CN114763541B - Oxidase and application thereof - Google Patents
Oxidase and application thereof Download PDFInfo
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- CN114763541B CN114763541B CN202110049830.3A CN202110049830A CN114763541B CN 114763541 B CN114763541 B CN 114763541B CN 202110049830 A CN202110049830 A CN 202110049830A CN 114763541 B CN114763541 B CN 114763541B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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Abstract
The invention relates to the technical fields of microorganism biotechnology, sewage treatment technology and genetic engineering. The oxidase disclosed in the present invention is a protein having one of the following amino acid sequences: 1) An amino acid sequence shown in a sequence table SEQ ID No. 1; 2) And (3) a protein with oxidase function, wherein the amino acid sequence of SEQ ID No.1 in the sequence table is subjected to substitution, deletion or addition of one to ten amino acid residues. The hydroxylamine reductase provided by the invention can convert ammonia nitrogen in sewage into nitrogen by taking hydroxylamine, mercaptoethylamine and the like as substrates, the conversion rate is more than 80%, and the hydroxylamine reductase plays an important role in hydroxylamine detoxification and ammonia nitrogen-containing wastewater treatment.
Description
Technical Field
The invention relates to the technical fields of microorganism biotechnology, sewage treatment technology and genetic engineering.
Background
Nitrogen is an important element that promotes sustainable development of agriculture. The conversion process from ammonia to nitrogen is the main mechanism of sewage denitrification treatment. Currently, four main routes for ammonia to nitrogen conversion are known, including: 1) Aerobic nitrification-anaerobic denitrification are the most widely known classical denitrification pathway; 2) Compared with the whole-process nitrification, the short-process nitrification-denitrification method not only reduces the aeration quantity so as to save energy consumption, but also reduces the organic matter consumption in the denitrification process so as to reduce the operation cost; 3) Anaerobic ammoxidation, in which nitrite is used as an electron acceptor to directly convert ammonia nitrogen into nitrogen; 4) The denitrification can be completed under aerobic conditions. The denitrification path can be completed by the cooperation of a plurality of strains or the conversion of aerobic-anaerobic conditions. How to find an enzyme which is environment-friendly and efficient has been a research hotspot and difficulty in this field.
Disclosure of Invention
The oxidase provided by the invention can directly oxidize hydroxylamine into nitrogen under the aerobic condition, has high conversion efficiency and low energy consumption, hardly accumulates greenhouse gas nitrous oxide, and enables the efficient and low-energy-consumption bioconversion of ammonia into nitrogen to be realized.
The invention aims to provide oxidase and a coding gene thereof.
The oxidase provided by the invention is derived from Alcaligenes (sp.) HO-1, the strain is preserved in the China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) registration number 16549 in the 9 th month 28 th year, the center is simply called CGMCC, and the center address is North Chen Silu No.1 and 3 in the Korean region of Beijing city.
First, the oxidase disclosed in the present invention is a protein having one of the following amino acid residue sequences;
1) The amino acid sequence in the sequence table SEQ ID No.1 consists of 371 amino acid residues;
2) And (3) a protein with oxidase function, wherein the amino acid sequence of SEQ ID No.1 in the sequence table is subjected to substitution, deletion or addition of one to ten amino acid residues.
Second, the oxidase gene disclosed in the present invention has one of the following nucleotide sequences:
1) The polynucleotide sequence of SEQ ID No.2 in the sequence table consists of 954bp base pairs;
2) A polynucleotide sequence for coding a protein SEQ ID No.1 in a sequence table.
The hydroxylamine reductase provided by the invention can convert ammonia nitrogen in sewage into nitrogen by taking hydroxylamine, mercaptoethylamine and the like as substrates, the conversion rate is more than 80%, and the hydroxylamine reductase plays an important role in hydroxylamine detoxification and ammonia nitrogen-containing wastewater treatment.
Drawings
FIG. 1 is a diagram showing the expression of oxidase in E.coli.
FIG. 2 is a graph showing the nitrogen formation per liter of the enzyme reaction system using hydroxylamine as a substrate.
FIG. 3 is a graph showing the conversion rate of nitrogen gas and the like of recombinant Escherichia coli by using ammonium sulfate as a substrate.
FIG. 4 is a graph showing the conversion rate of nitrogen gas or the like of HO 1 strain using ammonium sulfate as a substrate.
FIG. 5 is a graph showing the conversion rate of nitrogen gas or the like of strain HO 1 using glutamine as a substrate.
Detailed Description
Example 1
Oxidase of the present invention and production of Gene thereof
Alcaligenes sp HO-1, which has been deposited in China general microbiological culture Collection center, registration number CGMCC No.16549, which is abbreviated as CGMCC, at 9 and 28 months of 2018, is also provided.
Single colony of alcaligenes HO-1 strain on LB plate is picked into LB liquid culture medium, shake culture is carried out at 30 ℃, thallus is collected, genome DNA is extracted, and Epicentre CopyControl Fosmid Library Production Kit kit is used for constructing FOSMID library. Randomly cutting the extracted high-quality genome DNA into fragments (concentrated at 30-45K), blunt-end treating and 5' -phosphorylating the DNA fragments, connecting to a CopyControl pCC1FOS Vector, and transducing to E.coli EPl300-T1 R Host cells were incubated at 37℃for 30 min, plated onto solid LB plates containing 12.5. Mu.g.mL-1 chloramphenicol resistance, selected CopyControl Fosmid positive monoclonal onto 384 well plates and stored in a-80℃refrigerator。
Hydroxylamine has a peak of maximum absorbance at 705nm, and reference citation (Shurong Liu et al, geoderma 232-234 (2014) 117-122; the library was screened colorimetrically using hydroxylamine as the reaction product, and the green reaction was a positive clone producing hydroxylamine. Plasmid sequencing was performed by extracting one of the clones. ORF analysis was performed on the sequencing results, blastx alignment was performed on NCBI, and the gene sequence encoding oxidase was analyzed from the sequence.
The amino acid sequence of the oxidase had 27.9% homology with the oxidase of Streptomyces venezuelae (ATCC 10712/CBS 650.69/DSM40230/JCM 4526). The amino acid sequence of the oxidase is shown as SEQ No.1 in a sequence table, and the nucleotide sequence of the coding gene is shown as SEQ No.2 in the sequence table.
Example 2
Construction of the novel oxidase expression vector of the invention and expression in E.coli
Extracting genome DNA of alcaligenes HO-1 strain, and PCR amplifying the oxidase gene of the invention by taking the genome DNA as a template, wherein the oxidase primer is as follows: forward primer: 5'gcaaatgggtcgcggatccATGACTATCAAAAGCTACGAAACCG 3 'and reverse primer 5'tcgagtgcggccgcaagcttTTGCAGCGCCTCCTGTTG 3', the horizontal line is the sequence overlapping the plasmid.
The reaction system is as follows: 6-10ng/ul of template; dNTPMix (25 mM) 8ul, forward primer (10 mM) 1ul, reverse primer (10 mM) 1ul,10 Xbuffer 5ul,FastPfu 0.4ul,ddH2O supplemented to 50ul, where the high fidelity enzyme and buffer were purchased from full gold Biotechnology. The PCR conditions were pre-denatured at 95℃for 2min, 20sec at 95℃for 55℃for 30sec, and at 72℃for 1min for 30 cycles; thoroughly extend at 72℃for 10min. The PCR amplified products are subjected to 1% agarose gel electrophoresis, and the two PCR systems are respectively amplified to obtain about 1000 bands and about 1100 bands, wherein the sizes of the bands are consistent with the expected results. The method of cutting glue and recovering homologous recombination is cloned to pET21a (+), oxidase pET21a (+) -84 is constructed, oxidation reduction enzyme pET21a (+) -85 of oxidase is transformed to colibacillus DH5 alpha competence, ampicillin resistant LB culture medium is cultivated, clone colony PCR is selected for verification, and positive clone sequencing results show that amplified products are nucleotide sequences of SEQ ID No.2 and SEQ ID No.4 in a sequence table. pET21a (+) -84, pET21a (+) -85, recombinant plasmids were transformed into E.coli BL21 (DE 3), ampicillin-resistant LB cultured, and positive clones were verified by colony PCR. Positive transformants containing pET21a (+) -84 and pET21a (+) -85 were cultured with LB liquid medium at 37℃and 150rpm, respectively, to a bacterial concentration OD 600.8, and were induced overnight by adding 0.5mM IPTG to express oxidase (FIG. 1). The two enzymes were purified by His Tag column (His Bind Resin Chromatography, novagen Co., USA) with an expression level of 200-500mg/L, specific procedures were performed with reference to the product manual.
Example 3
Oxidase Activity assay of the present invention
Taking hydroxylamine as an example, a method for measuring oxidase activity was elucidated.
Under the catalysis of oxidase, hydroxylamine takes FAD/NADH, FD/FDR and the like as electron transfer bodies to form final product nitrogen. In the enzyme activity measuring system, hydroxylamine or 15 N-labeled hydroxylamine as a substrate can be determined by measuring N 2 ， 15 N 2 The oxidase activity was measured.
The reaction system comprises 10mM hydroxylamine or 15 N hydroxylamine, 20mM Tris HCL pH8.5,1-12mg oxidase protein, oxidase reductase protein, the total volume of the enzymatic reaction is 300. Mu.L, and the reaction is carried out at 30 ℃ for 100min. Enzyme activity of 1 unit was defined as the formation of 1. Mu. Mol N per minute 2 Or (b) 15 N 2 The amount of enzyme required.
After 10mM hydroxylamine was used as a substrate, 20mM Tris HCL pH8.5, 12mg of an oxidase system was added, 4mM FAD and 10mM NADH were used as electron transfer chains, and the reaction was carried out at 30℃for 100 minutes, the amount of nitrogen generated in 300. Mu.L of the enzymatic reaction system was determined by detection, and the novel hydroxylamine oxidoreductase system was able to convert more than 80% of hydroxylamine to nitrogen (FIG. 2).
Identification of oxidase-based catalytic products:
n was detected by GC/MS (model 7890A/5975C, agilent) 2 Or (b) 15 N 2 ，N 2 O and other gaseous products, 50% O is filled in the anaerobic tube 2 And injecting the mixed gas of/He into the anaerobic pipe through a syringe, and extracting the gas in the pipe through the syringe after the reaction. 15 N 2 The detection of (C) was performed using a CP-Molsive 5A (25 m. Times.0.32 mm. Times.30 μm, agilent, USA) column, N 2 The detection of O was carried out using a GS-Carbon Plot (30 m 0.32mm 3.0 μm, agilent, USA) column.
Example 4
The oxidase E.coli recombinant strain of the invention can convert ammonia molecules into nitrogen
E.coli recombinant strains containing pET21a (+) -84, pET21a (+) -85 and pET21a (+) -86 are cultured to a bacterial concentration OD by using HNM liquid culture medium at 37 ℃ and 150rpm under 50% oxygen concentration in an anaerobic bottle 600 At 0.8, the cells were induced to culture at 30℃for 5 days with the addition of 0.5mM IPTG to express oxidase.
By GS/MS detection of headspace gas, induced cultures can be grown 15 The N-labelled ammonium sulphate was converted to nitrogen with a conversion of about 4% (figure 3).
HNM medium formulation (g/L): (NH) 4 ) 2 SO 4 0.66, sodium succinate hexahydrate 7.88, KH 2 PO 4 0.5，Na 2 HPO 4 ·12H 2 O 1.25，MgSO 4 ·7H 2 O0.2, 2mL of trace element solution, pH 8.0.
Microelement solution formula (g/L): EDTA.2Na 57.1, znSO 4 ·7H 2 O 3.9，CaCl 2 ·2H 2 O 7.0，MnCl 2 ·4H 2 O 5.1，FeSO 4 ·7H 2 O 5.0，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1，CuSO 4 ·5H 2 O 1.6，CoCl 2 ·6H 2 O 1.6，pH6.0。
Example 5
The hydroxylamine oxidase-containing alcaligenes HO-1 wild strain of the invention can convert ammonia molecules into nitrogen
The oxidase-containing alcaligenes wild strain is cultured in an anaerobic bottle at 30deg.C with 50% oxygen concentration and 150rpm for 2 days, and HNM liquid culture medium is used to culture the strain 15 The N-labelled ammonium sulphate was converted to nitrogen with a conversion of up to about 50% (FIG. 4).
Example 6
The oxidase-containing alcaligenes HO-1 wild strain of the invention can convert glutamine into nitrogen
The oxidase-containing alcaligenes wild strain is cultured in an anaerobic bottle at 30deg.C with 50% oxygen concentration and 150rpm for 2 days, and HNM liquid culture medium is used to culture the strain 15 The conversion of N-labeled glutamine to nitrogen was about 30% (FIG. 5).
HNM medium formulation (g/L): glutamine 0.66, sodium succinate hexahydrate 7.88, KH 2 PO 4 0.5，Na 2 HPO 4 ·12H 2 O 1.25，MgSO 4 ·7H 2 O0.2, 2mL of trace element solution, pH 8.0.
Microelement solution formula (g/L): EDTA.2Na 57.1, znSO 4 ·7H 2 O 3.9，CaCl 2 ·2H 2 O 7.0，MnCl 2 ·4H 2 O 5.1，FeSO 4 ·7H 2 O 5.0，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1，CuSO 4 ·5H 2 O 1.6，CoCl 2 ·6H 2 O 1.6，pH6.0。
Sequence listing
<110> institute of microorganisms at national academy of sciences
<120> an oxidase and use thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 317
<212> PRT
<213> Alcaligenes sp.)
<400> 1
Met Thr Ile Lys Ser Tyr Glu Thr Asp Asp Ala Val Arg Asn Met Leu
1 5 10 15
Gln Lys Leu Ser Val Leu Trp Lys Asn Arg Ala Ala Val Asn Gln Glu
20 25 30
Leu Pro Asp Tyr Asn Asn Leu Ala Phe Asp Pro Asn Lys Ala Asp Phe
35 40 45
Ser Glu Cys Leu Leu Pro Phe Arg Glu His Gln Ala Trp Leu Glu Ala
50 55 60
Pro Glu Glu Leu Lys Ser Gln Cys Leu Ser Tyr Ala Trp Gly Ile Tyr
65 70 75 80
Asn Leu Lys Thr Ile Tyr Val Glu Cys Asn Val Val Thr Pro Ser Cys
85 90 95
Glu Asp Ile Ile Lys Thr Pro Pro Pro Ser Ala Asn Arg Asn Leu Leu
100 105 110
Gln Asp Val Met Ser Gln Ala Leu Leu Asp Glu Ala Leu His Thr Arg
115 120 125
Met Ser Ile Met Ala Cys Asn Tyr Ile Tyr Ser Met Arg Gly Leu Gln
130 135 140
Pro Leu Asp Phe Thr Asn Phe Asn Leu Val Gln Trp Arg Asn Asp Ile
145 150 155 160
Leu Ser Gln Cys Ser Ser Glu Ser Glu Arg Arg Leu Thr Arg Phe Ala
165 170 175
Ile Ala Cys Ala Ser Glu Thr Leu Ile Thr Asp Tyr Leu Lys Thr Met
180 185 190
Ala Glu Asp Lys Ser Ile Gln Thr Ile Cys His Glu Val Thr Arg Thr
195 200 205
His Ala Met Asp Glu Trp Ser His Ser Ser Val Phe Ser Phe Val Ala
210 215 220
Ser Asp Ile Ile His Gly Leu Ser Gln Lys Glu Arg Glu His Met Arg
225 230 235 240
Ala Val Ile Leu Arg Thr Val Glu Met Phe Ala Asn Asn Glu Met Gly
245 250 255
Ala Trp Glu Lys Val Phe Ser Met Val Asn Phe Pro Asn Ala Arg Asp
260 265 270
Ile Leu His Asp Thr Gly Asp Ser Asn Glu Ile Gly Val Tyr Thr Gly
275 280 285
Ser Val Glu Ser Leu Ile Glu Arg Ile Gly Leu Asn Ser Lys Ser Gly
290 295 300
Gln Ala Gln Ala Gln Asp Glu Gln Gln Glu Ala Leu Gln
305 310 315
<210> 2
<211> 954
<212> DNA
<213> Alcaligenes sp.)
<400> 2
atgactatca aaagctacga aaccgatgat gctgtacgta atatgctgca aaagctgtct 60
gtactttgga aaaaccgggc ggcggtcaat caggagctgc cggactacaa caatttggca 120
ttcgatccga acaaggccga tttcagcgaa tgtctgctgc ctttccgtga gcaccaggcc 180
tggctggaag cacctgagga attgaaatcg cagtgtctgt cttacgcttg ggggatctac 240
aacctcaaga ccatttatgt ggagtgcaac gtcgtcactc cgtcctgcga agacatcatc 300
aagacgccgc caccgagcgc caaccgcaat ctgctgcagg atgtgatgtc ccaggccttg 360
ctggatgaag ccctgcacac ccgcatgtcc atcatggcat gcaactacat ctattccatg 420
cgcggtctgc aacctttgga tttcaccaac ttcaacctgg tgcaatggcg caacgacatc 480
ctgagccagt gcagttctga atccgagcgt cgcctgaccc gctttgccat tgcctgcgcg 540
tccgaaaccc tgattaccga ctacctcaag accatggctg aggacaagag catccagact 600
atctgccatg aagtgacccg cacgcacgcc atggacgagt ggagccactc cagcgtgttc 660
agctttgtcg cctcggacat cattcacggt ctgagccaga aagagcgtga gcatatgcgt 720
gccgttatcc tgcgcaccgt ggaaatgttc gccaacaacg aaatgggcgc ctgggaaaaa 780
gtgttctcca tggtgaactt ccccaatgca cgcgacattt tgcacgacac cggcgattcc 840
aacgaaattg gcgtgtacac cggttcggtt gaaagcctga tcgagcgcat tggcttgaac 900
agcaaatcgg ggcaggcaca ggcccaagac gagcaacagg aggcgctgca atga 954
Claims (3)
1. An application of oxidase in converting glutamine into nitrogen, wherein the oxidase is protein with an amino acid sequence shown in a sequence table SEQ ID No. 1.
2. The use according to claim 1, wherein the oxidase gene has a nucleotide sequence consisting of 954bp base pairs as shown in sequence table SEQ ID No.2.
3. The use according to claim 1, wherein the amino acid sequence of the oxidase consists of 317 amino acid residues.
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| CN105018404A (en) * | 2015-07-24 | 2015-11-04 | 哈尔滨南森生物科技有限公司 | Genetic engineering aerobic nitrobacteria, and construction method and application thereof to process high-concentration ammonia nitrogen wastewater |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018404A (en) * | 2015-07-24 | 2015-11-04 | 哈尔滨南森生物科技有限公司 | Genetic engineering aerobic nitrobacteria, and construction method and application thereof to process high-concentration ammonia nitrogen wastewater |
Non-Patent Citations (1)
| Title |
|---|
| "NCBI Reference Sequence: WP_003803202.1";National Center for Biotechnology Information;National Center for Biotechnology Information;第1页 * |
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