Improvements in immobilized microbial nitrilase for the production of glycolic acid

专利摘要:

公开号:ES2562791T9
申请号:ES08845012.7T
申请日:2008-10-31
公开日:2016-04-18
发明作者:Robert Dicosimo；Arie Ben-Bassat
申请人:EI Du Pont de Nemours and Co；
IPC主号:

专利说明:

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SEQ ID NO: 38 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 201 (L201 K; Leu → Lys) .
SEQ ID NO: 39 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 38) comprising a single amino acid substitution at the residue of position 201 (Leu201 → Lys) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 40 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 201 (L201 N; Leu → Asn) .
SEQ ID NO: 41 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 40) comprising a single amino acid substitution at the residue of position 201 (Leu201 → Asn) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 42 is the nucleotide sequence of a mutant nitrilase of A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 201 (L201S; Leu → Ser).
SEQ ID NO: 43 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 42) comprising a single amino acid substitution at the residue of position 201 (Leu201 → Ser) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 44 is the nucleotide sequence of a mutant nitrilase of A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (F168K; Phe → Lys).
SEQ ID NO: 45 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 44) comprising a single amino acid substitution in the residue of position 168 (Phe168 → Lys) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 46 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (F168M; Phe → Met).
SEQ ID NO: 47 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 46) comprising a single amino acid substitution at the residue of position 168 (Phe168 → Met) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 48 is the nucleotide sequence of a mutant nitrilase of A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (F168T; Phe → Thr).
SEQ ID NO: 49 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 48) comprising a single amino acid substitution at the residue of position 168 (Phe168 → Thr) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 50 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (F168V; Phe → Val).
SEQ ID NO: 51 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 50) comprising a single amino acid substitution at the residue of position 168 (Phe168 → Val) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 52 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (T210A; Thr → Ala).
SEQ ID NO: 53 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 52) comprising a single amino acid substitution at the residue of position 210 (Thr210 → Ala) of the nitrilase from
A. Facilis 72W.
SEQ ID NO: 54 is the nucleotide sequence of a mutant nitrilase from A. facilis 72W comprising a codon change that resulted in a single amino acid substitution in the residue of position 168 (T210C; Thr → Cys).
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SEQ ID NO: 55 is the amino acid sequence deduced from the mutant nitrilase (SEQ ID NO: 54) comprising a single amino acid substitution at the residue of position 210 (Thr210 → Cys) of the nitrilase of
A. Facilis 72W.
SEQ ID NO: 56 is the nucleotide sequence of the nitrilase of A. facilis 72W expressed in E. coli strain i SS1001 (ATCC PTA-1177).
SEQ ID NO: 57 is the amino acid sequence deduced from the mutant nitrilase of A. facilis 72W expressed in E. coli strain SS1001 (ATCC PTA-1177).
Biological deposits
The following biological deposits have been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of the patent procedure:
Reference for identification of the International Designation of the Date of the deposit depositing deposit
Acidovorax facilis 72W ATCC 55746 March 8, 1996
E. coli SS1001 ATCC PTA-1177 January 11, 2000
As used herein, "ATCC" refers to the International Agency for the Deposit of American Type Culture Collection microorganisms domiciled at ATCC, 10801 University Blvd., Manassas, VA 20110-2209, USA. The "Designation of the International Deposit" is the access number to the crop deposited in ATCC.
The mentioned deposits will be kept in the indicated international deposit for at least thirty (30) years and will be made available to the public after the grant of a patent that describes them. The availability of a deposit does not constitute a license to practice the present invention in abrogation of the patent rights granted by the government action.
Detailed description of the invention
A method is provided to improve the specific activity of an immobilized, crosslinked and dehydrated enzyme catalyst, which has nitrilase activity for the hydrolysis of glycolonitrile to glycolic acid after rehydration. In particular, a method is provided for glutaraldehyde pretreatment of an enzyme catalyst having nitrilase activity, immobilizing the pretreated cells with glutaraldehyde and chemically crosslinking the immobilized cells before dehydration. After rehydration, the enzyme catalyst pretreated with glutaraldehyde, immobilized and crosslinked exhibits better nitrilase-specific activity compared to immobilized and cross-linked enzyme catalysts having nitrilase activity that are dehydrated and rehydrated without such treatment.
Definitions:
In this description, a series of terms and abbreviations are used. Unless otherwise specified, the following definitions apply.
As used herein, the term "comprising" means the presence of the characteristics, integers, stages or components indicated to which the claims refer, but which does not exclude the presence or addition of one or more of other characteristics, integers, stages, components or their groups.
As used herein, the term "approximately" that modifies the amount of an ingredient
or reactant of the invention employed refers to the variation in the numerical amount that can occur, for example, by the typical procedures for measuring and handling liquids used to prepare concentrates or solutions for use in the real world; for involuntary errors in these procedures; due to differences in the manufacture, source or purity of the ingredients used to prepare the compositions
or carry out the methods; and the like The term "approximately" also includes amounts that differ due to different equilibrium conditions for a composition that results from a particular initial mixture. Whether or not modified by the term "approximately", the claims include amounts equivalent. In one embodiment, the term "approximately" means within 10% of the described numerical value, preferably within 5% of the described numerical value.
As used herein, the term "glycolonitrile" is abbreviated as "GLN" and is synonymous with hydroxyacetonitrile, 2-hydroxyacetonitrile, hydroxymethylnitrile and all other synonyms of the registration number compound in the Chemical Abstracts Service (CAS) 107-16-4.
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Nitrilase source GenBank® access numberAmino acid sequence SEQ ID NO:Sequence of the distinctive remainder (positions of amino acid residues)
Synechocystis sp. PCC 6803 NP_442646.113GALACWEHYNPL (165-176)
Pseudomonas entomophila L48 YP_6090481.114GAAVCWENYMPL (161-172)
Zymomonas moblis YP_162942.1fifteenGAAICWENYMPV (161-172)
Bacillus sp. OxB-1 BAA90460.116GGLQCWEHFLPL (158-169)
Comamonas testosteroni AAA82085.117GGLQCWEHALPL (159-170)
Synechococcus sp. CC9605 YP_381420.118GALACWEHYNPL (156-167)
Pseudomonas fluorescens Pf-5 YP_260015.119GAVICWENMMPL (161-172)
Nocardia farcinica IFM 10152 YP_119480.1twentyGALCCWEHLQPL (159-170)
Alcaligenes faecalis 1650 AAY06506.1twenty-oneGALCCWEHLSPL (159-170)
Pseudomonas syringae pv. syringae B728a AAY35081.122GALCCWEHLQPL (157-168)
Bradyrhizobium sp. BTAil ZP_00859948.12. 3GALCCWEHLQPL (163-174)
Rhodococcus rhodochrous NCIMB 11216 CAC8823724GALNCWEHFQTL (161-172)
Rhodococcus rhodochrous ATCC 39484 ™ N / A25GALNCWEHFQTL (161-172)
Also described herein is the nitrilase catalyst comprising a polypeptide having nitrilase activity, isolated from a genus selected from the group consisting of Acidovorax, Rhodococcus, Nocardia, Bacillus and Alkalgenes. In another example, the nitrilase catalyst comprises a polypeptide having nitrilase activity isolated from a genus selected from the group consisting of Acidovorax and Rhodococcus.
In another embodiment, the polypeptide having nitrilase activity comes from Acidovorax facilis 72W (ATCC 55746)
or a polypeptide (having nitrilase activity) that is substantially similar to Acidovorax facilis 72W nitrilase (SEQ ID NO: 4) or the enzyme derived from A. facilis 72W represented by SEQ ID NO: 51.
In one embodiment, the nitrilase catalyst is a host microbial cell transformed to express at least one polypeptide having nitrilase activity. In one embodiment, the transformed host cell is selected from the group consisting of: Comamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp. ., Salmonella sp., Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Yarrowia sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonas sp., Rhizobium sp., And Streptomyces sp. In a preferred embodiment, the host microbial cell is selected from the group consisting of Bacillus sp., Pseudomonas sp. and Escherichia sp. In a preferred embodiment, the catalyst is an Escherichia coli host cell that recombinantly expresses one or more of the polypeptides having nitrilase activity.
Also described herein is the nitrilase catalyst comprising a polypeptide having nitrilase activity, wherein said polypeptide having nitrilase activity has at least 60% identity with SEQ ID NO: 51, preferably at least 70% of identity with SEQ ID NO: 51, even more preferably at least 80% identity with SEQ ID NO: 51, and even more preferably at least 90% identity with SEQ ID NO: 51, and most preferably at least 95% identity with SEQ ID NO: 51.
Working examples of various catalysts having nitrilase activity derived from various sources, including a nitrilase catalyst derived from A. facilis 72W, are described herein. Various mutants derived from the enzyme Acidovorax facilis 72W nitrilase have been described in the art (US Patents 7,148,051 and. 7,198,927).
In one embodiment, the polypeptide having nitrilase activity is selected from the group consisting of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57. In Another embodiment, the polypeptide having nitrilase activity is selected from the group consisting of SEQ ID NO: 4, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57. In
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Another embodiment, the polypeptide having nitrilase activity is selected from the group consisting of SEQ ID NO: 4, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57. In another embodiment, the polypeptide having nitrilase activity is selected from the group consisting of SEQ ID NO: 4, 24, 25 and 51. In another embodiment, the nitrilase catalyst comprises the SEQ polypeptide. ID NO SEQ ID NO: 51.
Acidovorax facilis 72W nitrilase (ATCC 55746)
A. facilis 72W nitrilase (EC 3.5.5.1) is a robust catalyst for the production of carboxylic acids from aliphatic or aromatic nitriles (WO 01/75077; U.S. Patent 6,870,038; and Chauhan et al. ., supra). It has also been shown to catalyze the conversion of α-hydroxynitriles (i.e. glycolonitrile) into α-hydroxycarboxylic acids (i.e., glycolic acid) (see U.S. Patents 6,383,786 and 6,416,980). However, nitrilase catalysts that have better nitrilase activity and / or stability (relative to A. facilis 72W nitrilase) when converting glycolonitrile to glycolic acid will reduce the cost of manufacturing glycolic acid. Therefore, a method for producing glycolic acid that uses a better nitrilase catalyst is useful for reducing the cost of manufacturing glycolic acid, however, A. facilis 72W nitrilase is an enzymatic catalyst for the purposes of the processes herein. invention, as well as said best nitrilase described in detail above.
Industrial production of the microbial catalyst
When commercial production of the enzyme catalysts described herein is desired, a variety of culture methodologies can be used. Fermentation operations can be carried out in batch, batch or continuous batch modes, said methods being well known in the art (Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second edition (1989) Sinauer Associates, Inc., Sunderland, MA, (1989); Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36 (3): 227-234 (1992)).
A classic batch culture method is a closed system where the composition of the media is adjusted at the beginning of the culture and is not subject to artificial disturbances during the cultivation procedure. Therefore, at the beginning of the culture procedure the medium is inoculated with the desired organism or organisms and the growth or metabolic activity is allowed to take place by adding nothing to the system. Typically, however, a "batch" culture is per batch with respect to the addition of the carbon source and attempts are often made to control factors such as pH and oxygen concentration. In batch systems, the system's metabolite and biomass compositions change constantly until the time when the crop is finished. Within batch cultures, the cells are moderated from a static logarithmic phase to a high growth logarithmic phase and finally to a stationary phase where the growth rate slows or stops. If left untreated, the cells in the stationary phase will eventually die. In some systems the cells in the logarithmic phase are often responsible for most of the production of the final product or intermediate product. In other systems a stationary or post-exponential phase production can be obtained.
A variation in the standard batch system is the powered batch system. The fed batch culture procedures are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Batch fed systems are useful when catabolite repression is apt to inhibit cell metabolism and when it is desirable to have limited amounts of substrate in the media. Measuring the actual concentration of the substrate in the fed batch systems is difficult and is therefore estimated based on changes in measurable factors, such as pH, dissolved oxygen and partial pressure of waste gases, such as CO2. Batch and batch fed methods are usual and well known in the art and examples can be found in Brock (supra) and Deshpande (supra).
Commercial production of the present enzyme catalysts having nitrilase activity can also be carried out by continuous culture. Continuous cultures are an open system in which a defined culture medium is continuously added to a bioreactor and an equal amount of conditioned medium is eliminated simultaneously during the procedure. Continuous cultures generally maintain the cells at a high and constant liquid phase density where the cells mainly develop in the logarithmic phase. Alternatively, continuous culture can be carried out with immobilized cells to which carbon and nutrients are continuously added and valuable products, by-products or residual products are continuously removed from the cell mass. Cellular immobilization can be performed using a wide range of solid supports composed of natural and / or synthetic materials.
Continuous or semi-continuous culture allows the modulation of a factor or any number of factors that affect cell growth or final cell concentration. For example, one method will keep a limiting nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to moderate. In other systems, a series of factors that affect growth can be continuously altered while the cell concentration is constant, measured by the turbidity of the medium. Continuous systems try to maintain steady-state growth conditions and, therefore, the
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Cell loss due to the media that are removed should be balanced with the rate of cell growth in the culture. Methods to modulate nutrients and growth factors in continuous culture procedures, as well as techniques to maximize cell formation speed, are well known in the art of industrial microbiology and a variety of methods have been detailed by Brock (supra).
The fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include, but are not limited to, monosaccharides, such as glucose and fructose, disaccharides, such as lactose or sucrose, polysaccharides, such as starch or cellulose, or mixtures thereof, and unpurified mixtures of renewable raw materials, such as liquid. Filtered cheese whey, fermented corn liquor, sugar beet molasses and barley malt. Therefore, it is considered that the carbon source used in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the choice of the organism.
Glutaraldehyde pretreatment of the enzyme catalyst before immobilization
Glutaraldehyde treatment of an enzyme catalyst fermentation culture can be a convenient way to kill the microbes in the culture, thus avoiding containment and safety problems in handling, conservation and transport associated with living recombinant cultures. It has now been discovered that glutaraldehyde pretreatment, or glutaraldehyde pretreatment followed by bisulfite treatment, can preserve nitrilase activity in suspended cells and in an immobilized form.
The preservation of nitrilase activity by glutaraldehyde pretreatment of an enzymatic catalyst is affected by time, temperature, glutaraldehyde concentration, pH and concentration of inhibitory products such as ammonia and other amines (for example, amino acids and peptides ) in the media that interact with glutaraldehyde. A preferred glutaraldehyde pretreatment method treats cells from high-density fermentation (100-150 DO550) with 5-10% by weight of glutaraldehyde in water which is preferably supplied as a suitable mixture of 50 mg to 500 mg of glutaraldehyde / L-min, more preferably supplied as a suitable mixture of 50 mg to 200 mg of glutaraldehyde / L-min, most preferably supplied as a suitable mixture of 50 mg to 100 mg of glutaraldehyde / L-min, resulting in a final concentration of about 3 g to about 5 g of glutaraldehyde / L (about 0.025 g to about 0.042 g of glutaraldehyde per OD550), more preferably about 3.6 g to about 5 g of glutaraldehyde / L (about 0.030 g to about 0.042 g of glutaraldehyde by DO550). The pretreated culture with glutaraldehyde can be maintained in the fermenter for approximately 1 to 5 hours. Next, a 10% by weight solution of sodium bisulfite in water at 1 g / L is optionally added to inactivate the residual glutaraldehyde.
The preferred pH for glutaraldehyde pretreatment of the enzyme catalyst in the fermentation broth or in cell suspension is from pH 5.0 to 9.0, more preferably from pH 5.0 to 8.0, even more preferably from pH 5, 0 to 7.0, still more preferably from pH 5.0 to 6.0, and most preferably from pH 5.0 to 5.5. The concentration of residual glutaraldehyde after pretreatment with glutaraldehyde is typically low, in the range of 10-200 ppm, and can be inactivated as indicated above, with the addition of sodium bisulfite to a final concentration of approximately 1 g / L . It was found that pretreatment with glutaraldehyde and bisulfite had no significant detrimental effect on nitrilase activity. The cell suspension pretreated with glutaraldehyde or glutaraldehyde / bisulfite is optionally cooled to 5-10 ° C, and optionally washed (by concentration and re-dilution of the cell suspension or fermentation broth) with water or a suitable preservation buffer to remove the residual bisulfite and unreacted glutaraldehyde.
Immobilization of the enzyme catalyst pretreated with glutaraldehyde and chemical crosslinking
Methods for immobilization of enzyme catalysts have been widely documented and are well known to those skilled in the art (Methods in Biotechnology, Vol. 1: Immobilization of enzymes and Cells; Gordon F. Bickerstaff, editor; Humana Press, Totowa, NJ , USA; 1997). The immobilization of the A. facilis 72W nitrilase catalyst (US Patent 6,870,038) has also been previously documented.
In addition, there is a method for immobilization in carrageenan and subsequent crosslinking with glutaraldehyde / polyethyleneimine of the immobilized enzyme catalyst (and as described in US Patent 6,870,038 and as described in detail in US Pat. US 6,551,804 B), however, one skilled in the art would readily recognize and apply variations to perform immobilization and crosslinking.
Such variations are contemplated herein and are within the scope of this procedure. In addition, the amounts or concentrations of the components used for immobilization and chemical crosslinking will vary depending on the amount and type of enzyme catalyst and the fermentation production of the enzyme catalyst. One skilled in the art would recognize these factors and adjust the immobilization and chemical cross-linking procedures accordingly. With regard to crosslinking with glutaraldehyde and polyethyleneimine, US Pat. 6,551,804 (supra), describes the processes and procedures
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to chemically crosslink cells immobilized with alginate. Said description also applies in the present invention for cells immobilized in carrageenan.
Dehydration / rehydration of the microbial enzyme catalyst immobilized in carrageenan and cross-linked with glutaraldehyde / polyethyleneimine
As indicated above, a particular problem related to the use of a microbial nitrilase catalyst addressed in the present application is the preservation and transport of the enzyme catalyst. Aspects of interest for the conservation and transport of enzymatic catalysts that have nitrilase activity include difficulties with the volume of the material and the inactivation of the enzymatic activity of the material over time. When immobilized in carrageenan and subsequently crosslinked with glutaraldehyde and polyethyleneimine, the resulting immobilized microbial nitrilase catalyst contained approximately 90% by weight of water, and the catalyst was typically stored at 5 ° C in an equivalent weight of aqueous buffer. A reduction in the amount of water present in the immobilized microbial nitrilase catalyst, and the removal of the aqueous buffer used to preserve the catalyst, would decrease the volume of the catalyst and the associated buffer necessary to be transported and stored before use, and would improve even more significantly the manufacturing economy of glycolic acid.
Dehydration of the immobilized and crosslinked enzyme catalyst with glutaraldehyde / polyethyleneimine can be carried out by any method known to those skilled in the art, including, but not limited to, air dehydration, dehydration in a stream of an inert gas, dehydration in a stove vacuum with or without purging with an inert gas (for example, nitrogen or argon) or lyophilization (freeze drying). The temperature for dehydration may preferably vary from about 5 ° C to about 60 ° C, more preferably from about 15 ° C to about 50 ° C and most preferably from about 20 ° C to about 40 ° C. The resulting dehydrated pearls can lose up to about 91% of their initial wet weight (when starting with beads consisting of about 5% by weight of dried cells of cells containing microbial nitrilase). The immobilized and dehydrated cell catalyst may be stored in air or under an inert atmosphere, and preferably at temperatures in the range of -25 ° C to 35 ° C, preferably 5 ° C to 25 ° C. The immobilized and dehydrated cell catalyst can be rehydrated by placing the dehydrated beads in water or in a suitable aqueous buffer, for example, a 0.10 M ammonium glycolate solution (pH 7.3), the rehydration temperature being preferably about 5 ° C to about 35 ° C. The resulting rehydrated beads can be used directly in a reaction for the production of glycolic acid from glycolonitrile, or stored in the rehydration liquid from about 5 ° C to about 35 ° C until use.
Hydrolysis of glycolonitrile to glycolic acid using a nitrilase catalyst
The enzymatic conversion of glycolonitrile to glycolic acid (in the form of acid and / or the corresponding ammonium salt) can be carried out by contacting an enzyme catalyst, an immobilized enzyme catalyst or an immobilized and cross-linked enzyme catalyst having nitrilase activity in suitable reaction conditions, as described below (ie, in an aqueous reaction mixture at certain pH ranges, temperatures, concentrations, etc.). In one embodiment, the entire recombinant microbial cells are pretreated with glutaraldehyde, immobilized in carrageenan, crosslinked, dehydrated and after rehydration the resulting enzyme catalyst is used directly for the conversion of glycolonitrile to glycolic acid, or the immobilized cells can be keep separate from the reaction mixture in bulk using hollow fiber membrane cartridges or ultrafiltration membranes. In a second embodiment, the entire recombinant microbial cells are immobilized on polyacrylamide gel, and the resulting enzyme catalyst is used directly for the conversion of glycolonitrile to glycolic acid.
The concentration of enzyme catalyst in the aqueous reaction mixture depends on the specific activity of the enzyme catalyst and is chosen to obtain the desired reaction rate. The weight of wet cells of the microbial cells used as a catalyst in hydrolysis reactions typically ranges from 0.001 grams to 0.250 grams of wet cells per mL of total reaction volume, preferably from 0.002 grams to 0.050 grams of wet cells per mL. The indicated% by weight of wet cells per volume of total reaction volume may be present in the reaction mixture in the form of an immobilized enzyme catalyst prepared as described above (supra), where the weight of wet cells as a percentage of the weight Total immobilized enzyme catalyst is known by the method of preparing the immobilized enzyme catalyst.
The glycolonitrile hydrolysis reaction temperature is chosen to control both the reaction rate and the stability of the enzyme catalyst activity. The reaction temperature may vary from just above the freezing point of the reaction mixture (about 0 ° C) to about 65 ° C, with a preferred reaction temperature range from about 5 ° C to about 35 ° C. The enzyme catalyst suspension can be prepared by suspending the immobilized and dehydrated cells in distilled water, or in an aqueous solution of a buffer that maintains the
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Initial reaction pH between about 5.0 and about 10.0, preferably between about 5.5 and about 8.0, more preferably between about 5.5 and about 7.7, and most preferably from about 6.0 up to approximately 7.7. As the reaction progresses, the pH of the reaction mixture may change due to the formation of an ammonium salt of the carboxylic acid from the corresponding nitrile functionality. The reaction can be performed to complete the conversion of glycolonitrile without pH control, or a suitable acid or base can be added during the course of the reaction to maintain the desired pH.
It was found that glycolonitrile was completely miscible with water in all proportions at 25 ° C. In cases where the reaction conditions are chosen, such that the solubility of the substrate (i.e. an α-hydroxynitrile) also depends on the temperature of the solution and / or the salt concentration (buffer or product Glycolic acid ammonium salt, also known as ammonium glycolate) in the aqueous phase, the reaction mixture may initially be composed of two phases: an aqueous phase containing the enzyme catalyst and dissolved α-hydroxynitrile, and an organic phase ( the undissolved α-hydroxynitrile). As the reaction progresses, the α-hydroxynitrile dissolves in the aqueous phase, and finally a mixture of product is obtained in a single phase. The reaction can also be performed by adding the α-hydroxynitrile to the reaction mixture at a rate approximately equal to the reaction rate of the enzymatic hydrolysis, thus maintaining an aqueous reaction mixture in a single phase, and avoiding the potential problem. of inhibition by the enzyme substrate at high concentrations of starting material.
The glycolic acid may be in the product mixture as a mixture of the protonized carboxylic acid and / or its corresponding ammonium salt (depending on the pH of the product mixture; the pKa of the glycolic acid is approximately 3.83) and may additionally be present as a salt of the carboxylic acid with any buffer that may additionally be present in the product mixture. Typically, the glycolic acid produced is primarily in the form of an ammonium salt (the pH of the glycolonitrile hydrolysis reaction is typically between about 5.5 and about 7.7). The glycolic acid product can be isolated from the reaction mixture as the protonized carboxylic acid, or as a salt of the carboxylic acid, as desired.
The final concentration of glycolic acid in the product mixture in the complete glycolonitrile conversion can vary from 0.001 M to the solubility limit of the glycolic acid product. In one embodiment, the concentration of glycolic acid will vary from about 0.10 M to about 5.0 M. In another embodiment, the concentration of glycolic acid will vary from about 0.2 M to about 3.0.
M.
Glycolic acid can be recovered in the form of the corresponding acid or salt using a variety of techniques including, but not limited to, ion exchange, electrodialysis, reactive solvent extraction, polymerization, thermal decomposition, alcoholysis and combinations thereof.
In addition, when a quantity, concentration or other value or parameter is given in the form of an interval, a preferred interval or a list of preferable higher values and lower preferable values, it should be understood that all intervals formed from a pair are specifically described. of any upper limit of the preferred range or value and any lower limit of the preferred range or value, regardless of whether the intervals are described separately. When a range of numerical values is listed herein, unless otherwise indicated, it is intended that the range include its extremes, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values cited when defining an interval.
General methods
The following examples are provided to demonstrate preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the following examples represent techniques discovered by the inventor to carry out the invention well, and therefore can be considered to be preferred modes of practice.
Suitable materials and methods for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples can be found described in Manual of Methods for General Bacteriology (1994) (Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G Briggs Phillips, eds.), American Society for Microbiology, Washington, DC.) Or by Thomas D. Brock, in Biotechnology: A Textbook of Industrial Microbiology, (1989) Second Edition, (Sinauer Associates, Inc., Sunderland, MA ). Methods for immobilizing enzymatic catalysts can be found in Bickerstaff, G. F., supra).
The procedures required for the preparation of genomic DNA, PCR amplification, DNA modifications by endo- and exo-nucleases to generate desired ends for DNA cloning, linkages and bacterial transformation, are well known in the art. Recombinant and DNA DNA techniques
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Table 2. Fermentation media, before sterilization.
(NH4) 2SO4 5.0 g / L
K2HPO4 4.0 g / L
KH2PO4 3.5 g / L
MgSO4.7H2O 0.6 g / L
Sodium Citrate. 2H2O 1.0 g / L
NZ Amine AS (Quest) 2.5 g / L
Antifoam -Biospumex 153K 0.25 mL / L
Table 3. Trace elements in fermentation
Concentration
Citric acid 10 g / L
CaCl2.2H2O 1.5 g / L
FeSO4.7H2O 5 g / L
ZnSO4.7H2O 0.39 g / L
CuSO4.5H2O 0.38 g / L
CoCl2.6H2O 0.2 g / L
MnCl2.4H2O 0.3 g / L
Table 4. Fermentation set points
Initial set points MinimumMaximum
Agitator (rpm) 4004001000
Air flow (standard liters per minute) 2210
pH 6.86.86.8
Pressure (kPa) 0.50.50.5
Dissolved oxygen (Od) 25%25%25%
Temperature ºC 303030
Table 5. Fermentation feeding protocol used with lactose induction 5
Fermentation time elapsed (h) Feed rate (g / min)Substratum
00 Glucose (in batches)
5 0.27Glucose (50% w / w)
14 1.3Lactose (25% w / w)
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Example 2
Immobilization of E. coli MG1655 / pNM18-168V in carrageenan beads crosslinked with GA / PEI
With rapid stirring, 12 g of carrageenan (FMC GP911) was slowly added to 228 g of distilled deionized water, at 50 ° C, the resulting mixture was heated to 80 ° C until the carrageenan dissolved completely, and the resulting solution was cooled with stirring until 52 ° C In a separate vessel equipped with a stir bar, 83.2 g of frozen E. coli MG1655 / pNM18-168V (25.2% in pcs) cells were added to 84.8 g of 0.35 M Na2HPO4 (pH 7.3) at approximately 25 ° C and mixed until the cells were suspended, and then a solution of deoxyribonuclease I (10 µL of 12,500 U / mL DNase (Sigma) / 100 mL of cell suspension) was added. The cell suspension was filtered consecutively by a 230 micron and 140 micron Nupro TF filter element and heated with stirring to 50 ° C. To the carrageenan solution at 52 ° C, 160.0 g of an E. coli MG1655 / pNM18168V cell suspension was added with stirring at 50 ° C, and the resulting cell / carrageenan suspension was pumped through a heated 20 gauge needle electrically at 47 ° C and 0.25M KHCO3 (pH = 7.3) was added dropwise with stirring at approximately 37-38 ° C; the flow rate through the needle was adjusted to 5-8 mL / min. The resulting pearls were allowed to harden in this same buffer for 1 hour at room temperature with stirring, and stored in 0.25 M potassium bicarbonate (pH 7.3).
Chemical crosslinking of immobilized cell / carrageenan beads was performed by adding 0.5 g of 25% glutaraldehyde (GA) in water (Sigma M 752-07) to 20 g of beads in suspension in 48 mL of bicarbonate of 0.25 M potassium (pH 7.3) and stirring for 1 hour at room temperature. To the pearl suspension was then added 2.0 g of 12.5% by weight polyethyleneimine (PEI, BASF LUPASOL PS) in water, and the pearl suspension was stirred for a further 18 hours at room temperature. The beads cross-linked with GA / PEI were recovered from the suspension, stirred twice for 15 minutes in 48 mL of 0.25 M potassium bicarbonate (pH 7.3), and then stored in 1.0 M ammonium bicarbonate (pH 7.3) at 5 ° C. Before being used as a catalyst for the conversion of glycolonitrile to glycolic acid (such as ammonium salt), the beads were washed twice for 15 minutes with 180 mL of 0.1 M ammonium glycolate (pH 7.3) at temperature environment to remove the 1.0 M ammonium bicarbonate preservation buffer (pH 7.3). The resulting immobilized cell catalyst was identified as immobilized NIT 60.
Example 3
Dehydration / rehydration of the E. coli MG1655 / pSW138-F168V transformant immobilized in carrageenan and crosslinked with glutaraldehyde / polyethyleneimine
E. coli MG1655 / pSW138-F168V transformant beads immobilized in carrageenan and crosslinked with glutaraldehyde / polyethyleneimine prepared as described in Example 2 were dehydrated in a vacuum oven (176 mm Hg) at 35 ° C with nitrogen purge during 24 hours. The ratio between the weight of dehydrated pearls and the weight of the original (non-dehydrated) pearls was 0.0914. Dehydrated pearls were subsequently rehydrated by placing the dehydrated pearls in a 20-fold (by weight) solution of 0.10 M ammonium glycolate (pH 7.3) at 5 ° C or 25 ° C for 18 hours. The resulting rehydrated pearls were washed twice with a solution 9 times (by weight) of 0.10 M ammonium glycolate (pH 7.3), and then weighed; the ratio between the weight of the rehydrated pearls and the weight of the original (non-dehydrated) pearls was 0.210 for the rehydrated pearls at 5 ° C, and the ratio between the weight of the rehydrated pearls and the weight of the original pearls was 0.212 for the pearls rehydrated at 25 ° C.
Example 4
Specific activity of the E. coli MG1655 / pSW138-F168V transformant immobilized in carrageenan and cross-linked with glutaraldehyde / polyethyleneimine before and after dehydration / rehydration
Batch reactions for the conversion of glycolonitrile to glycolic acid were carried out at 25 ° C in a controlled temperature water bath. A first reaction vessel equipped with a magnetic stir bar was charged with 8.0 g of E. coli beads MG1655 / pSW138-168V / carrageenan crosslinked with GA / PEI (dry cell weight 0.40 g, prepared as described in Example 2 without dehydration / rehydration), 6.0 mL of aqueous ammonium glycolate (4.0 M, pH 7.0) and 21.7 mL of deionized distilled water. A second reaction vessel equipped with a magnetic stir bar was charged with 1.71 g of E. coli beads MG1655 / pSW138-168V / carrageenan crosslinked with rehydrated GA / PEI (dry cell weight 0.41 g, prepared as described in Example 3 with dehydration at 35 ° C and rehydration at 5 ° C), 6.0 mL of aqueous ammonium glycolate (4.0 M, pH 7.0) and 28.0 mL of deionized distilled water. A third reaction vessel equipped with a magnetic stir bar was charged with 1.70 g of E. coli beads MG1655 / pSW138-168V / carrageenan crosslinked with rehydrated GA / PEI (weight dried cells 0.40 g, prepared as described in Example 2 with dehydration at 35 ° C and rehydration at 25 ° C), 6.0 mL of aqueous ammonium glycolate (4.0 M, pH 7.0) and 28.0 mL of deionized distilled water. Then 3.50 mL (3.75 g) of 60.8% glycolonitrile (GLN) was added simultaneously and with stirring to each reaction vessel in
image13


Table 7: Power Protocol
Feed time intervals, (h) Feed rate, g / minSubstratumStage
0 6.1350% w / w glucose1st
one 7.1350% w / w glucose1st
2 8.2850% w / w glucose1st
3 9.6250% w / w glucose1st
4 11.1850% w / w glucose1st
5 11.1850% w / w glucose1st
6 11.1850% w / w glucose1st
7 11.1850% w / w glucose1st
8 11.1850% w / w glucose1st
0 11.2225% w / w glucose2nd
2 24.4225% w / w glucose2nd
twenty 16.7225% w / w glucose2nd
30 18.725% w / w glucose2nd
40 18.725% w / w glucose2nd
At the end of the fermentation, the stirring was reduced to 150 rpm, the aeration was stopped and the temperature was maintained at 35 ° C. Part of the fermentation broth was removed, leaving approximately 180 kg in the fermenter. This remaining broth was titrated to pH 5.2 and maintained at this pH with 20% H2SO4 (20% w / w) and NaOH (50% 5 w / w), while 9.0 L of glutaraldehyde was added with stirring. aqueous (GA, 10% w / w) at a flow rate of ~ 90 mL / min; This addition rate was equivalent to 50 mg of glutaraldehyde / L of fermentation broth / min, and the final concentration of glutaraldehyde was approximately 5 g of glutaraldehyde / L (0.035 g of glutaraldehyde / DO550). After 5 h from the start of the addition of glutaraldehyde to the broth, the pH was adjusted to 7.0 and 1.8 L of aqueous sodium bisulfite (10% w / w, pH 7) was added with stirring (approximately 1 g of sodium bisulfite 10 / L final concentration), and the broth was stirred for a further 15 minutes. The temperature of the broth was then decreased to 10 ° C and the stirring was decreased to 100 rpm. The broth was concentrated to 40 kg of cell suspension using a Diskstack centrifuge (Alfa Laval), then 50 kg of DI water (20 ° C) was added to the suspension and the mixture was concentrated by centrifugation to produce 40 kg of washed cell suspension. The suspension (identified as NIT 188A-C2) was stored at 5 ° C, and a portion of the cell suspension was used directly for the preparation of an immobilized cell catalyst (Example 6). The specific activity of
Nitrilase during each stage of the procedure is summarized in Table 8.
Table 8: Nitrilase activity during the different stages of treatment with GA and bisulfite
Fermentation stage U of BZN / g pcs
Pretreatment with GA 2819
Post-treatment with GA 3300
NaHSO3 2493
Example 6
Immobilization of E. coli MG1655 / pNM18-168V pretreated with glutaraldehyde in carrageenan beads crosslinked with GA / PEI.
The final cell suspension concentrate recovered from the fermentation broth treated with sodium bisulfite and glutaraldehyde of Example 5 was centrifuged at 5 ° C. The resulting cell pellet was resuspended in an amount 5 times by weight of 0.35 M potassium phosphate buffer (pH 7.2), and centrifugation of the resulting cell suspension at 5 ° C produced a wet cell paste that was immobilized and chemically crosslinked
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权利要求:
Claims (1)
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同族专利:

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公开号 | 公开日

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US7867748B2|2011-01-11|

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JP5583586B2|2014-09-03|

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ES2562791T3|2016-03-08|

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CN102016018A|2011-04-13|

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US7741088B2|2010-06-22|

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EP2215226B1|2015-12-02|

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EP2215226B9|2016-03-02|

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PL2215226T3|2016-06-30|

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WO2009059104A1|2009-05-07|

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US20090111148A1|2009-04-30|

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DK2215226T3|2016-03-07|

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JP2011501970A|2011-01-20|

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US20100136656A1|2010-06-03|

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EP2215226A1|2010-08-11|

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CN102016018B|2013-09-25|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US11/930,744|US7741088B2|2007-10-31|2007-10-31|Immobilized microbial nitrilase for production of glycolic acid|

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US930744|2007-10-31|

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PCT/US2008/081952|WO2009059104A1|2007-10-31|2008-10-31|Improvement in immobilized microbial nitrilase for production of glycolic acid|

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