12

<211> 37

<212> DNA

<213> target-r(Artificial Sequence)

<400> 12

ctgcgggagc ggtgtcgctt actcagccag tttgaac 37

<210> 13

<211> 29

<212> DNA

<213> pK18-f(Artificial Sequence)

<400> 13

tcctcaagga cggggatcct ctagagtcg 29

<210> 14

<211> 29

<212> DNA

<213> pK18-r(Artificial Sequence)

<400> 14

ggtttcgccg gtaccgagct cgaattcgt 29

Claims

1. The application of glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase or a genetically engineered expression strain containing the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase in preparing corresponding sugar acid by oxidizing the anomeric carbon hydroxyl of lactose or galactose or xylose or arabinose, wherein the amino acid sequence of the glucose quinic acid shikimate family PQQ-dependent membrane-bound dehydrogenase is shown as SEQ ID No. 2.

2. The use according to claim 1, wherein the glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase is expressed by a genetically engineered expression strain, wherein the genetically engineered expression strain is obtained by transforming a recombinant expression vector into pseudomonas putida or integrating a glucose quinic acid shikimate family PQQ-dependent membrane bound dehydrogenase encoding gene into the genetically engineered pseudomonas putida genome; the recombinant expression vector comprises a coding gene for the PQQ-dependent membrane-bound dehydrogenase of the glucose quinic acid shikimate family.

3. The use according to claim 2, characterized in that the pseudomonas putida is KT2440, deposit No. ATCC No. 47054.

4. The use according to claim 2, wherein the genetically engineered pseudomonas putida has deleted a glucose dehydrogenase encoding gene on the genome of pseudomonas putidagcd。

5. The use according to claim 2, wherein the expression vector of the recombinant expression vector is a pBBRMCS-2 plasmid.

6. The use according to claim 1, wherein in the use, any one or more of lactose, galactose, xylose and arabinose is used as a substrate, the genetically engineered expression strain or the PQQ-dependent membrane-bound dehydrogenase of the glucose quinic acid shikimate family is used as a catalyst, and an acid neutralizer is added at the same time to perform a conversion reaction to obtain the corresponding sugar acid compound.

7. The use according to claim 6, wherein the acid neutralizer is calcium carbonate or sodium hydroxide.

8. The use according to claim 1, wherein the oxidation reaction conditions are: the reaction temperature is 25-40 ℃, the reaction pH is 5.5-7.5, and the reaction system contains metal ion Mg ²⁺ Or Fe (Fe) ³⁺ Or Ca ²⁺ 0.1~2.0 mM。