Technical Field
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The invention belongs to the field of biochemical engineering, and relates to a production method of L-glufosinate-ammonium or salt thereof; in particular to a method for racemizing DL-glufosinate-ammonium to generate L-glufosinate-ammonium by using enzyme catalytic reaction.
Background
Glufosinate (4- [ hydroxy (methyl) phosphonyl ] -DL-homoalanine) is the second to sell transgenic crop tolerant herbicide worldwide. It is a broad-spectrum contact herbicide, and its action mechanism is that it can inhibit the activity of L-glutamine synthetase in plant body, so that it can make nitrogen metabolism in plant body be disturbed, and finally can kill it. Compared with glyphosate, glufosinate-ammonium has the remarkable advantages of wide practicability, quick response, long persistent period, lower toxicity, safety and the like. Therefore, the sale amount of glufosinate-ammonium is rapidly increased, the market demand is huge in a future period of time, and the prospect is very wide.
However, the technical route of glufosinate-ammonium is complex, the technical difficulty of product production is high, and the high price becomes a barrier to quickly replace glyphosate. Currently marketed glufosinate is a racemic mixture containing equal amounts of the two optical isomers (DL-glufosinate), but only the L-configuration is physiologically active. If DL-glufosinate-ammonium can be efficiently and specifically descemized to generate L-glufosinate-ammonium and useless D-glufosinate-ammonium is converted into active L-glufosinate-ammonium, the herbicidal activity can be improved by nearly one time on the basis of not changing the existing production line and capacity of glufosinate-ammonium. Therefore, the racemization removal of DL-glufosinate-ammonium to prepare the chiral pure L-glufosinate-ammonium has important practical significance and becomes the hot direction for synthesizing the L-glufosinate-ammonium in recent years.
In recent years, numerous methods for producing L-glufosinate-ammonium from DL-glufosinate-ammonium have been reported. The traditional chemical modification resolution method has no competitiveness because of high cost and incapability of utilizing D-type glufosinate-ammonium. Several ways of converting D-glufosinate to L-glufosinate have been reported to date, mainly by the following representative routes:
selective resolution by N-acylated hydrolase.
In the chinese patent application CNA, DL-glufosinate-ammonium is prepared into N-acetyl glufosinate-ammonium, and the latter can selectively obtain L-glufosinate-ammonium by hydrolysis of carboxypeptidase; the D-substrate does not participate in hydrolysis, and can be recycled to the hydrolysis step after racemization by a chemical or enzymatic method. The disadvantage of this process is the need for multiple reactions and the need to separate the hydrolysed L-glufosinate from the N-acyl substrate.
2. After the D-glufosinate-ammonium is oxidized into 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid (PPO for short), the intermediate is reduced or transaminated to generate the L-glufosinate-ammonium. In most literature, the step of converting D-glufosinate-ammonium to PPO is an oxidation process catalyzed by D-amino acid oxidase (DAAO), and often hydrogen peroxide generated is removed by adding Catalase (CAT).
In the Chinese patent application CNA, D-glufosinate-ammonium in the racemate is oxidized by oxygen under the action of DAAO to generate PPO, then the PPO is reduced by formic acid under the catalysis of palladium-carbon to generate DL-glufosinate-ammonium, and the steps are repeated in such a way, and the DL-glufosinate-ammonium is gradually converted into L-glufosinate-ammonium by utilizing the stereoselectivity of DAAO. The scheme has the advantages of one-pot reaction and no need of additional separation when the conversion rate is high. However, the disadvantage is that the palladium-carbon catalyst is used in large amounts and the reduction reaction without stereoselectivity difference wastes the reaction raw materials (oxygen and ammonium formate).
In U.S. patent application No. A1, the same procedure is used for the conversion of D-glufosinate to PPO, which is followed by the stereoselective transamination reaction catalyzed by L-amino acid transaminase (L-TA) to convert PPO to L-glufosinate. The disadvantages of this process are also evident, the transamination step being an equilibrium reaction, the need to provide an excess of amino donor (amino acid or organic amine) to ensure a high conversion (3 equivalents of amino donor, 90% conversion), and these excess amino donor and corresponding by-products seriously affecting the subsequent isolation and purification steps.
In the Chinese patent application CNA, the conversion from PPO to L-glufosinate-ammonium is a stereoselective reduction catalyzed by L-amino acid dehydrogenase (L-AADH). The patent application uses a plurality of cofactor circulating systems such as formate dehydrogenase, glucose dehydrogenase, ketoreductase, etc., to achieve the deracemization of a 20mM racemized substrate. In this scheme, the second reaction is not in equilibrium and does not require a large excess of hydrogen donor, but the conversion concentration is relatively low. In a similar Chinese patent application CNA, the racemic substrate concentration was also only 50 mM. In addition, in the chinese patent application CNA, the conversion of a substrate (200mM) with a relatively high concentration was achieved by using bacterial cells expressing the functional protein as a catalyst, but the process loss was relatively large (about 17%).
Compared with the above route, the scheme of using D-amino acid oxidase to cooperate with L-amino acid dehydrogenase has potential cost advantage. However, in the case reported at present, the substrate concentration is not high, or the loss is too large, which results in too high production cost. The main factors that prevent conversion of higher concentrations of substrate may be: DAAO-catalyzed oxidation reactions are slow, with dissolved oxygen being an important rate-limiting factor. The reaction speed can be seriously limited only by the natural dissolution of oxygen molecules in the air above the liquid level; the currently reported oxygen dissolving method is a mode of blowing air under the liquid surface in combination with stirring (as in the chinese patent application CN A), but high-shear stirring and vigorous bubble tumbling easily cause in vitro enzyme inactivation, and also cause severe foaming, which is not suitable for scale-up production.
How to efficiently supply oxygen to realize racemization removal of high-concentration DL-glufosinate-ammonium becomes the bottleneck of the prior art.
Disclosure of Invention
DAAO-catalyzed oxidation of D-glufosinate to PPO is the rate-limiting step. Hydrogen peroxide is one of the products of DAAO. In previous literature reports, the accumulation of hydrogen peroxide from the reaction inhibited DAAO activity (enzymericrobTechnol. Aug1; 27(3-5): 234-.
In the prior art, hydrogen peroxide is not expected to accumulate in DAAO catalytic reaction and needs to be removed by adding Catalase, however, the invention unexpectedly discovers that oxygen released by catalyzing the decomposition of a large amount of hydrogen peroxide by Catalase (Catalase, CAT) can be used as an oxygen source required by DAAO oxidation reaction by additionally adding a large amount of hydrogen peroxide into a reaction system. The release of oxygen catalyzed by CAT occurs uniformly in aqueous solution, has a large contact area with water, and can be rapidly dissolved in water. The examples of the present invention further demonstrate: oxygen rapidly decomposed by hydrogen peroxide under the catalytic action of catalase can effectively support the rapid and stable oxidation action of oxidases such as DAAO and the like, so that the racemization of high-concentration DL-glufosinate ammonium salt is realized to generate the high-concentration L-glufosinate ammonium salt.
Based on the above findings, the present invention provides a method for producing a glufosinate-ammonium intermediate mixture, which comprises using DL-glufosinate-ammonium or a salt thereof as a raw material, and selectively oxidizing D-glufosinate-ammonium or a salt thereof to 2-carbonyl-4- (hydroxymethylphosphono) butyric acid or a salt thereof as an intermediate in the presence of an oxidase by providing an efficient oxygen supply means for supplying a mixture of the intermediate and L-glufosinate-ammonium or a salt thereof, wherein the efficient oxygen supply means is a means for supplying hydrogen peroxide and catalase to the reaction system;
preferably, the reaction system is closed, and the closed system can improve the oxygen partial pressure and reduce the loss of oxygen, thereby being beneficial to improving the reaction speed and efficiency;
preferably, the oxidase is a D-amino acid oxidase;
preferably, the mass concentration of the added hydrogen peroxide is 1-70%; the addition amount of catalase is 1-U/mL;
preferably, the initial concentration of the DL-glufosinate-ammonium or the salt thereof is 30-300 g/L;
the present invention further provides a process for producing L-glufosinate or a salt thereof, characterized in that, after the steps of the above process, 2-carbonyl-4- (hydroxymethylphosphono) butyric acid or a salt thereof in the above glufosinate intermediate mixture is converted into L-glufosinate or a salt thereof by a step of reduction or transamination;
preferably, the reduction step is a stereoselective reduction reaction catalyzed by an L-amino acid dehydrogenase (L-AADH);
preferably, the L-amino acid dehydrogenase is selected from L-glutamate dehydrogenase or L-valine dehydrogenase;
preferably, NAD is added+NADH or NADP+NADPH as coenzyme;
preferably, glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase is added as a system for regenerating the coenzyme;
preferably, the production method of the L-glufosinate-ammonium adopts a one-pot method to feed materials;
further, the present invention provides a method for supplying oxygen to an enzyme-catalyzed reaction using oxygen as a substrate, which comprises adding hydrogen peroxide and catalase.
The production method of the L-glufosinate-ammonium or the salt thereof is specifically described as follows:
oxidizing D-type glufosinate-ammonium to 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid (PPO) in the presence of oxygen, catalyzed by D-amino acid oxidase; the latter is reduced to L-glufosinate-ammonium in situ under the catalysis of L-amino acid dehydrogenase and corresponding coenzyme circulating system; the hydrogen peroxide produced during the oxidation process and the additionally added hydrogen peroxide are efficiently decomposed into water and oxygen by means of catalase. The whole process is as follows:
the net reaction equation can be written as:
d-glufosinate-ammonium + H2O2+ coenzyme regeneration substrate &#; L-glufosinate-ammonium + coenzyme regeneration product +2H2Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the invention supplies oxygen by in-situ decomposition of the added hydrogen peroxide, the oxygen is dissolved in the solution quickly, and the oxygen saturation is easily realized in the solution, so that the oxidation reaction speed is obviously accelerated.
(2) The oxygen supply mode of the invention is mild, rapid stirring is not needed, the foam is less, the enzyme inactivation is less, and the enzyme activity maintenance time is longer.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.
Catalase used in the examples is known and is available from Zizhuang Jingding Biotech Ltd under the trade designation QD-001; d-amino acid oxidase and L-glutamic acid dehydrogenase are known and those used in the above-mentioned prior arts CNA, CNA and CNA can be used, and D-amino acid oxidase used in the examples of the present invention is commercially available from Soviet navigation Biotech Ltd, trade name YH ; the L-glutamate dehydrogenase used was purchased from Suzhou pilotage Biotech, Inc., under the product number YH ; the D-glucose dehydrogenase used was purchased from Sozhou navigation Biotech Ltd, under the product number YH .
Example 1.
DAAO rapidly oxidizes D-glufosinate-ammonium (50g/L) under the condition of adding hydrogen peroxide
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To a 500mL three-necked flask was added 10g of glufosinate ammonium salt, 180mL of phosphate buffer (pH8.0,50mM) was added and stirring was turned on. After the temperature (30 &#; C.) had stabilized, 0.5ml of catalase and 150mg of D-amino acid oxidase were added in this order and the reaction was started. In the reaction process, hydrogen peroxide with the mass concentration of 10% is dripped to maintain that small bubbles are discharged in the bottle, ammonia water is used for controlling the pH value to be about 8.0, and a sample is taken 5 hours after the reaction and is compared with the D and L configuration proportion by a pre-column derivatization high performance liquid chromatography to determine the conversion rate. After the reaction is finished, no D-glufosinate-ammonium is left, and the conversion rate is 100%.
Comparative example 1.
Comparative example under the condition of aeration-agitation under liquid level
To a 500mL three-necked flask was added 10g of glufosinate ammonium salt, 180mL of phosphate buffer (pH8.0,50mM) was added and stirring was turned on. After the temperature (30 &#; C.) had stabilized, 0.5ml of catalase and 150mg of D-amino acid oxidase were added in this order and the reaction was started. Air is continuously introduced below the reaction liquid surface through an air duct in the reaction process, ammonia water is used for controlling the pH value to be about 8.0, a sample is taken 5 hours after the reaction, and the D and L configuration ratios are compared by using pre-column derivatization high performance liquid chromatography to determine the conversion rate. After the reaction was completed, D-glufosinate-ammonium accounted for 31.3% of the total glufosinate-ammonium, and the conversion was 37.4%.
Comparative example 2.
Comparative example under atmospheric open-air agitation
To a 500mL three-necked flask was added 10g of glufosinate ammonium salt, 180mL of phosphate buffer (pH8.0,50mM) was added and stirring was turned on. After the temperature (30 &#; C.) had stabilized, 0.5ml of catalase and 150mg of D-amino acid oxidase were added in this order and the reaction was started. In the reaction process, ammonia water is used for controlling the pH value to be about 8.0, a sample is taken 5 hours after the reaction, and the D and L configuration ratios are compared by using a pre-column derivatization high performance liquid chromatography to determine the conversion rate. After the reaction was completed, D-glufosinate-ammonium accounted for 43.85% of the total glufosinate-ammonium, and the conversion was 12.3%.
Example 2.
Deracemization of 50g/L glufosinate-ammonium, 200mL system.
To a 500mL three-necked flask was added 10g of glufosinate ammonium salt, 180mL of phosphate buffer (pH8.0,50mM) was added and stirring was turned on. After the temperature (30 &#;) is stabilized, 7.5g of glucose and 20mg of coenzyme NADP are added in sequence+0.5ml of catalase, 100mg of glucose dehydrogenase and 100mg of glutamate dehydrogenase were added to the system, and 200 mgD-amino acid oxidase was added to start the reaction. Dropwise adding substance in the reaction processHydrogen peroxide with the concentration of 10 percent is used for maintaining the release of small bubbles in the bottle, ammonia water is used for controlling the pH value to be about 8.0, a sample is taken after 7 hours of reaction, and the D and L configuration concentrations and the relative proportion are compared by pre-column derivatization high performance liquid chromatography to determine the conversion rate and ee percent. After the reaction is finished, D-glufosinate-ammonium is not detected, the ee value reaches 100%, and the conversion rate is 98.3%.
Example 3.
Deracemization of 200g/L glufosinate-ammonium, alcohol dehydrogenase circulation, 5L system.
g of glufosinate ammonium salt is added into a 10L fermentation tank, mL of deionized water is added for dissolution, stirring is started for 100r/min, and the pH is adjusted to 8.0 by a small amount of ammonia water. After the temperature (30 &#;) and pH (8.0) had stabilized, 500mg of coenzyme NADP were added in sequence+40ml of catalase, 750g of glucose, 10g of D-glucose dehydrogenase and 10g of glutamate dehydrogenase were added to the system, and finally 20g of D-amino acid oxidase was added to start the reaction. And (3) dropwise adding hydrogen peroxide with the mass concentration of 30% in the reaction process, and maintaining the dissolved oxygen level in the tank to be close to a saturation value. After 10 minutes of reaction, the tank body is closed, and the pH is controlled to be about 8.0 by ammonia water. Samples were taken 7 hours after reaction conversion and ee% were determined by comparing D and L configuration concentrations and relative proportions using pre-column derivatization hplc. After the reaction, the percent content of D-glufosinate-ammonium is detected to be 0.75%, the ee value reaches 98.5%, and the conversion rate is 95.2%.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
*No endorsement is implied in the referencing of trade names.
Glufosinate is a nonselective foliar herbicide used for broadcast burndown application before planting or prior to emergence of canola, corn, sweet corn, soybean, and sugar beet. It can also be applied post-emergence to glufosinate-tolerant canola, corn, sweet corn, and soybean; however, spray contact with non-tolerant crops can result in injury. Other labeled uses of glufosinate include directed postemergence applications around trees, vines, and berries, as well as potato vine desiccation. In addition to agricultural uses, some glufosinate products can be used to control plants along landscape borders and around ornamental trees.
Glufosinate provides control of many annual broadleaf and grass weeds; however, control of large or well-tillered annual grasses, such as yellow foxtail, wild oat, or volunteer cereals, can be marginal. Glufosinate also provides suppression of some perennial weeds. Glufosinate has no soil activity.
Approximately 800,000 pounds of glufosinate were sold in Minnesota in .1 A USDA:NASS Survey, indicated that glufosinate was applied to 1% of corn acres, and 2% of soybean acres in the state.2 It was applied on up to 11% of the acres in specific counties (Wilkins). As glyphosate resistant weeds become more prevalent, glufosinate use may increase as an alternative herbicide option.3
Glufosinate controls weeds by inhibiting glutamine synthetase (herbicide site of action 10), an enzyme involved in the incorporation of ammonium into the amino acid glutamine. Inhibition of this enzyme causes a buildup of phytotoxic ammonia in plants which disrupts cell membranes. Glufosinate is a contact herbicide with limited translocation within the plant. Control is best when weeds are actively growing and not under stress.
Due to its contact activity, thorough spray coverage is needed for effective weed control. This is achieved by:
Glufosinate tolerant cultivars of corn, sweet corn, soybeans, and canola are available for use in Minnesota and marketed under the LibertyLink® name. In addition, Enlist E3 soybean varieties are available which are tolerant to glufosinate as well as glyphosate and 2,4-D. Only 2,4-D choline products, Enlist One® and Enlist Duo®, can be applied on LibertyLink® crops. Check the pesticide labels carefully prior to application.
Herbicide resistance is the inherited ability of a plant, such as weeds, to survive an herbicide application that the original population was susceptible to. The development of resistance to herbicides is a growing concern for weed management because it can lead to the loss of herbicide options, which can have important economic and environmental consequences.3
Glufosinate resistant Italian ryegrass, Lolium perenne ssp. Multiflorum, has been reported in Oregon and California.4 Resistance in other weed species such as perennial ryegrass (Lolium perenne), ridged ryegrass (Lolium rigidum), and goosegrass (Eleusine indica) have been found in other parts of the world. Glufosinate resistant weed species have not been reported in Minnesota. To prevent the development of resistant weeds, utilize practices such as combing and rotating herbicides sites-of-action and mechanical weed control.3
Glufosinate movement in soil depends on both soil properties and pesticides properties. The leaching potential of glufosinate is reduced with increasing soil clay and organic matter content. According to the National Pesticide Information Center&#;s Herbicide Properties Tool, glufosinate is likely to reach shallow groundwater in sandy soils (Koc = 10) but not in silty loam soils (Koc = 250).5 However, in soil column experiments, glufosinate and its degradants did not leach further than 6 inches in loam or clay soils, or further than 24 inches in sandy soils.6
Movement of glufosinate to surface water can occur dissolved in runoff water or adsorbed to eroding soil. Runoff loss is greatest if a surface water runoff event occurs shortly after application. Glufosinate may also move offsite via drift during application.
The MDA started monitoring for this pesticide in . Water Quality Portal7 data indicated that glufosinate was detected in 0.7% of groundwater samples and 1.1% of surface water samples in the United States. The highest concentrations detected in groundwater and surface water on the Water Quality Portal were 4.5 and 3.2 ug/L, respectively. The Minnesota Department of Health has established a drinking water reference value for glufosinate of 5 µg/L. The lowest Environmental Protection Agency (EPA) OPP (Office of Pesticide Products) aquatic life benchmark for glufosinate is 72 ug/L for non-vascular plants.
Glufosinate is very toxic to non-target plants. Since it is a contact herbicide, drift of a small volume of spray can result in necrotic vegetation. However, thorough spray coverage would be needed to kill plants, especially if larger in size.
Glufosinate is moderately toxic to fish and aquatic invertebrates with a lethal concentration, LC50, of > ug/L for both rainbow trout and water flea.7 Glufosinate is slightly toxic to mammals and birds through ingestion with acute oral LD50 values > mg/kg. Glufosinate is classified as practically non-toxic to adult honeybees on an acute contact and oral exposure basis, LD50 >100 μg ai/bee. However, the EPA states in their interim decision for glufosinate that "the Agency is unable to assess risks to pollinators at the present time" and may require additional pollinator studies related to honey bee chronic and larval toxicity testing before a final registration review decision is made for glufosinate. The toxicity of glufosinate degradates is similar to or lower than the parent compound.
Evaluation of human dietary exposure to glufosinate and its breakdown products by the EPA showed that the greatest risk from glufosinate was from contamination of drinking water. However, observed levels of exposure were found to be below levels of concern and were not considered a reasonable adverse health risk. Based on rodent studies, glufosinate is classified as not likely to be carcinogenic in humans.8
Glufosinate is a slight skin irritant and a severe eye irritant.9 EPA evaluation determined that short and intermediate term dermal exposure was not a concern if label personal protective equipment requirements, such as goggles and gloves, are utilized. The restricted entry interval for glufosinate treated areas varies among products, crops, and type of field activity.
1Minnesota Department of Agriculture. . Pesticide Sales Database. Accessed March 25, .
2Minnesota Department of Agriculture, USDA: NASS Minnesota Field Office, . Pesticide Usage on Four Major Crops in Minnesota.
3Ohio State University Extension. Weed Control Guide for Ohio, Indiana and Illinois. Bulletin 789.
4 Heap, I. The International Herbicide-Resistant Weed Database. www.weedscience.org. Accessed March 30, .
5National Pesticide Information Center. Herbicide Properties Tool. http://npic.orst.edu/HPT/. Accessed March 30, .
6United States Environmental Protections Agency. Fate and Ecological Risk Assessment for the Registration Review of Glufosinate.
7National Water Quality Monitoring Council. Water Quality Portal. Accessed March 25, .
8Donovan, W.H., D. Dotson, and K. Rury. June 19, . USEPA Memorandum: Glufosinate ammonium. Updated of revised acute and chronic aggregate dietary assessment (food and drinking water) exposure assessment in support of the petition proposing tolerances for residues of glufosinate ammonium in citrus fruits, pome fruits, stone fruits, olives, and sweet corn. CAS No: -82-2. Decision No. . https://deq.mt.gov/Portals/112/Water/WQPB/Standards/Glufosinate.pdf?ver=-12-23--317
9United States Environmental Protection Agency. Reregistration Review of Glufosinate Ammonium (PC Code ). Docket ID: EPA-HQ-OPP--. . Accessed 4/12/20.
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