CN109022412A - A kind of method of fixed l-amino acid deaminase - Google Patents

A kind of method of fixed l-amino acid deaminase Download PDF

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Publication number
CN109022412A
CN109022412A CN201810905503.1A CN201810905503A CN109022412A CN 109022412 A CN109022412 A CN 109022412A CN 201810905503 A CN201810905503 A CN 201810905503A CN 109022412 A CN109022412 A CN 109022412A
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Prior art keywords
amino acid
acid deaminase
fixed
deaminase
binding domain
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Pending
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CN201810905503.1A
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Chinese (zh)
Inventor
吴黎诚
郭小雷
章权
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Zhejiang Zhengshuo Biotechnology Co Ltd
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Zhejiang Zhengshuo Biotechnology Co Ltd
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Priority to CN201810905503.1A priority Critical patent/CN109022412A/en
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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12—Cellulose or derivatives thereof
    • 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
    • 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
    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03002—L-Amino-acid oxidase (1.4.3.2)

Abstract

The invention belongs to field of biotechnology, it is related to a kind of method of fixed l-amino acid deaminase, by cellulose binding domain (cellulose binding domain, CBD) 3 ' or 5 ' ends that genetic fragment passes through the short chain link of section of DNA to l-amino acid deaminase (L-amino acid deaminase, LAAD) genetic fragment.L-amino acid deaminase is merged cellulose binding domain by the present invention, it is fixed in conjunction with microcrystalline cellulose, since microcrystalline cellulose is cheap and easy to get, immobilization is at low cost, and the specificity of fixation procedure plays the role of to enzyme purification, so that the by-product of enzymatic is few.Immobilization L-AAD-CBD is used multiple times due at low cost, has biggish industrialization applying value.

Description

A kind of method of fixed l-amino acid deaminase
Technical field
The invention belongs to field of biotechnology, are related to a kind of method of fixed l-amino acid deaminase.
Background technique
L-amino acid deaminase (LAAD, L-Amino Acid Deaminase), may also be referred to as L-amino acid oxidase (LAAO, L-Amino Acid Oxidase, EC 1.4.3.2) is a kind of flavoprotein (flavoenzyme) to natural and non- Natural l-amino acid has the position the α deamination activity of wide spectrum.LAAD aoxidizes l-amino acid in the presence of oxygen and takes off alpha-amido, generates Ketone acid and ammonia, and generate by-product hydrogen peroxide.The amino that l-amino acid deaminase can be used for aoxidizing l-amino acid prepares corresponding 2-ketoacid prepares D- amino acid for aoxidizing the l-amino acid in dl aminoadipic acid.Either 2-ketoacid or D- amino acid be all It is important medicine intermediate.
L-amino acid deamination enzyme source multiple-microorganism, such asProteus、ProÍidencia、MorganellaBelong to, in addition L-amino acid deaminase is also commercialized utilization in snake venom, only too expensive and cannot be widely used.
Microbe-derived l-amino acid deaminase is easier to carry out genetic manipulation, and can obtain enzyme by fermentation.But L- The enzyme activity of amino acid deaminase is lower, and the cost for industrializing utilization is relatively high, needs using immobilised enzymes technique, repeated multiple times to make With enzyme, to reduce the use cost of enzyme.
Summary of the invention
The present invention overcomes existing microbe-derived l-amino acid deaminase enzyme activity lower, industrializes the cost ratio of utilization Higher defect provides a kind of method of fixed l-amino acid deaminase.
The present invention adopts the following technical scheme that:
A kind of method of fixed l-amino acid deaminase, by cellulose binding domain (cellulose binding domain, CBD) It is fused to the C-terminal or N-terminal of l-amino acid deaminase (L-amino acid deaminase, LAAD).
Preferably, cellulose binding domain from Clostridium thermocellum (Clostridium thermocellum) fiber Plain binding domain (cellulose binding domain, CBD) (genbank accession number AF283517.1).
Preferably, l-amino acid deaminase from proteus mirabilis (Proteus mirabilis) L- amino Sour deaminase (L-amino acid deaminase, LAAD) (genbank accession number EU669819.1).
Preferably, cellulose binding domain (cellulose binding domain, CBD) genetic fragment passes through one section The short chain link of DNA to l-amino acid deaminase (L-amino acid deaminase, LAAD) genetic fragment 3 ' or 5 ' end.
The sequence of the short chain of DNA are as follows: GGAGGAGGAGGAGGAGGAGGAGGAGGA.
Specifically operating procedure includes:
1. recombinant bacterium constructs
Will from Clostridium thermocellum (Clostridium thermocellum) cellulose binding domain (cellulose Binding domain, CBD) (genbank accession number AF283517.1) be fused to from proteus mirabilis (Proteus mirabilis) l-amino acid deaminase (L-amino acid deaminase, LAAD) (genbank accession number EU669819.1 C-terminal or N-terminal).
Full genome synthesizes two genetic fragments, and CBD genetic fragment passes through the short chain of section of DNA (GGAGGAGGAGGAGGAGGAGGAGGAGGA) it is connected to LAAD genetic fragment and obtains 3 ' or 5 ' ends, and be connected into pET24a carrier and (adopt Double digestion is carried out with I/Hind of Nde), plasmid LAAD-CBD is constructed, conversion to E.Coli BL21(DE3) constructs recombinant bacterium PMLAAD-CBD。
2. recombinant bacterium is expressed
Recombinant bacterium PMLAAD-CBD is fermented using TB.
1) culture medium
(1) LB:
(2) agar LB-Kan plate (g/L):
Note: medium sterilization is cooled to 50-60 DEG C, and 100ml LB adds 100 μ l Kan solution (100mg/ml), mixes, inverted plate (25-30ml/6cm plate).
(3) TB (g/L):
2) fermentation process
(1) actication of culture: bacterium is connect to LB agar plate (Kan, 100mg/L), 37 DEG C of culture 12-14h from glycerol tube or milk pipe.
(2) first order seed: picking from the plate single bacterium to 4ml LB test tube, adds Kan to final concentration of 100mg/L, and 37 DEG C, 220rpm overnight incubation (16h).
(3) secondary seed: fresh bacterium solution is taken to be inoculated in LB(100ml/500ml by 2% inoculum concentration) shaking flask, add Kan dense to end Degree is 100mg/L, and 37 DEG C, 220rpm cultivates 3.0-3.5h, and OD600 is 1.0 or so.
(4) the TB fermentation of 1L/5L: the inoculum concentration of secondary seed 2%-10% accesses TB culture medium (1L/5L fermentation shake flask), adds Kan to 25mg/L 37 DEG C, 220rpm, cultivates 3h, starts 25 DEG C of cooling；Add IPTG to final concentration (0.1mM), 220rpm, 25 Degree, overnight incubation.
(5) ferment 20-24h, and thalline were collected by centrifugation by 3500rpm*25min.
(6) -30 DEG C of refrigerator freezings of thallus are for 24 hours.
3. microcrystalline cellulose immobilization
1) thallus adds the water of 5-6 times of volume, is uniformly dispersed with high-speed shearing machine, then squeezes broken born of the same parents twice with high pressure homogenizer；
2) high speed centrifugation, clear enzyme solution in acquisition；
3) microcrystalline cellulose with biomass equivalent is added in enzyme solution, and 20-30 DEG C of concussion mixes 2h；
4) it filters, collects the thick enzyme of immobilization, washed repeatedly with kaliumphosphate buffer (20-100mM, pH7-8.5), filtering must consolidate Surely change l-amino acid deaminase.
By implementing above-mentioned technical proposal, l-amino acid deaminase is merged cellulose binding domain by the present invention, with crystallite fibre Dimension element in conjunction with and fix, since microcrystalline cellulose is cheap and easy to get, immobilization is at low cost, and the specificity of fixation procedure plays Effect to enzyme purification, so that the by-product of enzymatic is few.Immobilization L-AAD-CBD is used multiple times due at low cost, is had There is biggish industrialization applying value.
Specific embodiment
Combined with specific embodiments below, invention is further described in detail.
A kind of method of fixed l-amino acid deaminase, includes the following steps:
1. recombinant bacterium constructs
Will from Clostridium thermocellum (Clostridium thermocellum) cellulose binding domain (cellulose Binding domain, CBD) (genbank accession number AF283517.1) be fused to from proteus mirabilis (Proteus mirabilis) l-amino acid deaminase (L-amino acid deaminase, LAAD) (genbank accession number EU669819.1 C-terminal or N-terminal).
Full genome synthesizes two genetic fragments, and CBD genetic fragment passes through the short chain of section of DNA (GGAGGAGGAGGAGGAGGAGGAGGAGGA) it is connected to LAAD genetic fragment and obtains 3 ' or 5 ' ends, and be connected into pET24a carrier and (adopt Double digestion is carried out with I/Hind of Nde), plasmid LAAD-CBD is constructed, conversion to E.Coli BL21(DE3) constructs recombinant bacterium PMLAAD-CBD。
2. recombinant bacterium is expressed
Recombinant bacterium PMLAAD-CBD is fermented using TB.
1) culture medium
(1) LB:
(2) agar LB-Kan plate (g/L):
Note: medium sterilization is cooled to 50-60 DEG C, and 100ml LB adds 100 μ l Kan solution (100mg/ml), mixes, inverted plate (25-30ml/6cm plate).
(3) TB (g/L):
2) fermentation process
(1) actication of culture: bacterium is connect to LB agar plate (Kan, 100mg/L), 37 DEG C of culture 12- from glycerol tube or milk pipe 14h。
(2) first order seed: picking from the plate single bacterium to 4ml LB test tube, adds Kan to final concentration of 100mg/L, and 37 DEG C, 220rpm overnight incubation (16h).
(3) secondary seed: fresh bacterium solution is taken to be inoculated in LB(100ml/500ml by 2% inoculum concentration) shaking flask, add Kan dense to end Degree is 100mg/L, and 37 DEG C, 220rpm cultivates 3.0-3.5h, and OD600 is 1.0 or so.
(4) the TB fermentation of 1L/5L: the inoculum concentration of secondary seed 2%-10% accesses TB culture medium (1L/5L fermentation shake flask), adds Kan to 25mg/L 37 DEG C, 220rpm, cultivates 3h, starts 25 DEG C of cooling；Add IPTG to final concentration (0.1mM), 220rpm, 25 Degree, overnight incubation.
(5) ferment 20-24h, and thalline were collected by centrifugation by 3500rpm*25min.
(6) -30 DEG C of refrigerator freezings of thallus are for 24 hours.
3. microcrystalline cellulose immobilization
1) thallus adds the water of 5-6 times of volume, is uniformly dispersed with high-speed shearing machine, then squeezes broken born of the same parents twice with high pressure homogenizer；
2) high speed centrifugation, clear enzyme solution in acquisition；
3) microcrystalline cellulose with biomass equivalent is added in enzyme solution, and 20-30 DEG C of concussion mixes 2h；
4) it filters, collects the thick enzyme of immobilization, washed repeatedly with kaliumphosphate buffer (20-100mM, pH7-8.5), filtering must consolidate Surely change l-amino acid deaminase.
Enzyme activity determination
The phosphate buffer of the 100mM pH6.5 of 8ml is added into the shaking flask of 250ml, the immobilization L- ammonia of 2g is then added Base acid deaminase is dispersed with stirring uniformly, is placed in 220rpm in 30 DEG C of shaking tables, preheats 30min.Then the bottom L-Phe of 10ml is added Object solution (20g/L, pH6.5,30 DEG C of preheating 30min) is placed in 30 DEG C of shaking tables, and 250rpm reacts 1h, after reaction, sampling, 20 times are diluted with phosphate aqueous solution (pH3.0).Centrifuging and taking supernatant, HPLC detect product ketone concentration of phenylalanine.
As a result: the concentration of product ketone phenylalanine is 2.5g/L.Illustrate the success of immobilization l-amino acid deaminase.
Immobilised enzymes uses the investigation of batch
Immobilization l-amino acid deaminase, reaction to no L-Phe remains according to the above scheme, and centrifugation recycling immobilised enzymes is further continued for Put into next round reaction.15 batches are used repeatedly, and the reaction time by initial 6h, extends to 12h.

Claims (7)

1. a kind of method of fixed l-amino acid deaminase, which is characterized in that cellulose binding domain is fused to l-amino acid and is taken off The C-terminal or N-terminal of adnosine deaminase.
2. a kind of method of fixed l-amino acid deaminase according to claim 1, which is characterized in that cellulose binding domain comes Derived from the cellulose binding domain of Clostridium thermocellum, genbank accession number AF283517.1.
3. a kind of method of fixed l-amino acid deaminase according to claim 1 or claim 2, which is characterized in that l-amino acid is de- Adnosine deaminase derives from the l-amino acid deaminase of proteus mirabilis, genbank accession number EU669819.1.
4. a kind of method of fixed l-amino acid deaminase according to claim 3, which is characterized in that cellulose binding domain (cellulose binding domain, CBD) genetic fragment passes through the short chain link of section of DNA to l-amino acid deaminase (L- Amino acid deaminase, LAAD) genetic fragment 3 ' or 5 ' end.
5. a kind of method of fixed l-amino acid deaminase according to claim 4, which is characterized in that the sequence of the short chain of DNA Are as follows: GGAGGAGGAGGAGGAGGAGGAGGAGGA.
6. a kind of method of fixed l-amino acid deaminase according to claim 4, which is characterized in that comprising steps of 1. weights Group bacterium building: full genome synthesizes two genetic fragments LAAD and CBD, and CBD genetic fragment passes through the short chain link of section of DNA to LAAD Genetic fragment obtains 3 ' or 5 ' ends, and is connected into pET24a carrier, constructs plasmid LAAD-CBD, converts to E.Coli BL21(DE3), Construct recombinant bacterium PMLAAD-CBD；2. recombinant bacterium is expressed；3. immobilization.
7. a kind of method of fixed l-amino acid deaminase according to claim 6, which is characterized in that step 3 immobilization Concrete operations are as follows:
1) thallus adds the water of 5-6 times of volume, is uniformly dispersed, then broken born of the same parents are twice；
2) high speed centrifugation, clear enzyme solution in acquisition；
3) microcrystalline cellulose with biomass equivalent is added in enzyme solution, and 20-30 DEG C of concussion mixes 2h；
4) it filters, collects the thick enzyme of immobilization, washed, filtered with the kaliumphosphate buffer of 20-100mM, pH7-8.5, obtain immobilization L-amino acid deaminase.
CN201810905503.1A 2018-08-10 2018-08-10 A kind of method of fixed l-amino acid deaminase Pending CN109022412A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662658A (en) * 2021-01-20 2021-04-16 江南大学 Production of L-phenylpyruvic acid by immobilized recombinant escherichia coli using L-phenylalanine
CN114958878A (en) * 2022-02-22 2022-08-30 山东蓝康生物科技有限公司 Immobilized enzyme and application thereof in synthesizing NMN

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5856201A (en) * 1993-04-14 1999-01-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Methods of detection using a cellulose binding domain fusion product
CN101899463A (en) * 2010-07-05 2010-12-01 山东大学 Beta-galactosidase with cellulose adsorption zone and application thereof
US20110045557A1 (en) * 2007-07-27 2011-02-24 Mohammed Ataai Novel Fusion Carbonic Anhydrase/Cellulose Binding Polypeptide Encoded by a Novel Hybrid Gene, and Method of Creating and Using the Same
CN102719500A (en) * 2012-07-06 2012-10-10 天津启仁医药科技有限公司 Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme
CN105802951A (en) * 2014-12-30 2016-07-27 丰益(上海)生物技术研发中心有限公司 Immobilized lipase as well as preparation method and application thereof
CN106479999A (en) * 2016-09-30 2017-03-08 苏州埃德蒙生物技术有限公司 A kind of L dglutamic oxidase CBD merges enzyme and its expressing gene and application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5856201A (en) * 1993-04-14 1999-01-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Methods of detection using a cellulose binding domain fusion product
US20110045557A1 (en) * 2007-07-27 2011-02-24 Mohammed Ataai Novel Fusion Carbonic Anhydrase/Cellulose Binding Polypeptide Encoded by a Novel Hybrid Gene, and Method of Creating and Using the Same
CN101899463A (en) * 2010-07-05 2010-12-01 山东大学 Beta-galactosidase with cellulose adsorption zone and application thereof
CN102719500A (en) * 2012-07-06 2012-10-10 天津启仁医药科技有限公司 Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme
CN105802951A (en) * 2014-12-30 2016-07-27 丰益(上海)生物技术研发中心有限公司 Immobilized lipase as well as preparation method and application thereof
CN106479999A (en) * 2016-09-30 2017-03-08 苏州埃德蒙生物技术有限公司 A kind of L dglutamic oxidase CBD merges enzyme and its expressing gene and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIN-OH BAEK等: "Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli", 《JOURNAL OF BASIC MICROBIOLOGY》 *
LAN DONGMING等: "Efficient purification of native recombinant proteins using proteases immobilized on cellulose", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 *
WANG JIANJUN等: "CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (−) γ-lactam production", 《BMC BIOTECHNOLOGY》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662658A (en) * 2021-01-20 2021-04-16 江南大学 Production of L-phenylpyruvic acid by immobilized recombinant escherichia coli using L-phenylalanine
CN114958878A (en) * 2022-02-22 2022-08-30 山东蓝康生物科技有限公司 Immobilized enzyme and application thereof in synthesizing NMN
CN114958878B (en) * 2022-02-22 2023-10-13 山东蓝康药业有限公司 Immobilized enzyme and application thereof in synthesis of NMN

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Application publication date: 20181218