CN102367430B - Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid - Google Patents
Pseudoalteromonas sp.B3 and application thereof in biological oxidation of L-amino acid Download PDFInfo
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Abstract
The invention discloses pseudoalteromonas sp.B3 and application thereof to biological oxidation of L-amino acid. During the application of the pseudoalteromonas sp.B3, an enzyme cultured by fermenting the pseudoalteromonas sp.B3 is taken as an enzyme source, the L-amino acid is taken as a substrate, and the enzyme source and the substrate react for 0.5-2h at a temperature of 25-50 DEG C and the pH value of 6-8 so as to oxidize the L-amino acid and release hydrogen peroxide. The invention provides a novel microbial strain for producing L-amino acid oxidase, and the strain can be use for biologically oxidizing the L-amino acid, has the characteristics of wide substrate spectrum, mild reaction, environment friendliness and the like, and has a wide application prospect in the aspects of quantitative analysis of the L-amino acid, splitting of the DL-amino acid and the like.
Description
(1) technical field
The present invention relates to a kind of new bacterial strain that produces L-amino acid oxidase, particularly pseudoalteromonas B3 and the application in bio-oxidation L-amino acid thereof.
(2) background technology
(L-amino acid oxidase is called for short LAAO to L-amino acid oxidase, and zymetology is numbered: EC1.4.3.2) be a kind of flavin protease take flavin adenine dinucleotide (FAD) as prothetic group.The amino acid whose oxidative deamination of this endonuclease capable specificity catalysis L-generates alpha-ketoacid, ammonia and hydrogen peroxide.Therefore, it can be applied to bio-oxidation L-amino acid.In recent years bibliographical information is arranged LAAO have effect with thrombocyte interaction, cytotoxicity and cell death inducing.Also report it have hemorrhage or hemolytic activity, cause oedema and antibiotic, AIDS virus resisting (Zhang Yun etc. the application of snake venom L-amino acid oxidase in preparation treating AIDS medicine. application number: the 03117417.5. applying date: the characteristic such as 2004-09-08.).LAAO has extremely wide application prospect in antibiotic, the fields such as protozoon, antitumor and AIDS virus resisting of killing.
LAAO is found extensively to be present in the multiple organisms such as the Skin mucus, mouse, sea hare of microorganism, snake venom, fish.Up to the present, derive from some LAAO of different plant species by successfully separation, purifying and evaluation, the physico-chemical properties such as enzymic activity, substrate characteristic, enzyme stability are studied, the determined and announcement of the gene order of some LAAO.Correlative study shows, derive from the dimer that the present known LAAO of different plant species all is comprised of two non-covalent subunits in cell, molecular weight is approximately 120kDa, and the molecular weight of each subunit is about 60kDa, is that a kind of FAD of needs is the albumen that contains glycosylation site of prothetic group; Most ofly in the biological activity such as antibiotic of LAAO produce hydrogen peroxide with LAAO catalysis and have direct or indirect contacting, catalase can suppress the biological activity such as antibiotic of LAAO, but, studies show that, LAAO is secreted into the extracellular, and this facilitates the separation and purification with LAAO.Have the LAAO of the Skin mucus that derives from fish by success clonal expression in intestinal bacteria, and the existing LAAO of snake venom that derives from is by preliminary crystallization.These provide solid foundation and theoretical direction all for the correlative study of carrying out LAAO in a deep going way.Simultaneously, both domestic and external studies show that, the substrate specificity of the LAAO in different plant species source and adaptive differ greatly.Some LAAO can act on several even tens seed amino acids, and as the muddy rhodococcus LAAO of foreign literature report, it can not only 20 kinds of L-amino acid of catalysis, and can their some derivatives of catalysis; But some can only act on a seed amino acid, and silent Salmonella LAAO only can strictly act on 1B as the sea.Some species had both contained the wide LAAO of substrate specificity, also contained simultaneously the narrow LAAO of substrate specificity.Many LAAO are higher to the catalytic activity of hydrophobicity and neutral amino acids, and some LAAO is higher to the amino acid active of other types.It is important organic synthesis and biosynthetic intermediate that L-amino acid generates corresponding alpha-ketoacid after by the LAAO catalyzed oxidation, in industries such as food, medicine and chemical industry the important application prospect is arranged.Although, LAAO is had the research in three, 40 years abroad, the Different L AAO such as Skin mucus that derive from microorganism, snake venom, fish are all had dabble, relevant report also is not many especially, research mainly concentrates on venin-derived LAAO.Domestic research to LAAO only has the time of more than ten years, only has the LAAO to deriving from snake venom to possess some special knowledge, to deriving from the report that has no of other species, and, domesticly also have no report to utilizing microbial source LAAO to carry out bio-oxidation L-amino acid.
(3) summary of the invention
The object of the invention provides a kind of new bacterial strain that produces L-amino acid oxidase---pseudoalteromonas B3, and the application of described bacterial strain in oxidation L-amino acid, has substrate spectrum, reaction temperature and, the characteristics such as environmental friendliness.
The technical solution used in the present invention is:
Pseudoalteromonas B3 (Pseudoalteromonas sp.B3), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, culture presevation number is: CGMCC NO.5353, preservation date are on October 17th, 2011.
Screening and the evaluation of pseudoalteromonas B3 bacterial classification of the present invention:
A) bacterial screening
(1) bacterial classification source: separation screening obtains in the ooze of (30.03 ° of N, 122.11 ° of E) from marine site, Ningbo of Zhejiang Dinghai;
(2) bacterial screening: from marine site, Dinghai, Ningbo (30.03 ° of N, 122.11 ° E) the following ooze of collecting of sea level 50cm places aseptic sample bottle to bring to the laboratory, then takes by weighing 10g ooze sample, joining in the aseptic seawater of 90mL, namely is that concentration is 10 behind the mixing
-1Sample, then according to a conventional method respectively gradient dilution to become concentration be 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Sample; The sample 100 μ L that get respectively different concns coat MM solid medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 20g/L; PH 7.2, and solvent is water) on the flat board, in 28 ℃ of lower 4d that cultivate, obtain single bacterium colony; Then the single bacterium colony that primary dcreening operation is obtained is at MM solid medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 20g/L; PH 7.2, and solvent is water) carry out twice line on the flat board and sieve again; Single bacterium colony that then will sieve again rear acquisition is inoculated in respectively MM liquid nutrient medium (yeast extract paste 3g/L, peptone 5g/L, the sea salt 30g/L of 5mL; PH 7.2, and solvent is water) in, in 28 ℃, shaking table is cultivated 4d under the 200rpm rotating speed, and the centrifugal 5min of 8000rpm collects fermented supernatant fluid; Get the 3mL fermented supernatant fluid as enzyme liquid, L-Phe (L-Phe) reaction with 150mmol/L, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, find that pseudoalteromonas B3 can make hydrogen peroxide detection reagent generation color reaction, pseudoalteromonas B3 energy catalyzed oxidation L-Phe is described and discharges hydrogen peroxide, thereby explanation pseudoalteromonas B3 can produce L-amino acid oxidase (LAAO).
(3) strain morphology feature and physiological and biochemical property:
Described pseudoalteromonas B3 morphological specificity is: bacillus pumilis, and diameter 1.0 μ m, long 3 μ m, behind 28 ℃ of cultivation 24h, bacterium colony is point-like, circle, moistening; Gram-negative, physiological and biochemical property is shown in Table 1:
The physio-biochemical characteristics of table 1 bacterial strain B3 ("+" is positive in the table, and "-" is negative)
B) 16S rDNA Sequence Identification:
Upstream primer 5 '-GAGTTTGATCCTGGCTCAG-3 '
Downstream primer 5 '-AGAAAGGAGGTGATCCAGCC-3 '
Amplification program is: then 94 ℃ of sex change 5min are 30 circulations, and each circulation comprises 94 ℃ of sex change 1min, 55 ℃ of annealing 50s, and 72 ℃ are extended 90s, and after the loop ends, 72 ℃ are extended 10min again, then are kept under 4 ℃.After amplification finishes, be after 1.0% agarose gel electrophoresis is proved conclusively PCR product size, to send Sangon Biotech (Shanghai) Co., Ltd. to carry out dna sequencing with concentration.After order-checking finishes, the 16S rDNA sequence of bacterial strain B3 is carried out sequence analysis with other species in the NCBI website, then use MegaV4.0.2 software development Phylogenetic tree, the result as shown in Figure 5, the homology of finding some bacterium in bacterial strain B3 and the pseudoalteromonas genus is higher, especially with Pseudoalteromonas rubra G31a 98.5% homology is arranged, therefore, with bacterial strain B3 called after pseudoalteromonas B3 (Pseudoalteromonas sp.B3).
16S rDNA sequence is:
AGTCGAGCGGTA ACATTTCTAGCTTGCTAGA AGATGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACCATTGGAAACGATGGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTGATTAAGCGAGATGTGAAAGCCCCGGGCTTAACCTGGGAACTGCATTTCGAACTGGTCAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTAGGAGCTGGGGTCTTCGGACAACTTTTCCAAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTACCAGAGATGGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGATTCGGTCGGGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCATATACAGAGTGCTGCGAACTAGCGATAGTAAGCGAATCACTTAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTGGATAGCTTAACCTTCGGGAGGGCGTCA
The application of pseudoalteromonas B3 of the present invention in bio-oxidation L-amino acid.
Further, described pseudoalteromonas B3 being applied as in bio-oxidation L-amino acid: the enzyme that obtains take pseudoalteromonas B3 fermentation culture is as the enzyme source, take L-amino acid as substrate, 25～50 ℃, pH 6～8, reaction 0.5～2h, make the L-amino-acid oxidase, discharge hydrogen peroxide.
Described enzyme source is that centrifugal the discarding of wet thallus fermented liquid that contain that obtains after the pseudoalteromonas B3 fermentation culture precipitated the rear supernatant liquor that obtains, wet thallus concentration is 10～25mg/L in the described pseudoalteromonas B3 fermented liquid, described L-amino acid initial substrate concentration is 30～300mmol/L, described supernatant liquor consumption is counted 0.04～0.8mg wet thallus/mmol substrate to contain in the wet thallus fermented liquid wet thallus quality, and the described wet thallus fermented liquid that contains refers to contain the wet thallus fermented liquid before centrifugal.
Described L-amino acid is preferably L-Leu, 1B, TYR, altheine, L-glutaminate, METHIONINE, CYSTINE, L-arginine, L-Trp or Cys.
Described enzyme source prepares as follows: (1) slant culture: pseudoalteromonas B3 is inoculated in slant medium (being the MM solid medium), cultivate 12～24h for 20～37 ℃, obtain the inclined-plane thalline, described slant medium final concentration consists of: yeast extract paste 1.5～6g/L, peptone 2.5～10g/L, sea salt 15～45g/L, agar 15～25g/L, pH 7～8, and solvent is water; (2) seed culture: from inclined-plane thalline picking one transfering loop inoculation to seed culture medium (being the MM liquid nutrient medium), cultivate 18～24h for 20～37 ℃, obtain seed liquor, described seed culture medium final concentration consists of: yeast extract paste 1.5～6g/L, peptone 2.5～10g/L, sea salt 15～45g/L, pH 7～8, and solvent is water; (3) fermentation culture: seed liquor is seeded to fermention medium (being the MM liquid nutrient medium) with 1～10% inoculum size, cultivate 48～120h for 20～37 ℃, obtain fermented liquid, with fermented liquid through 8000～12000rpm, behind centrifugal 5～10min, discard precipitation, collect supernatant liquor, obtain described enzyme source; Described fermention medium final concentration consists of: yeast extract paste 1.5～6g/L, and peptone 2.5～10g/L, sea salt 15～45g/L, pH 7～8, and solvent is water.
Further, described pseudoalteromonas B3 being applied as in bio-oxidation L-amino acid: the supernatant liquor that contains the centrifugal acquisition of wet thallus fermented liquid after the pseudoalteromonas B3 fermentation culture is as the enzyme source, take L-amino acid as substrate, 30 ℃, pH7.0, reaction 0.5h makes the L-amino-acid oxidase discharge hydrogen peroxide, detects hydrogen peroxide with the hydrogen peroxide agent box; Wet thallus concentration is 10～25mg/L in the described pseudoalteromonas B3 fermented liquid, described L-amino acid initial substrate concentration is 30～300mmol/L, and described supernatant liquor consumption is counted 0.08～0.8mg wet thallus/mmol substrate to contain in the wet thallus fermented liquid wet thallus quality.
The L-amino acid oxidase that bacterial strain B3 produces among the present invention is to be secreted into extracellularly, and therefore, what obtain after the pseudoalteromonas B3 fermentation culture contains the centrifugal rear removal thalline of wet thallus fermented liquid, contains enzyme in the supernatant liquor of acquisition, i.e. the enzyme source.So supernatant liquor rather than wet thallus with the centrifugal rear acquisition of pseudoalteromonas B3 fermented liquid among the present invention carry out enzymatic reaction.The consumption of described supernatant liquor comes in the quality that contains wet thallus in the wet thallus fermented liquid before centrifugal.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the microbial strains that the new product L-amino acid oxidase of a strain is provided, this bacterial classification energy bio-oxidation L-amino acid, and have substrate wide spectrum, reaction temperature and, the characteristics such as environmentally friendly, have broad application prospects at aspects such as the amino acid whose quantitative analysis of L-, the fractionations of DL-amino acid.
(4) description of drawings
The amino acid whose checking of pseudoalteromonas B3 bio-oxidation L-: A-group 1, B-group 2, C-group 3, D-group 4 among Fig. 1 embodiment 2;
The amino acid whose checking of pseudoalteromonas B3 bio-oxidation L-among Fig. 2 embodiment 3: A-experimental group, B-positive controls, C-negative control group;
Fig. 3 pseudoalteromonas B3 colony characteristics photo;
Fig. 4 pseudoalteromonas B3 thalline Photomicrograph, A: Sarcina lutea; B: pseudoalteromonas B3;
Fig. 5 pseudoalteromonas B3 phylogenetic tree;
Fig. 6 pseudoalteromonas B3 is to Different L-amino acid whose oxidation property;
Fig. 7 temperature is on the amino acid whose impact of pseudoalteromonas B3 bio-oxidation L-;
Fig. 8 pH is on the amino acid whose impact of pseudoalteromonas B3 bio-oxidation L-;
Fig. 9 metal ion is on the amino acid whose impact of pseudoalteromonas B3 bio-oxidation L-.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1 bacterial screening and evaluation
A) bacterial screening
From collecting ooze (30.03 ° of N, 122.11 ° of E) below the sea level 50cm of marine site, Dinghai, Ningbo, place aseptic sample bottle, then take by weighing 10g ooze sample, join in the aseptic seawater of 90mL, namely be that concentration is 10 behind the mixing
-1Sample, with aseptic seawater according to a conventional method respectively gradient dilution to become concentration be 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Sample; The sample 100 μ L that get respectively different concns coat MM solid medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 20g/L; PH 7.2, and solvent is water) on the flat board, in 28 ℃ of lower 4d that cultivate, obtain single bacterium colony; Then the single bacterium colony that primary dcreening operation is obtained is at MM solid medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 20g/L; PH 7.2, and solvent is water) carry out twice line on the flat board and sieve again; Single bacterium colony that then will sieve again rear acquisition is inoculated in respectively MM liquid nutrient medium (yeast extract paste 3g/L, peptone 5g/L, the sea salt 30g/L of 5mL; PH 7.2) in, in 28 ℃, shaking table is cultivated 4d under the 200rpm rotating speed, and the centrifugal 5min of 8000rpm collects fermented supernatant fluid, obtains the enzyme source; (wet thallus concentration: 0.1mg wet thallus/mmol substrate) above-mentioned supernatant liquor is as enzyme liquid to get 3mL, L-Phe (L-Phe) 3mL reaction 0.5h with 300mmol/L, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, find that pseudoalteromonas B3 can make hydrogen peroxide detection reagent generation color reaction, pseudoalteromonas B3 energy catalyzed oxidation L-Phe is described and discharges hydrogen peroxide (seeing Table 1), thereby explanation pseudoalteromonas B3 can produce LAAO.
Table 1 uses the hydrogen peroxide detection kit to the detection of the hydrogen peroxide of reaction solution generation
| Reaction | The concentration of hydrogen peroxide (mmol/L) | Relative reactivity (%) |
| 1 | 184 | 100 |
| 2 | 14 | 7.61 |
| 3 | 4 | 2.17 |
Reaction 1: the L-Phe that adds 150mmol/L in the bacterial strain B3 fermented supernatant fluid
Reaction 2: do not add L-Phe in the bacterial strain B3 fermented supernatant fluid
Reaction 3: bacterial strain B3 fermented supernatant fluid adds the L-Phe of 150mmol/L after with 95 ℃ of pyroprocessing 10min again
B) strain identification
(1) bacterial classification morphological specificity and physiological and biochemical property:
Pseudoalteromonas B3 is inoculated in MM solid medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 30g/L; PH 7.2, and solvent is water) on the flat board, in 28 ℃ of lower 4d that cultivate, obtain single bacterium colony and observe its colony growth characteristic, as shown in Figure 3.
With single colony inoculation of pseudoalteromonas B3 in the MM of 5mL liquid nutrient medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L; PH 7.2, solvent is water) in, in 28 ℃, after shaking table is cultivated 4d under the 200rpm rotating speed, carry out gramstaining with Sarcina lutea (Sarcina lutea, CPCC160021, Chinese Microbial resources storehouse) for contrast, the microscope of its single colonial morphology (oily mirror, 1500 *) photo such as Fig. 4 (A: Sarcina lutea; B: pseudoalteromonas B3).
(2) 16S rDNA sequencing:
With single colony inoculation of bacterial strain B3 in the MM of 5mL liquid nutrient medium (yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L; PH 7.2, solvent is water) in, in 28 ℃, after shaking table is cultivated 4d under the 200rpm rotating speed, get the 1.5mL nutrient solution, abandon supernatant behind the centrifugal 1min of 12000rpm, collect thalline, add 125 μ L concentration and be the ethylenediamine tetraacetic acid (EDTA) (EDTA) of 0.5M (pH8.0), 10 μ L concentration are the RNase A of 200 μ g/mL, behind the concuss suspension thalline, behind 28 ℃ of water bath processing 30min, add 70 μ L concentration and be 10% sodium lauryl sulphate (SDS), 5 μ L concentration are the Proteinase K of 10mg/mL, behind the mixing, multigelation three times, then adding 70 μ L concentration is the NaCl of 5M, and mixing is placed on 30min on ice, the centrifugal 20min of 12000rpm, get supernatant, the chloroform that adds 1 times of volume: primary isoamyl alcohol (24: 1), behind the mixing, the centrifugal 10min of 12000rpm, remove supernatant, the dehydrated alcohol that adds 2 times of volumes, behind-20 ℃ of lower preservation 10min, the centrifugal 10min of 12000rpm, abandon supernatant, ethanol with an amount of 75% cleans 3 times, after drying, adds the TE solution dissolving bacterial strain B3 genomic dna of 50 μ L.
Prepare the PCR reaction mixture of 50 μ L, include: 37 μ L sterilized waters, 5 μ L, 10 * Taq dna polymerase buffer liquid, 4 μ L concentration are the dNTPs of 2.5mM, 100nM upstream primer 27F (5 '-GAGTTTGATCCTGGCTCAG-3 '), 100nM downstream primer 1527R (5 '-AGAAAGGAGGTGATCCAGCC-3 '), 1ng bacterial strain B3 genomic dna and 1UTaq archaeal dna polymerase.Then reaction mixture is placed the 16SrDNA of amplification bacterial strain B3 on the PCR instrument, amplification program is: 94 ℃ of sex change 5min, then be 30 circulations, each circulation comprises 94 ℃ of sex change 1min, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, after the loop ends, 72 ℃ are extended 10min again, then are kept under 4 ℃.After amplification finishes, be after 1.0% agarose gel electrophoresis is proved conclusively PCR product size, to send Sangon Biotech (Shanghai) Co., Ltd. to carry out dna sequencing with concentration.After order-checking finishes, the 16S rDNA sequence of bacterial strain B3 is carried out sequence analysis with other species in the NCBI website, then use MegaV4.0.2 software development Phylogenetic tree, the result as shown in Figure 5, the homology of finding some bacterium in bacterial strain B3 and the pseudoalteromonas genus is higher, especially with Pseudoalteromonas rubra G31a 98.5% homology is arranged, therefore with bacterial strain B3 called after pseudoalteromonas B3 (Pseudoalteromonas sp.B3).
The amino acid whose checking of embodiment 2 pseudoalteromonas B3 bio-oxidation L----the detection of ketone acid
Single bacterium colony of bacterial classification B3 is inoculated in respectively MM liquid nutrient medium (yeast extract paste 3g/L, peptone 5g/L, the sea salt 30g/L of 5mL; PH 7.2, and solvent is water) in, in 28 ℃, shaking table is cultivated 4d under the 200rpm rotating speed, and the centrifugal 5min of 8000rpm collects fermented supernatant fluid, obtains the enzyme source;
Reaction is divided into 4 groups: organize 1 positive control group and (directly use α-phenyl-pyruvic acid and 2, the 4-dinitrophenylhydrazine reaction), group 2 is experimental group, group 3 is not for (to add substrate L-amino acid without the substrate group, other operate same experimental group), group 4 is enzyme deactivation group (the supernatant liquor high-temperature inactivation that will contain enzyme, other operate same experimental group);
Group 1 is operating as: the 1.5mL volumetric concentration be 0.1% 2, the 4-dinitrophenylhydrazine aqueous solution and 1.5mL concentration are after 0.2mmol/L α-phenyl-pyruvic acid aqueous solution at room temperature reacts 10min, be the NaOH termination reaction of 2.5M with 6mL concentration, the centrifugal 5min of 13000rpm collects supernatant, detects 520nm wavelength (OD with spectrophotometer
520) under light absorption value.The result shows (Figure 1A): reaction solution has obvious colour-change (from the faint yellow reddish-brown that becomes), and, at 520nm wavelength (OD
520) under higher light absorption value (OD is arranged
520=0.202);
Group 2 is operating as: get the above-mentioned fermented supernatant fluid of 1.5mL as enzyme liquid (wet thallus concentration: 0.1mg wet thallus/mmol substrate), add the L-Phe 1.5mL of 300mmol/L and the catalase of 2000U, after at room temperature reacting 30min, add the 1.5mL volumetric concentration and be 0.1% 2, the 4-dinitrophenylhydrazine aqueous solution, after at room temperature reacting 10min, be the NaOH termination reaction of 2.5M with 6mL concentration, the centrifugal 5min of 13000rpm collects supernatant, detects 520nm wavelength (OD with spectrophotometer
520) under light absorption value, the result as shown in Figure 1, the fermented supernatant fluid of the pseudoalteromonas B3 of pyroprocessing and added reaction solution (group 2 in the reaction tubes of substrate L-Phe not, Figure 1B) obvious colour-change (from the faint yellow reddish-brown that becomes) is arranged, and, at 520nm wavelength (OD
520) under higher light absorption value (OD is arranged
520=0.145), the consistent (OD of this result and positive control (group 1, Figure 1A is directly with α-phenyl-pyruvic acid and 2,4 dinitrophenyl hydrazine reaction)
520=0.202), illustrates after the fermented supernatant fluid of pseudoalteromonas B3 and the L-Phe reaction, have α-phenyl-pyruvic acid to generate.Accordingly, if do not add substrate L-Phe (group 3, Fig. 1 C), still be faint yellow after the reaction, OD
520Value is lower (OD also
520=0.038), illustrates do not have alpha-ketoacid to generate; Same, after the fermented supernatant fluid pyroprocessing deactivation with pseudoalteromonas B3, add again L-Phe (group 4, Fig. 1 D) first, also still be faint yellow after the reaction, OD
520Also lower (OD
520=0.051), illustrates also do not have α-the phenyl-pyruvic acid generation.The result is with theoretical consistent, illustrate that pseudoalteromonas B3 ferments after, produced LAAO.
The amino acid whose checking of embodiment 3 pseudoalteromonas B3 bio-oxidation L----the detection of ammonia
Experiment divides 3 groups, experimental group, positive controls and negative control group.
Adding 3mL volumetric concentration is 4% ammoniacal liquor in the uncultivated MM liquid nutrient medium of positive controls: 47mL, behind the room temperature preservation 30min, the NaOH aqueous solution 0.5mL that adds 1M, reaction 1min, pH test strip color becomes green (alkalescence) by yellow (neutrality);
The uncultivated MM liquid nutrient medium of negative control group: 50mL is as enzyme liquid, and other operate same experimental group;
Experimental group: collect supernatant liquor according to embodiment 2 methods, get pseudoalteromonas B3 supernatant liquor (the wet thallus concentration: 0.1mg wet thallus/mmol substrate) as enzyme liquid of 50mL, and the Erlenmeyer flask of the 250mL that packs into, with the L-Phe 50mL of 300mmol/L behind room temperature reaction 30min, (NaOH is highly basic, can make the ammonia (NH in the solution to add 0.5mL concentration and be the NaOH aqueous solution of 1M
4 +) with ammonia (NH
3) discharge), reaction 1min, in the release of Erlenmeyer flask bottleneck with pH ELISA test strip ammonia, the result is shown in Fig. 2 A (experimental group), the reaction system that contains pseudoalteromonas B3 has ammonia to discharge, so that pH test strip color becomes green (alkalescence) by yellow (neutrality), its result and positive control (Fig. 2 B, in the ammoniacal liquor directly the NaOH aqueous solution of adding 1M) are consistent; And the pH test strip of negative control (Fig. 2 C, uncultivated MM liquid nutrient medium as enzyme liquid, the same experimental group of other conditions) does not have colour-change, still is yellow.Illustrate that pseudoalteromonas B3 can produce LAAO, thereby act on L-Phe and produce ammonia (NH
4 +).
Embodiment 4 pseudoalteromonas B3 are to the oxidation property of different substrates
(1) slant culture: pseudoalteromonas B3 is inoculated in slant medium, cultivate 18h for 25 ℃, obtain inclined-plane thalline (MM solid medium), described slant medium final concentration consists of: yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, agar 20g/L, pH 7.2, and solvent is water;
(2) seed culture: from inclined-plane thalline picking one transfering loop inoculation to seed culture medium (MM liquid nutrient medium), cultivate 24h for 25 ℃, obtain seed liquor, described seed culture medium final concentration consists of: yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, pH 7.2, and solvent is water; (3) fermentation culture: seed liquor is seeded to fermention medium (MM liquid nutrient medium) with 10% inoculum size, cultivate 120h for 25 ℃, obtain fermented liquid, with fermented liquid through 12000rpm, behind the centrifugal 5min, collect supernatant liquor, described fermention medium final concentration consists of: yeast extract paste 3g/L, peptone 5g/L, sea salt 30g/L, pH 7.2, and solvent is water; (4) bio-oxidation: get respectively the above-mentioned fermented supernatant fluid of 3mL as enzyme liquid (wet thallus concentration: the 0.1mg/mmol substrate), respectively with L-Leu (L-Leu) 3mL of 300mmol/L, 300mmol/L 1B (L-Lys) 3mL, 300mmol/LL-tyrosine (L-Tyr) 3mL, 300mmol/L altheine (L-Asn) 3mL, 300mmol/LL-glutamine (L-Gln) 3mL, 300mmol/L METHIONINE (L-Met) 3mL, 300mmol/L CYSTINE (L-cystine) 3mL, 300mmol/L L-arginine (L-Arg) 3mL, 300mmol/L L-Trp (L-Trp), 3mL300mmol/L Valine (L-Val) 3mL, 300mmol/L Pidolidone (L-Glu) 3mL, 300mmol/L Serine (L-Ser) 3mL, 300mmol/L L-PROLINE (L-Pro) 3mL, 300mmol/L Beta-L-Ala (Beta-Ala) 3mL, 300mmol/L DL-aspartic acid (DL-Asp) 3mL and 300mmol/L Cys (L-Cys) 3mL are 25 ℃ of temperature, 7 times reactions of pH 0.5h, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, the result as shown in Figure 6, find that pseudoalteromonas B3 can carry out biooxidation reactions as substrate with multiple L-amino acid, and make hydrogen peroxide detection reagent generation color reaction, the substrate that enzymic activity is the highest is L-Leu, next is 1B, TYR, altheine, L-glutaminate, METHIONINE, CYSTINE, L-arginine, L-Trp; The substrate that enzymic activity is minimum is Cys.
The oxidation property of pseudoalteromonas B3 under embodiment 5 differing tempss
Obtain the preparation method in enzyme source after the pseudoalteromonas B3 fermentation culture with embodiment 4.
Get respectively the 3mL fermented supernatant fluid as enzyme liquid (wet thallus concentration: 0.1mg wet thallus/mmol substrate), L-Phe 3mL with 300mmol/L (regulates pH with the 50mmol/L potassium phosphate buffer) under the pH7.0 condition successively, respectively at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, under 45 ℃ and 50 ℃, reaction 0.5h, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, the result as shown in Figure 7, find that pseudoalteromonas B3 fermented supernatant fluid can keep preferably oxidation vigor under 25～50 ℃, wherein, vigor is the highest under 30 ℃ of conditions.
The oxidation property of the lower pseudoalteromonas B3 of embodiment 6 different pH values
The preparation method in the enzyme source that obtains after the pseudoalteromonas B3 fermentation culture is with embodiment 4.
Get respectively the 3mL fermented supernatant fluid as enzyme liquid (wet thallus concentration: 0.1mg wet thallus/mmol substrate), successively with the L-Phe 3mL of 300mmol/L, under 30 ℃ of conditions, respectively at pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, (regulate pH with the 50mmol/L potassium phosphate buffer) under pH7.5 and the pH8.0, reaction 0.5h, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, the result as shown in Figure 8, find that pseudoalteromonas B3 fermented supernatant fluid can keep preferably oxidation vigor under pH6.0～8.0 conditions, wherein, vigor is the highest under the pH7.0 condition.
The impact of embodiment 7 different metal ion pair pseudoalteromonas B3 oxidation properties
The preparation method in the enzyme source that obtains after the pseudoalteromonas B3 fermentation culture is with embodiment 4.
Get respectively the 3mL fermented supernatant fluid as enzyme liquid (wet thallus concentration: 0.1mg wet thallus/mmol substrate), successively with the L-Phe 3mL of 300mmol/L at 30 ℃, (regulate pH with the 50mmol/L potassium phosphate buffer) under the pH7.0 condition, add respectively the zinc sulfate solution 0.6mL of 100mmol/L, 100mmol/L manganese sulfate solution 0.6mL, 100mmol/L copper sulfate solution 0.6mL, 100mmol/L calcium chloride water 0.6mL, 100mmol/L cobalt chloride solution 0.6mL, 100mmol/L magnesium sulfate solution 0.6mL, 100mmol/L ferrous sulfate aqueous solution 0.6mL, 100mmol/L potassium chloride solution 0.6mL, 100mmol/L cadmium nitrate aqueous solution 0.6mL and 100mmol/L barium chloride solution 0.6mL, reaction 0.5h, with hydrogen peroxide detection kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay kit, Invitrogen, USA) detect the hydrogen peroxide production, the result as shown in Figure 9, find zinc sulfate, calcium chloride, sal epsom, Repone K and cadmium nitrate can improve the oxidation vigor of pseudoalteromonas B3 fermented supernatant fluid, wherein, the oxidation vigor is the highest under the interpolation zinc sulfate condition.And other metal ions all can suppress the oxidation vigor of B3 fermented supernatant fluid to some extent, and wherein, it is the most obvious that ferrous sulfate and bariumchloride suppress.
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University
＜120〉pseudoalteromonas B3 and the application in bio-oxidation L-amino acid thereof
<130>
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 1396
<212> DNA
<213> Pseudoalteromonas sp.
<400> 1
agtcgagcgg taacatttct agcttgctag aagatgacga gcggcggacg ggtgagtaat 60
gcttgggaac atgcctttag gtgggggaca accattggaa acgatggcta ataccgcata 120
atgtctacgg accaaagggg gcttcggctc tcgcctttag attggcccaa gtgggattag 180
ctagttggtg aggtaacggc tcaccaaggc gacgatccct agctggtttg agaggatgat 240
cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 300
tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt gtgaagaagg ccttcgggtt 360
gtaaagcact ttcagtcagg aggaaaggtt agtagttaat acctgctagc tgtgacgtta 420
ctgacagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcga 480
gcgttaatcg gaattactgg gcgtaaagcg tacgcaggcg gttgattaag cgagatgtga 540
aagccccggg cttaacctgg gaactgcatt tcgaactggt caactagagt gtgatagagg 600
gtggtagaat ttcaggtgta gcggtgaaat gcgtagagat ctgaaggaat accgatggcg 660
aaggcagcca cctgggtcaa cactgacgct catgtacgaa agcgtgggga gcaaacagga 720
ttagataccc tggtagtcca cgccgtaaac gatgtctact aggagctggg gtcttcggac 780
aacttttcca aagctaacgc attaagtaga ccgcctgggg agtacggccg caaggttaaa 840
actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgatgca 900
acgcgaagaa ccttacctac acttgacata cagagaactt accagagatg gtttggtgcc 960
ttcgggaact ctgatacagg tgctgcatgg ctgtcgtcag ctcgtgttgt gagatgttgg 1020
gttaagtccc gcaacgagcg caacccttat ccttagttgc cagcgattcg gtcgggaact 1080
ctaaggagac tgccggtgat aaaccggagg aaggtgggga cgacgtcaag tcatcatggc 1140
ccttacgtgt agggctacac acgtgctaca atggcatata cagagtgctg cgaactagcg 1200
atagtaagcg aatcacttaa agtatgtcgt agtccggatt ggagtctgca actcgactcc 1260
atgaagtcgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggc 1320
cttgtacaca ccgcccgtca caccatggga gtgggttgct ccagaagtgg atagcttaac 1380
cttcgggagg gcgtca 1396
Claims (7)
1. pseudoalteromonas B3(Pseudoalteromonas sp. B3), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, culture presevation number is: CGMCC NO.5353, preservation date are on October 17th, 2011.
2. pseudoalteromonas B3 as claimed in claim 1 is characterized in that the 16S rDNA sequence of described pseudoalteromonas B3 is:
AGTCGAGCGGTAACATTTCTAGCTTGCTAGAAGATGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTTAGGTGGGGGACAACCATTGGAAACGATGGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTGATTAAGCGAGATGTGAAAGCCCCGGGCTTAACCTGGGAACTGCATTTCGAACTGGTCAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTAGGAGCTGGGGTCTTCGGACAACTTTTCCAAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTACCAGAGATGGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGATTCGGTCGGGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCATATACAGAGTGCTGCGAACTAGCGATAGTAAGCGAATCACTTAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTGGATAGCTTAACCTTCGGGAGGGCGTCA。
3. the application of pseudoalteromonas B3 claimed in claim 1 in bio-oxidation L-amino acid, described L-amino acid is L-Leu, 1B, TYR, altheine, L-glutaminate, METHIONINE, CYSTINE, L-arginine, L-Trp or Cys.
4. the application of pseudoalteromonas B3 as claimed in claim 3 in bio-oxidation L-amino acid, it is characterized in that described being applied as: the enzyme that obtains take pseudoalteromonas B3 fermentation culture is as the enzyme source, take L-amino acid as substrate, 25～50 ℃, pH 6～8, reaction 0.5～2h makes the L-amino-acid oxidase, discharges hydrogen peroxide.
5. the application of pseudoalteromonas B3 as claimed in claim 4 in bio-oxidation L-amino acid, it is characterized in that described enzyme source is that centrifugal the discarding of wet thallus fermented liquid that contain that obtains after the pseudoalteromonas B3 fermentation culture precipitated the rear supernatant liquor that obtains, described L-amino acid concentration is 30～300mmol/L, and described supernatant liquor consumption is counted 0.04～0.8mg wet thallus/mmol substrate to contain in the centrifugal primary fermentation liquid of wet thallus fermented liquid the wet thallus quality.
6. the application of pseudoalteromonas B3 as claimed in claim 4 in bio-oxidation L-amino acid, it is characterized in that described enzyme source prepares as follows: (1) slant culture: pseudoalteromonas B3 is inoculated in slant medium, cultivate 12～24h for 20～37 ℃, obtain the inclined-plane thalline, described slant medium final concentration consists of: yeast extract paste 1.5～6 g/L, peptone 2.5～10 g/L, sea salt 15～45 g/L, agar 15～25 g/L, pH 7～8, and solvent is water; (2) seed culture: from inclined-plane thalline picking one transfering loop inoculation to seed culture medium, cultivate 18～24h for 20～37 ℃, obtain seed liquor, described seed culture medium final concentration consists of: yeast extract paste 1.5～6 g/L, peptone 2.5～10 g/L, sea salt 15～45 g/L, pH 7～8, and solvent is water; (3) fermentation culture: seed liquor is seeded to fermention medium with 1～10% inoculum size, cultivate 48～120h for 20～37 ℃, obtain fermented liquid, fermented liquid is centrifugal, discard precipitation, collect supernatant liquor, obtain described enzyme source, described fermention medium final concentration consists of: yeast extract paste 1.5～6 g/L, peptone 2.5～10 g/L, sea salt 15～45 g/L, pH 7～8, and solvent is water.
7. the application of pseudoalteromonas B3 as claimed in claim 4 in bio-oxidation L-amino acid, it is characterized in that described being applied as: the supernatant liquor that contains the centrifugal acquisition of wet thallus fermented liquid after the pseudoalteromonas B3 fermentation culture is as the enzyme source, take L-amino acid as substrate, 30 ℃, pH 7.0, reaction 0.5h makes the L-amino-acid oxidase discharge hydrogen peroxide; Wet thallus concentration is 10～25mg/L in the described pseudoalteromonas B3 fermented liquid, described L-amino acid substrate concentration is 30～300mmol/L, and described supernatant liquor consumption is counted 0.08～0.8mg wet thallus/mmol substrate to contain in the centrifugal primary fermentation liquid of wet thallus fermented liquid the wet thallus quality.
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| WO1994025574A1 (en) * | 1993-04-30 | 1994-11-10 | Novo Nordisk A/S | L-amino acid oxidase |
| CN1526445A (en) * | 2003-03-04 | 2004-09-08 | 中国科学院昆明动物研究所 | Application of snake venom L-amino acid oxidase in preparing AIDS treating medicine |
| CN102121020A (en) * | 2010-12-07 | 2011-07-13 | 中山大学 | Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof |
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| WO1994025574A1 (en) * | 1993-04-30 | 1994-11-10 | Novo Nordisk A/S | L-amino acid oxidase |
| CN1526445A (en) * | 2003-03-04 | 2004-09-08 | 中国科学院昆明动物研究所 | Application of snake venom L-amino acid oxidase in preparing AIDS treating medicine |
| CN102121020A (en) * | 2010-12-07 | 2011-07-13 | 中山大学 | Gene complete sequence of siganus oramin L-amino acid oxidase and application thereof |
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