CN107937376A

CN107937376A - A kind of general raw bacterium acid amides enzyme mutant, gene, engineering bacteria and its application

Info

Publication number: CN107937376A
Application number: CN201710974605.4A
Authority: CN
Inventors: 郑仁朝; 郑裕国; 金建强; 吴哲明; 汤晓玲
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2017-10-19
Filing date: 2017-10-19
Publication date: 2018-04-20
Anticipated expiration: 2037-10-19
Also published as: CN107937376B

Abstract

The invention discloses a kind of general raw bacterium acid amides enzyme mutant, gene, engineering bacteria and its application, the acid amides enzyme mutant is by general raw bacterium amidase amino acid sequence the 105th, 175,301,305 or 309 carries out single mutation shown in SEQ ID No.2 or multimutation obtains.General raw bacterium acid amides enzyme mutant provided by the invention improves 2~3 times compared with parent's vigor, to the tolerance of 2 chloro-nicotinamide of substrate more than 200mM, is even more than 1M by feed supplement, and conversion ratio still is able to be maintained at more than 95%.It can effectively mitigate Product inhibiton effect of 2 chlorine apellagrins to amidase or thalline using heretofore described feed process, while greater catalytic efficiency is ensured 2 chlorine apellagrin of product be separated out as much as possible, facilitate the extraction purification of product.Therefore, acid amides enzyme mutant of the invention can be used for 2 chlorine apellagrin of enzyme process industrialized production.

Description

A kind of general raw bacterium acid amides enzyme mutant, gene, engineering bacteria and its application

(1) technical field

The present invention relates to a kind of preparation method of 2- chlorine apellagrins, more particularly to a kind of general raw bacterium acid amides enzyme mutant, coding Gene, and its application in hydrolysis 2- chloro-nicotinamides prepare pesticide, medicine intermediate 2- chlorine apellagrins.

(2) background technology

2- chlorine apellagrins (2-Chloronicotinic Acid, abbreviation 2-CA), also known as 2- chloro-3-pyridyl formic acid, belong to cigarette 2 chlorinated compounds of acid.2- chlorine apellagrins are a kind of important fine-chemical intermediates, are widely used in pesticide and medical work Industry.In pesticide field, 2- chlorine apellagrins can be used for synthesizing fungicide, insecticide and herbicide etc., such as nicosulfuron, Diflufenican (pesticide, 2003,42,5-8；Pesticide, 2013,565-567)；In field of medicaments, available for synthesis Multiple Classes of Antibiotics, cardiovascular disease Sick medicine etc., such as anti-AIDS drug nevirapine, anti-depression medicine Midanping, non-steroidal anti-inflammatory analgesics Buddhist nun's fluorine goes out (chemistry and bioengineering, the 2008,25,5-8 such as acid, pranoprofen and mefenamic acid；Study of pharmacy, 2009,28,485- 487；US5,569,760；Fine-chemical intermediate, 2009,39,37-39).The demand of country's 2- chlorine apellagrins in 2016 reaches 3350 tons (synthesis chemistry, 2016,620-623), the market demand is huge, has a extensive future.

At present, the industrial production of 2- chlorine apellagrins mainly uses chemical method, can be divided into two big methods (synthesis chemistry, 2011, 19,285-286)：First, it is based on existing pyridine ring parent (CN101817781；CN104513198；CN103848783), directly Tap into row chlorination or introduce corresponding functional group；Second, using appropriate active straight chain compound, closed by grafting and ring-closure reaction Into (CN102993092；CN103193705；CN104592104).However, both the above chemical method is superfluous there are processing step The problems such as long, condition is harsh, and yield is low, and accessory substance is more, environmental pressure weight.In recent years, living things catalysis is because its is environmental-friendly, selection Property it is high, energy consumption is low, catalyst is degradable the advantages that, developed the important method as the manufacture of large and fine chemicals rapidly. Therefore, exploitation can efficiently prepare the biocatalysts of 2- chlorine apellagrins and be of great significance.

At present, related bioanalysis prepare 2- chlorine apellagrins research it is less, mainly utilize acid amides enzyme hydrolysis 2- chloro-nicotinamides Prepare 2- chlorine apellagrins (CN101857889；New Biotechnol.,2011,28,610-615；Catal.Commun.,2013, 38,6-9；Protein Expres.Purif.,2016,126,16-25；Enzyme Microb.Technol.,2016,86, 93-102).Amidase (Amidase, EC 3.5.1.x) is a kind of hydrolysis that can be catalyzed amido link fracture and generate corresponding carboxylic acid Enzyme, substrate spectrum is wide, can efficient catalytic various aliphatic, aromatic series and heterocyclic amide hydrolysises.Amidase has well because of it Chemistry and stereoselectivity, the great potential in medicine, in terms of pesticide intermediate preparation, is increasingly subject to industrial quarters attention.So And existing research report shows that the catalytic efficiency of acid amides enzyme hydrolysis 2- chloro-nicotinamides is generally relatively low.Therefore, amidase is improved The hydrolysis efficiency of 2- chloro-nicotinamides is catalyzed, to realize that commercial Application is very necessary.

(3) content of the invention

The purpose of the present invention is pinpoint amidase (Pa-Ami) of the saturation mutation technology to general raw bacterium source by protein Transformed, there is provided a kind of mutant protein, improves its hydrolysis vigor to 2- chloro-nicotinamides, exist so as to be conducive to the amidase Application in the synthesis of 2- chlorine apellagrins.

The technical solution adopted by the present invention is：

The present invention is by the general raw bacterium amidase base from Pantoea sp. (GenBank No.WP008109374) Because carrying out clonal expression, fixed point saturation is carried out to the expression vector comprising general raw bacterium amidase gene using full plasmid pcr Mutation, builds mutated library.Screened using the 96 orifice plate high flux screening models based on true substrate, it is a series of right to obtain The acid amides enzyme mutant that 2- chloro-nicotinamide vigor is obviously improved, efficiently synthesizes 2- chlorine apellagrins.The final present invention provides a kind of source In general raw bacterium acid amides enzyme mutant, acid amides enzyme mutant is by general raw bacterium amidase amino acid sequence shown in SEQ ID No.2 What 105,175,301,305 or 309 progress single mutation or multimutation obtained.

Further, preferably described general raw bacterium acid amides enzyme mutant be by amino acid sequence shown in SEQ ID No.2 carry out with Under point mutation：The glycine mutation of (1) the 175th is into alanine；The threonine of (2) the 301st sports leucine；(3) The alanine mutation of the 305th is threonine；The mutant serine of (4) the 309th is tyrosine；The glycine of (5) the 175th The threonine of the 301st is sported into leucine while being mutated into alanine；The glycine mutation of (6) the 175th is into the third ammonia By the alanine mutation of the 305th it is threonine while sour；The glycine mutation of (7) the 175th is incited somebody to action into while alanine The mutant serine of the 309th is tyrosine；By the third of the 305th while the threonine of (8) the 301st sports leucine Histidine mutations are threonine；It is by the mutant serine of the 309th while the threonine of (9) the 301st sports leucine Tyrosine；By the mutant serine of the 309th it is tyrosine while the alanine mutation of (10) the 305th is threonine； The glycine mutation of (11) the 175th into alanine and the threonine of the 301st sport leucine while by the 305th Alanine mutation be threonine；The glycine mutation of (12) the 175th is sported into alanine and the threonine of the 301st By the mutant serine of the 309th it is tyrosine while leucine；The glycine mutation of (13) the 175th into alanine and By the mutant serine of the 309th it is tyrosine while the alanine mutation of the 305th is threonine；(14) the 301st Threonine is sported the mutant serine of the 309th while leucine and the alanine mutation of the 305th are threonine For tyrosine；The glycine mutation of (15) the 175th sports leucine and into alanine and the threonine of the 301st By the mutant serine of the 309th it is tyrosine while the alanine mutation of 305 is threonine.

Further, preferably described acid amides enzyme mutant is general raw bacterium amidase amino acid sequence shown in SEQ ID No.2 175 glycine mutations are alanine, and amino acid sequence is SEQ ID No.4, and nucleotides sequence is classified as SEQ ID No.3.

Further, preferably described acid amides enzyme mutant is general raw bacterium amidase amino acid sequence shown in SEQ ID No.2 175th glycine mutation is alanine, while is threonine by 305 alanine mutations, and amino acid sequence is SEQ ID No.6, nucleotides sequence are classified as SEQ ID No.5.

The present invention also provides the restructuring base that a kind of general raw bacterium amidase mutant code gene and encoding gene are built Because of engineering bacteria.

The invention further relates to a kind of general raw bacterium acid amides enzyme mutant in catalysis 2- chloro-nicotinamides prepare 2- chlorine apellagrins Application, the application is obtained wet with the fermented culture of the engineering bacteria containing general raw bacterium amidase mutant code gene Thalline is catalyst, using 2- chloro-nicotinamides as substrate, using pH as 7.5~8.5 (preferable ph 8.0) buffer solution (preferably Tris-HCl buffer solutions) it is that reaction medium forms reaction system, (preferably 40 under the conditions of 30~60 DEG C, 150~500r/min DEG C, 200r/min) carry out conversion reaction, extract reaction solution isolate and purify after reaction, obtain 2- chlorine apellagrins.The reaction system In, initial substrate concentration is 50~300mM (preferably 200mM), and the dosage of the catalyst is final concentration of in terms of wet thallus weight 1~10g/L reaction systems (preferably 10g/L).

Further, the substrate is added with supplemental forms, when substrate residual concentration (is mended less than the last time in reaction system Total addition before material) addition 20% when (every 10~60min (preferably 15min)), add 50~200mM's of final concentration Substrate (preferably 100mM).

Wet thallus of the present invention is prepared as follows：By the engineering bacteria containing general raw bacterium amidase mutant code gene It is inoculated into the LB fluid nutrient mediums containing final concentration 50mg/L kanamycins, 37 DEG C, 150r/min culture 12h, then with body Product 1% inoculum concentration of concentration is transferred in the LB fluid nutrient mediums of the fresh kanamycins of 50mg/L containing final concentration, 37 DEG C, 150r/ Min is cultivated to cell concentration OD₆₀₀For 0.4~0.8, then the IPTG of final concentration of 0.1~1mM is added (preferably into culture medium 0.1mM), 28 DEG C, 150r/min Fiber differentiation 12h, take culture to centrifuge, and collect precipitation and obtain wet thallus.LB Liquid Cultures Base composition is (g/L)：Peptone 10, yeast extract 5, NaCl 10, solvent are deionized water, pH value 7.0；LB tablets are trained It is (g/L) to support base composition：Peptone 10, yeast extract 5, NaCl 10, agar 15, solvent are deionized water, pH value 7.0.

General raw bacterium acid amides enzyme mutant of the present invention can be used with the full cellular forms of engineering bacteria, can also be without pure The thick enzyme form changed uses, and can also be and is used through the zymoprotein form of partial purification or Economical Purification.If desired, can be with Immobilised enzymes or immobilized cell shape is made in the acid amides enzyme mutant of the present invention using immobilization technology known in the art Formula is used.

The acid amides enzyme mutant of the present invention is greatly improved compared with parent's vigor, complete in the crude extract using the enzyme or engineering bacteria When cell is catalyzed, reaction rate still maintains higher state.In addition, the acid amides enzyme mutant of the present invention can adapt to 30~ 55 DEG C of catalytic temperature.

Compared with prior art, the beneficial effects are mainly as follows：General raw bacterium amidase provided by the invention is dashed forward Variation improves 2~3 times compared with parent's vigor, to the tolerances of substrate 2- chloro-nicotinamides more than 200mM, is even more than by feed supplement 1M, and conversion ratio still is able to be maintained at more than 95%.It can effectively mitigate 2- chlorine using heretofore described feed process Nicotinic acid causes product 2- chlorine apellagrins to the greatest extent may be used the Product inhibiton effect of amidase or thalline while greater catalytic efficiency is ensured Energy ground separates out, and facilitates the extraction purification of product.Therefore, acid amides enzyme mutant of the invention can be used for enzyme process industrialized production 2- chlorine Nicotinic acid.

(4) illustrate

Fig. 1 is acid amides enzyme mutant G175A and acid amides enzyme mutant G175A/A305T and parent's amidase whole-cell catalytic 2- chloro-nicotinamides (200mM) prepare the reaction process contrast of 2- chlorine apellagrins.

Fig. 2 prepares 2- chlorine cigarettes for acid amides enzyme mutant G175A whole-cell catalytic 2- chloro-nicotinamides (initial concentration 200mM) The feed supplement reaction process of acid, is feed supplement time point at arrow.

Fig. 3 is prepared for acid amides enzyme mutant G175A/A305T whole-cell catalytic 2- chloro-nicotinamides (initial concentration 200mM) The feed supplement reaction process of 2- chlorine apellagrins, is feed supplement time point at arrow.

(5) embodiment

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：The fixed point saturation mutation of amidase and screening

Pinpoint saturation mutation Technical Reference (Appl.Microbiol.Biotechnol., 2014,98,2473-2483) Description, the high flux screening model of Positive mutants refer to (CN100370034；Appl.Microbiol.Biotechnol., Description 2007,74,256-262).Detailed process is as follows：

To the general raw bacterium amidase Pa-Ami (amino from Pantoea sp. (GenBank No.WP008109374) Acid sequence is shown in SEQ ID No.2, nucleotides sequence is classified as shown in SEQ ID No.1) the 175th site in amino acid sequence Glycine (Gly, G), the threonine (Thr, T) of the 301st, the alanine (Ala, A) of the 305th and the silk ammonia of the 309th Sour (Ser, S) carries out saturation mutation respectively, designs each mutant primer (being shown in Table 1), has general raw bacterium amidase Pa-Ami to compile with clone The plasmid pET28-Pa-Ami of code gene is template, carries out full plasmid amplification.PCR system is：2×phanta Max buffer 25 μ L, dNTP mixture (10mM) 0.75 μ L, are mutated sense primer (50 μM) and anti-sense primer (50 μM) each 1 μ L, plasmid 0.5 μ L, Phanta Max archaeal dna polymerases of pET28-Pa-Ami 0.5 μ L, ddH₂O is mended to 50 μ L.PCR conditions are 95 DEG C of pre- changes Property 2min；95 DEG C of denaturation 15s, 55-65 DEG C of annealing 15s, 72 DEG C of extension 6.5min, carry out 30 circulations；Last 72 DEG C of extensions 10min.Through 0.9% agarose gel electrophoresis analysis PCR product after positive, to take 20 μ L of PCR product, 1 μ L Dpn I are added, 37 DEG C digestion 2-3h removes template plasmid DNA, and 10min, transformed competence colibacillus cell E.coli BL21 (DE3) are inactivated at 65 DEG C, applies Cloth contains the LB tablets of kanamycins (50mg/L), and 37 DEG C of overnight incubations, obtain the mutant library more than 300 clones.Picking list Bacterium colony is inoculated into 96 well culture plates (the LB culture mediums that 1mL kanamycins containing 50mg/L is housed) respectively, 37 DEG C, 150rpm cultures To OD₆₀₀About 0.5 or so, the IPTG of final concentration of 0.1mM is then added, 12h is induced at 28 DEG C.96 new well culture plates are taken, point The bacterium solution that hole-specifically 100 μ L of corresponding addition are not cultivated, with 2- chloro-nicotinamides (50mM) and hydroxylamine hydrochloride (100mM) for substrate, in 50 30min is reacted at DEG C.After reaction, 100 μ L reaction solutions are taken to be added to 200 μ L nitrite ions (355mM FeCl₃It is dissolved in 0.65M HCl in), using the engineering bacteria cell before mutation as reference, according to the speed of color development system color change (yellow green → peony) To be judged, filter out color change and be faster than the bacterial strain of control group as primary dcreening operation positive bacteria.The positive colony warp that screening obtains After vitality test verification, from wherein extracting plasmid, determine that point mutation that each positive clone molecule introduces is respectively the through DNA sequencing The glycine mutation of 175 is alanine (G175A), and the threonine of the 301st sports leucine (T301L), the 305th Alanine mutation is threonine (A305T) and the mutant serine of the 309th is tyrosine (S309Y), specifically such as the institute of table 2 Show.Wherein, the vigor highest of G175A, is derived from acid amides enzyme mutant engineering bacteria E.coli BL21 (DE3)/pET28- G175A。

Table 1：Primer

Note：N=A/G/C/T, K=G/T, M=A/C, B=G/C/T, H=A/C/T, Y=C/T, R=A/G, D=A/G/T, V=A/G/C, W=A/T, S=G/C.

Embodiment 2：More sites superposition saturation mutation of acid amides enzyme mutant G175A

The high-throughput screening method of saturation mutation technology and Positive mutants is the same as embodiment 1.Detailed process is as follows：

To dashing forward (Thr, T) including the 301st threonine on the basis of acid amides enzyme mutant G175A, the 305th alanine (Ala, A) and the 309th serine (Ser, S) equipotential point carry out fixed point saturation mutation, with the matter of acid amides enzyme mutant G175A Grain pET28-G175A is template, carries out full plasmid amplification.PCR system is：2 × phanta Max buffer 25 μ L, dNTP 0.75 μ L of mixture (10mM), are mutated sense primer (50 μM) and anti-sense primer (50 μM) (primer sequence is shown in Table 1) each 1 μ L, matter 0.5 μ L, Phanta Max archaeal dna polymerases of grain pET28-G175A 0.5 μ L, ddH₂O is mended to 50 μ L.PCR conditions are 95 DEG C of pre- changes Property 2min；95 DEG C of denaturation 15s, 55-65 DEG C of annealing 15s, 72 DEG C of extension 6.5min, carry out 30 circulations；Last 72 DEG C of extensions 10min.Through 0.9% agarose gel electrophoresis analysis PCR product after positive, to take 20 μ L of PCR product, 1 μ L Dpn I are added, 37 DEG C digestion 2-3h removes template plasmid DNA, and 10min, transformed competence colibacillus cell E.coli BL21 (DE3) are inactivated at 65 DEG C, applies Cloth contains the LB tablets of kanamycins (50mg/L), and 37 DEG C of overnight incubations, obtain the mutant library more than 300 clones.Each saturation The screening of mutation library is with embodiment 1, the difference is that the G175A mutant cells that one group of embodiment 1 of control increase used is obtained. The positive colony that screening obtains extracts plasmid after vitality test verifies (table 2), the point mutation for determining to introduce through DNA sequencing, its The Ala that the middle highest positive colony DNA sequencing of vigor is shown as the 305th sports Thr (A305T), is derived from amidase Mutant engineering bacteria E.coli BL21 (DE3)/pET28-G175A/A305T.

Embodiment 3：Parent's amidase and acid amides enzyme mutant engineering bacteria induced expression and purifying

(1) induced expression

It will include starting strain E.coli BL21 (DE3)/pET28-Pa-Ami and mutant strain E.coli BL21 (DE3) including/pET28-G175A (embodiment 1), E.coli BL21 (DE3)/pET28-G175A/A305T (embodiment 2) Each Positive mutants body bacterial strain is inoculated into the LB fluid nutrient mediums of the kanamycins containing 50mg/L respectively, 37 DEG C, train under 150r/min 12h is supported, is then transferred to 1% (v/v) inoculum concentration in the LB fluid nutrient mediums of the fresh kanamycins containing 50mg/L, 37 DEG C, 150r/min is cultivated to cell concentration OD₆₀₀For 0.4~0.8, then the IPTG of final concentration of 0.1mM is added into culture medium, 28 DEG C, 150r/min Fiber differentiation 12h, take 4 DEG C of culture, 8000rpm centrifugation 15min, collect wet thallus, available for enzyme activity determination 2- chlorine apellagrins are prepared with living things catalysis.

(2) protein purification and concentration mensuration

5g wet thallus is respectively taken to be suspended in 50mL Tris-HCl buffer solutions (20mM, pH 8.0), vibration utilizes super after shaking up Sound wave cell crushing instrument smudge cells 20min.By bacterial cell disruption liquid at 4 DEG C, it is broken that 12,000rpm centrifugation 20min remove cell Piece, collects supernatant (crude enzyme liquid) and is used for follow-up isolate and purify.Using BioLogic LP low pressure chromatography systems, pass through Ni- NTA columns are purified, and start to collect destination protein after there is absworption peak (AUFS values are more than 0.1), until appearance terminates.Then The enzyme liquid of elution is fitted into bag filter (molecular cut off 14kD) and is placed in dialysis in 20mM Tris-HCl buffer solutions (pH 8.0) Desalination 24h.With bovine serum albumin (BSA) for standard protein, the albumen of enzyme liquid after dying method with coomassie brilliant blue measure dialysis is utilized Content (Anal.Biochem., 1976,25,248-256.).

Embodiment 4：The determination of activity of parent's amidase and acid amides enzyme mutant

The work of the parent's amidase and acid amides enzyme mutant of the purifying obtained in embodiment 3 to 2- chloro-nicotinamides is measured respectively Power.According to the zymoprotein concentration measured in embodiment 3, with 20mM Tris-HCl buffer solutions (pH 8.0) by corresponding zymoprotein It is diluted to 0.5mg/mL.Reaction system forms (1mL)：20mM Tris-HCl (pH 8.0), 50mM 2- chloro-nicotinamides and 10 μ L, 0.5mg/mL zymoproteins dilution, reacts 10min at 50 DEG C, then samples 100 μ L, and it is molten to add the HCl that 20 μ L concentration are 1M Liquid terminates reaction, and adds ultra-pure water to 1mL, after 0.22 μm of filtering with microporous membrane of via hole diameter, carries out liquid-phase chromatographic analysis.It is used Liquid chromatograph is Hitachi Primaide；Chromatographic column type：C18 columns, 5 μ m 250mm × 4.6mm；Mobile phase：Acetonitrile：Water：Phosphorus Acid=250：750：1(v/v/v)；Chromatographic condition：40 DEG C, Detection wavelength 270nm, flow velocity 1mL/min of column temperature.Enzyme-activity unit (U) Definition：Under the conditions of 50 DEG C, pH 8.0, the enzyme amount that catalysis 2- chloro-nicotinamides per minute generate needed for 1 micromole's 2- chlorine apellagrins is made For a unit of activity (U).Parent's amidase Pa-Ami and fractional mutations vitality of subject are shown in Table 2, and mutation vitality of subject is obvious compared with parent Raising, the wherein vigor of double-mutant G175A/A305T are 3.7 times of parent.

Table 2：The vigour of acid amides enzyme mutant and parent's amidase

Embodiment 5：Acid amides enzyme mutant whole-cell catalytic 2- chloro-nicotinamides prepare 2- chlorine apellagrins (one)

Acid amides enzyme mutant E.coli BL21 (the DE3)/pET28-G175A wet thallus cell obtained using in embodiment 3 as Catalyst, 2- chlorine apellagrins are prepared by substrate of 2- chloro-nicotinamides.10mL reaction systems are：200mM Tris-HCl buffer solutions (pH 8.0), 200mM 2- chloro-nicotinamides, 10g/L wet thallus, reacts under 40 DEG C, 200r/min.(100 μ L) is sampled during reaction, Using liquid chromatographic detection tracking reaction process (see Fig. 1), liquid chromatographic detection condition is the same as embodiment 4.As shown in Figure 1, it is dense eventually Spend for 10g/L acid amides enzyme mutant G175A wet thallus can quick catalysis 200mM 2- chloro-nicotinamides hydrolysis, it is only necessary to 20min Left and right conversion ratio is just more than 98%.

Embodiment 6：Acid amides enzyme mutant whole-cell catalytic 2- chloro-nicotinamides prepare 2- chlorine apellagrins (two)

With amidase double-mutant bacterial strain E.coli BL21 (the DE3)/pET28-G175A/A305T obtained in embodiment 3 Wet thallus cell is catalyst, using 2- chloro-nicotinamides as substrate preparation 2- chlorine apellagrins, the starting strain E.coli under similarity condition BL21 (DE3)/pET28-Pa-Ami and amidase mutants which had E.coli BL21 (DE3)/pET28-G175A is as control. Reaction condition is with embodiment 5, and testing conditions are the same as embodiment 4.As shown in Figure 1, parent's amidase Pa-Ami catalytic efficiencies are relatively low, Still not less than 98%, and under same substrate concentration, double-mutant G175A/A305T is only needed conversion ratio when reaction reaches 120min 15min is catalyzed, conversion ratio just reaches 98.6%.In addition, hydrolysis abilities of the double-mutant G175A/A305T to 2- chloro-nicotinamides Better than single mutant G175A.

Embodiment 7：Acid amides enzyme mutant whole-cell catalytic 2- chloro-nicotinamides prepare 2- chlorine apellagrins (three)

With amidase starting strain E.coli BL21 (the DE3)/pET28-Pa-Ami (control group) obtained in embodiment 3 It is catalyst with mutant E.coli BL21 (DE3)/pET28-G175A wet thallus cell, is prepared by substrate of 2- chloro-nicotinamides 2- chlorine apellagrins.10mL reaction systems are：200mM Tris-HCl buffer solutions (pH 8.0), initial concentration are the 2- chlorine cigarettes of 200mM Acid amides, 10g/L wet thallus, is reacted under 40 DEG C, 200r/min.When substrate residual concentration is less than last addition 20% When, add the 2- chloro-nicotinamides of final concentration 100mM.Finally, control group feed supplement 2 times, 2- chloro-nicotinamides is final tired in system Meter total amount is 400mM；And mutant G175A carries out 9 feed supplements altogether, the finally accumulative total amount of 2- chloro-nicotinamides is in system 1100mM.(100 μ L) is sampled during reaction, utilizes liquid chromatographic detection tracking reaction process (see Fig. 2), liquid chromatographic detection bar Part is the same as embodiment 4.As shown in Figure 2, the ultimate cumulative recovery of mutant G175A catalysis 2- chlorine apellagrins is up to 1055mM, total conversion Rate reaches 95.9%；And in control group, Pa-Ami is catalyzed the ultimate cumulative recovery only 370mM of 2- chlorine apellagrins, and overall conversion is 94.2%.

Embodiment 8：Acid amides enzyme mutant whole-cell catalytic 2- chloro-nicotinamides prepare 2- chlorine apellagrins (four)

With amidase double-mutant bacterial strain E.coli BL21 (the DE3)/pET28-G175A/A305T obtained in embodiment 3 Wet thallus cell is catalyst, and 2- chlorine apellagrins are prepared by substrate of 2- chloro-nicotinamides.10mL reaction systems are：200mM Tris- HCl buffer solutions (pH 8.0), initial concentration be 200mM 2- chloro-nicotinamides, 10g/L wet thallus, under 40 DEG C, 200r/min into Row reaction.When substrate residual concentration is less than last addition 20%, the 2- chloro-nicotinamides of final concentration 100mM are added, altogether 11 feed supplements are carried out, the finally accumulative total amount of 2- chloro-nicotinamides is 1300mM in system.(100 μ L) is sampled during reaction and carries out liquid Phase chromatography detection reaction process (see Fig. 3), liquid chromatographic detection condition is the same as embodiment 4.From the figure 3, it may be seen that 2- chlorine apellagrins is final Cumulative production is up to 1250mM, and total conversion reaches 96.1%.

The present invention is not specifically limited text, and the present invention can make in the range of claims are summarized Various changes, these change within the scope of the present invention.

Sequence table

<110>Zhejiang Polytechnical University

<120>A kind of general raw bacterium acid amides enzyme mutant, gene, engineering bacteria and its application

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<211> 471

<212> PRT

<213>General Pseudomonas (Pantoea sp.)

<400> 2

Met Asn Asn Leu His Tyr Lys Ser Leu Leu Glu Ile Gly Arg Leu Ile

1 5 10 15

Gln Ser Gly Glu Ile Ser Ser Val Glu Val Thr Gln Thr Leu Leu Thr

20 25 30

Arg Ile Asp Thr Leu Asp Arg Asp Leu His Ser Tyr Phe Tyr Val Met

35 40 45

Arg Asp Ser Ala Leu Gln Gln Ala Ala Glu Ala Asp Ala Glu Ile Ala

50 55 60

Gln Gly Lys Met Arg Gly Pro Leu His Gly Val Pro Ile Ala Leu Lys

65 70 75 80

Asp Leu Ile Trp Thr Lys Asp Ala Pro Thr Ser His Gly Met Ile Ile

85 90 95

His Lys Asp Arg Tyr Pro Thr Glu Asp Ser Thr Val Val Glu Arg Phe

100 105 110

Arg Ala Ala Gly Ala Val Ile Leu Gly Lys Leu Thr Gln Thr Glu Ser

115 120 125

Ala Phe Ala Asp His His Pro Asp Ile Ala Arg Pro Asn Asn Pro Trp

130 135 140

Gly Ser Ala Leu Trp Thr Gly Ala Ser Ser Ser Gly Ser Gly Val Ala

145 150 155 160

Thr Ala Ala Gly Leu Cys Phe Gly Ser Ile Gly Thr Asp Thr Gly Gly

165 170 175

Ser Ile Arg Phe Pro Ser Asn Ala Asn Gly Leu Thr Gly Ile Lys Pro

180 185 190

Thr Trp Ser Arg Val Thr Arg His Gly Ala Cys Glu Leu Ala Ala Ser

195 200 205

Leu Asp His Ile Gly Pro Met Ala Arg Asn Ala Ala Asp Ala Ala Ala

210 215 220

Met Leu Gln Ala Ile Ala Gly Arg Asp Asp Lys Asp Pro Thr Ser Ser

225 230 235 240

Ser Glu Pro Val Pro Asp Tyr Leu Ala Leu Met Thr Arg Gly Ile Ser

245 250 255

Lys Met Arg Ile Gly Val Asp Lys Ser Trp Ala Leu Glu Lys Val Asp

260 265 270

Glu Glu Thr Arg Ala Ala Leu Gln Ser Ala Ile Ala Thr Leu Ser Ser

275 280 285

Leu Gly Ala Thr Leu Val Asp Ile Thr Leu Pro Asp Thr Glu Lys Ala

290 295 300

Ala Ala Glu Trp Ser Ala Leu Cys Ala Val Glu Thr Ala Leu Ala His

305 310 315 320

Glu Asp Thr Tyr Pro Ala Gln Lys Asp Gln Tyr Gly Pro Gly Leu Ala

325 330 335

Gly Leu Leu Asp Leu Gly His Ser Ile Thr Ala Leu Glu Tyr Gln Arg

340 345 350

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

355 360 365

Thr Gln Val Asp Leu Ile Leu Ala Pro Ala Thr Ala Tyr Ala Gly Leu

370 375 380

Thr Trp Asp Thr Met Thr Arg Phe Gly Thr Asp Gln Ala Leu Phe Asn

385 390 395 400

Gly Val Leu Arg Tyr Thr Ser Ala Phe Asp Ala Ser Gly His Pro Thr

405 410 415

Ile Thr Leu Pro Cys Gly Lys Thr Ala Ser Gly Ala Pro Ile Gly Phe

420 425 430

Gln Leu Val Ala Ala His Phe Ala Glu Thr Thr Met Ile Gln Gly Ala

435 440 445

Trp Ala Phe Gln Gln Val Thr Asp Trp His Lys Gln His Pro Ala Leu

450 455 460

His His His His His His His

465 470

<210> 3

<211> 1416

<212> DNA

<213>General Pseudomonas (Pantoea sp.)

<400> 3

atgaataacc tgcattacaa atctctgctg gaaatcggtc gcctgatcca atctggtgaa 60

atctcctctg ttgaagtgac gcaaaccctg ctgacgcgta ttgataccct ggatcgcgac 120

ctgcatagct atttttacgt gatgcgtgat tctgcactgc agcaagcggc cgaagcagac 180

gctgaaatcg ctcagggcaa aatgcgtggt ccgctgcacg gcgttccgat tgcactgaaa 240

gatctgatct ggaccaaaga cgctccgacg tcacatggta tgattatcca caaagatcgt 300

tatccgaccg aagactcgac ggtggttgaa cgttttcgcg cagctggtgc ggtgattctg 360

ggcaaactga cccagacgga aagtgcgttt gcggatcatc acccggacat cgcacgtccg 420

aacaatccgt ggggtagcgc cctgtggacc ggtgccagct ctagtggctc tggtgttgcc 480

accgccgcgg gtctgtgctt tggcagcatt ggcaccgata cggcgggtag tatccgtttc 540

ccgtccaacg cgaatggcct gaccggtatt aaaccgacgt ggagtcgtgt cacccgtcat 600

ggcgcctgtg aactggcagc ttccctggat cacattggcc cgatggcacg taatgccgcg 660

gatgcagctg cgatgctgca ggcaatcgct ggtcgcgatg acaaagatcc gaccagcagc 720

agcgaaccgg ttccggacta tctggcgctg atgacccgtg gtattagtaa aatgcgcatc 780

ggcgtcgata aatcctgggc cctggaaaaa gtggacgaag aaacccgtgc cgcactgcag 840

agcgcgattg cgaccctgag ctctctgggt gccaccctgg tggatattac cctgccggac 900

acggaaaaag ctgcggccga atggtctgca ctgtgcgctg tcgaaacggc actggctcat 960

gaagatacct atccggcgca gaaagaccaa tatggtccgg gtctggcggg tctgctggat 1020

ctgggccact ccattaccgc actggaatac cagcgcctgc tgctgtcacg tgcagctctg 1080

cgcggtgata tttcggccct gtttacccaa gttgacctga ttctggcccc ggcaaccgcg 1140

tatgcgggtc tgacctggga taccatgacg cgttttggta cggaccaggc gctgttcaat 1200

ggcgtgctgc gctacacctc agcgttcgat gcctcgggtc atccgaccat tacgctgccg 1260

tgtggcaaaa ccgcatctgg tgctccgatc ggctttcaac tggtggcggc ccacttcgcg 1320

gaaaccacga tgatccaggg tgcgtgggcc ttccaacagg tcaccgattg gcataaacag 1380

catccggcac tgcatcatca tcaccatcat cactga 1416

<210> 4

<211> 471

<212> PRT

<213>General Pseudomonas (Pantoea sp.)

<400> 4

Met Asn Asn Leu His Tyr Lys Ser Leu Leu Glu Ile Gly Arg Leu Ile

1 5 10 15

Gln Ser Gly Glu Ile Ser Ser Val Glu Val Thr Gln Thr Leu Leu Thr

20 25 30

Arg Ile Asp Thr Leu Asp Arg Asp Leu His Ser Tyr Phe Tyr Val Met

35 40 45

Arg Asp Ser Ala Leu Gln Gln Ala Ala Glu Ala Asp Ala Glu Ile Ala

50 55 60

Gln Gly Lys Met Arg Gly Pro Leu His Gly Val Pro Ile Ala Leu Lys

65 70 75 80

Asp Leu Ile Trp Thr Lys Asp Ala Pro Thr Ser His Gly Met Ile Ile

85 90 95

His Lys Asp Arg Tyr Pro Thr Glu Asp Ser Thr Val Val Glu Arg Phe

100 105 110

Arg Ala Ala Gly Ala Val Ile Leu Gly Lys Leu Thr Gln Thr Glu Ser

115 120 125

Ala Phe Ala Asp His His Pro Asp Ile Ala Arg Pro Asn Asn Pro Trp

130 135 140

Gly Ser Ala Leu Trp Thr Gly Ala Ser Ser Ser Gly Ser Gly Val Ala

145 150 155 160

Thr Ala Ala Gly Leu Cys Phe Gly Ser Ile Gly Thr Asp Thr Ala Gly

165 170 175

Ser Ile Arg Phe Pro Ser Asn Ala Asn Gly Leu Thr Gly Ile Lys Pro

180 185 190

Thr Trp Ser Arg Val Thr Arg His Gly Ala Cys Glu Leu Ala Ala Ser

195 200 205

Leu Asp His Ile Gly Pro Met Ala Arg Asn Ala Ala Asp Ala Ala Ala

210 215 220

Met Leu Gln Ala Ile Ala Gly Arg Asp Asp Lys Asp Pro Thr Ser Ser

225 230 235 240

Ser Glu Pro Val Pro Asp Tyr Leu Ala Leu Met Thr Arg Gly Ile Ser

245 250 255

Lys Met Arg Ile Gly Val Asp Lys Ser Trp Ala Leu Glu Lys Val Asp

260 265 270

Glu Glu Thr Arg Ala Ala Leu Gln Ser Ala Ile Ala Thr Leu Ser Ser

275 280 285

Leu Gly Ala Thr Leu Val Asp Ile Thr Leu Pro Asp Thr Glu Lys Ala

290 295 300

Ala Ala Glu Trp Ser Ala Leu Cys Ala Val Glu Thr Ala Leu Ala His

305 310 315 320

Glu Asp Thr Tyr Pro Ala Gln Lys Asp Gln Tyr Gly Pro Gly Leu Ala

325 330 335

Gly Leu Leu Asp Leu Gly His Ser Ile Thr Ala Leu Glu Tyr Gln Arg

340 345 350

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

355 360 365

Thr Gln Val Asp Leu Ile Leu Ala Pro Ala Thr Ala Tyr Ala Gly Leu

370 375 380

Thr Trp Asp Thr Met Thr Arg Phe Gly Thr Asp Gln Ala Leu Phe Asn

385 390 395 400

Gly Val Leu Arg Tyr Thr Ser Ala Phe Asp Ala Ser Gly His Pro Thr

405 410 415

Ile Thr Leu Pro Cys Gly Lys Thr Ala Ser Gly Ala Pro Ile Gly Phe

420 425 430

Gln Leu Val Ala Ala His Phe Ala Glu Thr Thr Met Ile Gln Gly Ala

435 440 445

Trp Ala Phe Gln Gln Val Thr Asp Trp His Lys Gln His Pro Ala Leu

450 455 460

His His His His His His His

465 470

<210> 5

<211> 1416

<212> DNA

<213>General Pseudomonas (Pantoea sp.)

<400> 5

atgaataacc tgcattacaa atctctgctg gaaatcggtc gcctgatcca atctggtgaa 60

atctcctctg ttgaagtgac gcaaaccctg ctgacgcgta ttgataccct ggatcgcgac 120

ctgcatagct atttttacgt gatgcgtgat tctgcactgc agcaagcggc cgaagcagac 180

gctgaaatcg ctcagggcaa aatgcgtggt ccgctgcacg gcgttccgat tgcactgaaa 240

gatctgatct ggaccaaaga cgctccgacg tcacatggta tgattatcca caaagatcgt 300

tatccgaccg aagactcgac ggtggttgaa cgttttcgcg cagctggtgc ggtgattctg 360

ggcaaactga cccagacgga aagtgcgttt gcggatcatc acccggacat cgcacgtccg 420

aacaatccgt ggggtagcgc cctgtggacc ggtgccagct ctagtggctc tggtgttgcc 480

accgccgcgg gtctgtgctt tggcagcatt ggcaccgata cggcgggtag tatccgtttc 540

ccgtccaacg cgaatggcct gaccggtatt aaaccgacgt ggagtcgtgt cacccgtcat 600

ggcgcctgtg aactggcagc ttccctggat cacattggcc cgatggcacg taatgccgcg 660

gatgcagctg cgatgctgca ggcaatcgct ggtcgcgatg acaaagatcc gaccagcagc 720

agcgaaccgg ttccggacta tctggcgctg atgacccgtg gtattagtaa aatgcgcatc 780

ggcgtcgata aatcctgggc cctggaaaaa gtggacgaag aaacccgtgc cgcactgcag 840

agcgcgattg cgaccctgag ctctctgggt gccaccctgg tggatattac cctgccggac 900

acggaaaaag ctaccgccga atggtctgca ctgtgcgctg tcgaaacggc actggctcat 960

gaagatacct atccggcgca gaaagaccaa tatggtccgg gtctggcggg tctgctggat 1020

ctgggccact ccattaccgc actggaatac cagcgcctgc tgctgtcacg tgcagctctg 1080

cgcggtgata tttcggccct gtttacccaa gttgacctga ttctggcccc ggcaaccgcg 1140

tatgcgggtc tgacctggga taccatgacg cgttttggta cggaccaggc gctgttcaat 1200

ggcgtgctgc gctacacctc agcgttcgat gcctcgggtc atccgaccat tacgctgccg 1260

tgtggcaaaa ccgcatctgg tgctccgatc ggctttcaac tggtggcggc ccacttcgcg 1320

gaaaccacga tgatccaggg tgcgtgggcc ttccaacagg tcaccgattg gcataaacag 1380

catccggcac tgcatcatca tcaccatcat cactga 1416

<210> 6

<211> 471

<212> PRT

<213>General Pseudomonas (Pantoea sp.)

<400> 6

Met Asn Asn Leu His Tyr Lys Ser Leu Leu Glu Ile Gly Arg Leu Ile

1 5 10 15

Gln Ser Gly Glu Ile Ser Ser Val Glu Val Thr Gln Thr Leu Leu Thr

20 25 30

Arg Ile Asp Thr Leu Asp Arg Asp Leu His Ser Tyr Phe Tyr Val Met

35 40 45

Arg Asp Ser Ala Leu Gln Gln Ala Ala Glu Ala Asp Ala Glu Ile Ala

50 55 60

Gln Gly Lys Met Arg Gly Pro Leu His Gly Val Pro Ile Ala Leu Lys

65 70 75 80

Asp Leu Ile Trp Thr Lys Asp Ala Pro Thr Ser His Gly Met Ile Ile

85 90 95

His Lys Asp Arg Tyr Pro Thr Glu Asp Ser Thr Val Val Glu Arg Phe

100 105 110

Arg Ala Ala Gly Ala Val Ile Leu Gly Lys Leu Thr Gln Thr Glu Ser

115 120 125

Ala Phe Ala Asp His His Pro Asp Ile Ala Arg Pro Asn Asn Pro Trp

130 135 140

Gly Ser Ala Leu Trp Thr Gly Ala Ser Ser Ser Gly Ser Gly Val Ala

145 150 155 160

Thr Ala Ala Gly Leu Cys Phe Gly Ser Ile Gly Thr Asp Thr Ala Gly

165 170 175

Ser Ile Arg Phe Pro Ser Asn Ala Asn Gly Leu Thr Gly Ile Lys Pro

180 185 190

Thr Trp Ser Arg Val Thr Arg His Gly Ala Cys Glu Leu Ala Ala Ser

195 200 205

Leu Asp His Ile Gly Pro Met Ala Arg Asn Ala Ala Asp Ala Ala Ala

210 215 220

Met Leu Gln Ala Ile Ala Gly Arg Asp Asp Lys Asp Pro Thr Ser Ser

225 230 235 240

Ser Glu Pro Val Pro Asp Tyr Leu Ala Leu Met Thr Arg Gly Ile Ser

245 250 255

Lys Met Arg Ile Gly Val Asp Lys Ser Trp Ala Leu Glu Lys Val Asp

260 265 270

Glu Glu Thr Arg Ala Ala Leu Gln Ser Ala Ile Ala Thr Leu Ser Ser

275 280 285

Leu Gly Ala Thr Leu Val Asp Ile Thr Leu Pro Asp Thr Glu Lys Ala

290 295 300

Thr Ala Glu Trp Ser Ala Leu Cys Ala Val Glu Thr Ala Leu Ala His

305 310 315 320

Glu Asp Thr Tyr Pro Ala Gln Lys Asp Gln Tyr Gly Pro Gly Leu Ala

325 330 335

Gly Leu Leu Asp Leu Gly His Ser Ile Thr Ala Leu Glu Tyr Gln Arg

340 345 350

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

355 360 365

Thr Gln Val Asp Leu Ile Leu Ala Pro Ala Thr Ala Tyr Ala Gly Leu

370 375 380

Thr Trp Asp Thr Met Thr Arg Phe Gly Thr Asp Gln Ala Leu Phe Asn

385 390 395 400

Gly Val Leu Arg Tyr Thr Ser Ala Phe Asp Ala Ser Gly His Pro Thr

405 410 415

Ile Thr Leu Pro Cys Gly Lys Thr Ala Ser Gly Ala Pro Ile Gly Phe

420 425 430

Gln Leu Val Ala Ala His Phe Ala Glu Thr Thr Met Ile Gln Gly Ala

435 440 445

Trp Ala Phe Gln Gln Val Thr Asp Trp His Lys Gln His Pro Ala Leu

450 455 460

His His His His His His His

465 470

Claims

1. a kind of general raw bacterium acid amides enzyme mutant, it is characterised in that the acid amides enzyme mutant is will be general shown in SEQ ID No.2 What raw the 105th, 175,301,305 or 309 progress single mutation of bacterium amidase amino acid sequence or multimutation obtained.

2. general raw bacterium acid amides enzyme mutant as claimed in claim 1, it is characterised in that the acid amides enzyme mutant is SEQ ID General raw the 175th glycine mutation of bacterium amidase amino acid sequence is alanine shown in No.2.

3. general raw bacterium acid amides enzyme mutant as claimed in claim 1, it is characterised in that the acid amides enzyme mutant is SEQ ID General raw the 175th glycine mutation of bacterium amidase amino acid sequence is alanine shown in No.2, while by 305 alanine mutations For threonine.

A kind of 4. general raw bacterium amidase mutant code gene described in claim 1.

A kind of 5. gene constructed recombination engineering bacteria of general raw bacterium amidase mutant code described in claim 4.

A kind of 6. the answering in catalysis 2- chloro-nicotinamides prepare 2- chlorine apellagrins of general raw bacterium acid amides enzyme mutant described in claim 1 With.

7. application as claimed in claim 6, it is characterised in that the application is with containing general raw bacterium amidase mutant code The wet thallus that the fermented culture of engineering bacteria of gene obtains is catalyst, using 2- chloro-nicotinamides as substrate, using pH as 7.5~8.5 Buffer solution for reaction medium form reaction system, conversion reaction is carried out under the conditions of 30~60 DEG C, 150~500r/min, instead Extract reaction solution and isolate and purify after answering, obtain 2- chlorine apellagrins.

8. application as claimed in claim 7, it is characterised in that in the reaction system, the initial concentration of substrate for 50~ 300mM, the dosage of the catalyst are calculated as 1~10g/L reaction systems with wet thallus weight.

9. application as claimed in claim 8, it is characterised in that the substrate is added with supplemental forms, when substrate in reaction system When residual concentration is less than addition 20%, the substrate of 50~200mM of final concentration is added.

10. application as claimed in claim 7, it is characterised in that the catalyst is prepared as follows：General raw bacterium acyl will be contained The engineering bacteria of amine enzyme mutant encoding gene is inoculated into the LB fluid nutrient mediums containing final concentration 50mg/L kanamycins, and 37 DEG C, 150r/min culture 12h, the fresh kanamycins of 50mg/L containing final concentration is then transferred to 1% inoculum concentration of volumetric concentration LB fluid nutrient mediums in, 37 DEG C, 150r/min cultivated to cell concentration OD₆₀₀For 0.4~0.8, then the addition end into culture medium Concentration is the IPTG of 0.1~1mM, and 28 DEG C, 150r/min Fiber differentiation 12h, take culture to centrifuge, and collects precipitation and obtains wet bacterium Body；The LB fluid nutrient mediums form：Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, solvent is deionization Water, pH value 7.0.