WO2020043077A1 - 制备l-苏式/赤式-对甲砜基苯丝氨酸的方法以及用于该方法的酶 - Google Patents

制备l-苏式/赤式-对甲砜基苯丝氨酸的方法以及用于该方法的酶 Download PDF

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WO2020043077A1
WO2020043077A1 PCT/CN2019/102718 CN2019102718W WO2020043077A1 WO 2020043077 A1 WO2020043077 A1 WO 2020043077A1 CN 2019102718 W CN2019102718 W CN 2019102718W WO 2020043077 A1 WO2020043077 A1 WO 2020043077A1
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amino acid
phenylserine
enzyme
seq
hydroxy
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PCT/CN2019/102718
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English (en)
French (fr)
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罗晖
常雁红
孙宏旭
胡清清
吕慧新
王艺达
肖莹
田珺玮
汪月
苏静宜
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北京科技大学
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Priority claimed from CN201810992381.4A external-priority patent/CN110872585B/zh
Priority claimed from CN201810992202.7A external-priority patent/CN110872604B/zh
Priority claimed from CN201810992214.XA external-priority patent/CN110872605B/zh
Application filed by 北京科技大学 filed Critical 北京科技大学
Publication of WO2020043077A1 publication Critical patent/WO2020043077A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • the present invention relates to a method for preparing L-threonine / erythro-p-methylsulfone phenylserine by enzyme catalysis, especially to catalyze the preparation of L- from glycine and p-methylsulfone phenylserine by using L- ⁇ -hydroxy- ⁇ -amino acid synthetase. Threo / erythro-p-sulfone phenylserine method.
  • the present invention also relates to a preparation system for use in the method of the present invention and an L- ⁇ -hydroxy- ⁇ -amino acid synthetase, including an L- ⁇ -hydroxy- ⁇ -amino acid synthetase cyclized with SpyTag-SpyCatcher.
  • L-threo-p-sulfosalyl phenylserine is the raw material for the production of methanesulfomycin and its fluorinated derivative, flumethromycin (flufenicol). Precursor substance. Both methanesulfomycin and flufenicol are broad-spectrum antibiotics with a wide range of uses. In recent years, people's demand for them is increasing year by year.
  • L-threo-p-methylsulfone phenylserine is mainly synthesized by chemical methods (for example, see Han Yuying et al. Research on the synthesis of copper p-sulfone phenylserine). Chemical Industry and Engineering, 2011, 28 (2): 29-34; and Han Yuying et al. Synthesis and resolution of DL-threo-p-methylsulfone phenylserine ethyl ester. Fine Chemicals, 2011, 28 (6): 599-602, 619), but the chemical method has reaction steps Long, harsh reaction conditions, high production costs, and unfriendly to the environment.
  • L-erythro-p-sulfone phenylserine is a diastereomer of L-threo-p-methylsulfone phenylserine.
  • L-threo-p-sulfosalyl phenylserine is the raw material for the preparation of methanesulfomycin and its fluorinated derivative, flumethromycin (flufenicol) Precursor substance.
  • L-threo-p-methylsulfone phenylserine ethyl ester directly determines the quality and cost of florfenicol, because L-erythro-p-methylsulfone phenylserine can be chemically converted to obtain L-threo- If p-methylsulfone phenylserine can be used to prepare L-erythro-p-methylsulfone phenylserine at a very simple and low cost, it will also have very important market development value.
  • L- ⁇ -hydroxy- ⁇ -amino acid synthetase (including L-threonine aldolase, L-phenylserine aldolase, serine hydroxymethyltransferase, etc.) can be used to catalyze glycine and corresponding aldehyde compounds in one step L- ⁇ -hydroxy- ⁇ -amino acid was synthesized under mild reaction conditions.
  • the first aspect of the present invention relates to a method for preparing L-threo-p-methylsulfone phenylserine by enzyme catalysis, by which the method can be prepared under mild and environmentally friendly reaction conditions and L-threo-pair can be obtained by simple separation. Methylsulfone phenylserine.
  • a second aspect of the present invention relates to a method for preparing L-threo-p-methylsulfonylphenylserine by using the waste produced by the method of the first aspect.
  • the preparation method realizes the repeated use of the reaction system.
  • the third aspect of the present invention relates to a method for enzymatically preparing L-erythro-p-methylsulfone phenylserine, by which the method can be prepared under mild and environmentally friendly reaction conditions and L-erythro-pair can be obtained by simple separation. Methylsulfone phenylserine.
  • a fourth aspect of the present invention relates to a production system for the methods of the first to third aspects described above.
  • a fifth aspect of the present invention relates to an L- ⁇ -hydroxy- ⁇ -amino acid synthetase that can be used in the methods of the first to third aspects described above, including L- ⁇ -hydroxy- ⁇ -amino acids cyclized with SpyTag-SpyCatcher Synthetase.
  • the first aspect of the present invention relates to a method for enzymatically preparing L-threo-p-sulfone phenylserine, and a method for preparing L-threo-p-sulfone phenylserine by simple separation.
  • the structural formula of the L-threo-p-methylsulfanylphenylserine is as follows:
  • the present invention provides an enzyme-catalyzed method for preparing L-threo-p-methylsulfone phenylserine, which method includes the following steps:
  • step (c) cooling the liquid phase obtained in step (b) to precipitate L-threo-p-sulfone phenylserine, and
  • step (a) the following reactions are performed in the presence of L- ⁇ -hydroxy- ⁇ -amino acid synthetase with glycine and p-methylsulfanylbenzaldehyde:
  • L-threo-p-methylsulfone phenylserine and L-erythro-p-methylsulfone phenylserine are generated. Among them, L-erythro-p-sulfone phenylserine cannot be directly used to prepare products with biological activity. .
  • it refers to an enzyme capable of catalyzing a condensation reaction between glycine and p-methylsulfonylidene to form L-threo / erythro-p-sulfone phenylserine
  • L- ⁇ -hydroxy- ⁇ -amino acid synthetase includes L- Threonine aldolase, L-phenyls
  • L-threo / erythro-p-sulfone phenylserine refers to L-threo-p-sulfone phenylserine and / or L-erythro-p-sulfone phenylserine.
  • the numerical points given include appropriate deviations, for example, the given values are ⁇ 10%, preferably ⁇ 5%, more preferably ⁇ 3%, and more preferably ⁇ 1%.
  • the amount of L- ⁇ -hydroxy- ⁇ -amino acid synthetase used in the reaction is not particularly required.
  • the amount is small, the reaction proceeds slowly, and the required reaction time is long; the amount is large, the reaction proceeds fast, and the required reaction time is short.
  • the amount of L- ⁇ -hydroxy- ⁇ -amino acid synthetase can be selected and adjusted according to the needs of the reaction.
  • the activity of L- ⁇ -hydroxy- ⁇ -amino acid synthetase is measured by its ability to catalyze the decomposition of L-phenylserine to benzaldehyde and glycine.
  • the activity of L- ⁇ -hydroxy- ⁇ -amino acid synthetase is defined as: catalyzing L- per minute under the conditions of about 30 ° C, pH of about 8.5 and L-phenylserine concentration of about 10 mmol / L
  • the amount of enzyme required for phenylserine to produce 1 ⁇ mol of benzaldehyde is 1 viable unit (U).
  • the activity of L- ⁇ -hydroxy- ⁇ -amino acid synthetase is determined as follows:
  • reaction conditions Take 10 ⁇ l of enzyme solution, 190 ⁇ l of deionized water, 200 ⁇ l of substrate solution and mix in a 1.5 ml centrifuge tube, and time the reaction at 30 ° C. When the reaction is 10 minutes, add 400 ⁇ l of 1.7% phosphoric acid stop solution to mix and terminate. reaction. Take the terminated reaction solution to measure its absorbance at 290nm, and compare it with the benzaldehyde concentration-290nm absorbance standard curve to get the benzaldehyde concentration in the reaction solution, and then calculate the activity of L- ⁇ -hydroxy- ⁇ -amino acid synthetase unit.
  • Synthetic enzymes (such as D-threonine aldolase) are catalyzed because D- ⁇ -hydroxy- ⁇ -amino acid synthetase catalyzes the reaction of glycine with p-methylsulfonylidene to form the chiral enantiomers of the two products mentioned above D-threo-p-sulfone phenylserine and D-erythro-p-sulfone phenylserine.
  • the active precursor for the production of sulfomycin and its fluorinated derivatives is L-threo-p-methylsulfone phenylserine, its D configuration enantiomer is not biologically active and needs to be obtained by complex chemical retransformation Product of the L configuration.
  • D- ⁇ -hydroxy- ⁇ -amino acid synthetase can not be used to obtain L-threo / erythro-p-methylsulfone phenylserine by simple isolation in production, that is, synthesis using D- ⁇ -hydroxy- ⁇ -amino acid Enzymes catalyzing the reaction of glycine with p-methylsulfonylbenzaldehyde cannot achieve the object of the present invention.
  • glycine is easily soluble in water, and the solubility of p-methylsulfonylbenzaldehyde in water is very low.
  • a cosolvent needs to be added to increase the solubility of p-methylsulfonylbenzaldehyde in water.
  • both p-methylsulfanylbenzaldehyde and glycine are dissolved in the reaction system in a saturated state.
  • p-methylsulfanylbenzaldehyde is added to the reaction system in an amount much greater than its solubility.
  • the molar amount of glycine added to the reaction system is greater than the molar amount of p-methanesulfonylbenzaldehyde.
  • glycine is added to the reaction system at a concentration of 0.5-2 mol / L, such as 0.6-1.5 mol / L, 0.8-1.2 mol / L, and p-methylsulfone benzaldehyde at 0.1-0.8 mol / L, such as 0.2-0.6 mol / L, 0.2-0.5 mol / L is added to the reaction system.
  • the co-solvent that can be used is selected from one or more of the following: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, t-butanol, 1 -Pentyl alcohol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2 -Butanol, 2,2-dimethylpropanol, ethylene glycol, glycerol, mercaptoethanol, ethyl acetate, butyl acetate, polyethylene glycol 6000 (PEG 6000), polyethylene glycol octylbenzene Ether (Triton X-100), acetonitrile, acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylethanolamine (DMAE), ethylene
  • the volume concentration of the co-solvent in the aqueous solution is not more than 60%, preferably not more than 55%, not more than 50%, not more than 45%, not more than 40%, and not more than 35%.
  • the volume concentration of the co-solvent is at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 22%, at least 25%, at least 30%.
  • Pyridoxal 5-phosphate can be added to the reaction system in step (a). Pyridoxal 5-phosphate can be used as a coenzyme of L- ⁇ -hydroxy- ⁇ -amino acid synthetase to increase the enzyme activity. However, pyridoxal 5-phosphate is not necessary in the method of the present invention. In the case of pyridoxal 5-phosphate, the amount of pyridoxal 5-phosphate can be not more than 200 ⁇ mol / L, for example, not more than 100 ⁇ mol / L, not more than 80 ⁇ mol / L, not more than 60 ⁇ mol / L, not more than 50 ⁇ mol / L.
  • the reaction of step (a) can be performed under a wide range of temperature conditions, for example, a temperature range of 4-50 ° C, a temperature range of 10-30 ° C, and a temperature range of 15-28 ° C.
  • a temperature range of 4-50 ° C a temperature range of 4-50 ° C
  • a temperature range of 10-30 ° C a temperature range of 15-28 ° C.
  • Formula-Formation of p-methylsulfanylphenylserine is selected according to the characteristics of the catalytic reaction of the enzyme and the desired final desired product, but usually in the temperature range of 4-50 ° C.
  • the pH of the reaction system in step (a) may be in the range of 5-10, for example, in the range of 6-9, in the range of 6-8, and in the range of 6-7.
  • a buffer may or may not be used in the reaction system. From the viewpoint of simplifying the process, it is preferable not to use a buffer.
  • the reaction in step (a) is preferably performed under stirring, and the stirring may be performed continuously or intermittently.
  • the stirring speed There is no particular limitation on the stirring speed, as long as the reaction system is in a substantially uniformly mixed state. In different reaction vessels, the appropriate stirring speed can be adjusted as required.
  • the reaction of step (a) is performed until the reaction of the reaction system reaches an equilibrium state.
  • the reaction time can be selected within a wide range, for example, the reaction can be performed for 2-75 hours, such as 5-70 hours, 10-68 hours, 10-45 Hours, 10-40 hours.
  • Increasing the selectivity of the enzyme is a method, that is, making the L- ⁇ -hydroxy- ⁇ -amino acid synthetase selectively catalyze the synthesis of L-threo-p-methylsulfone phenylserine without catalysis or as little as possible Catalyzed synthesis of L-erythro-p-methylsulfone phenylserine.
  • this method is still in the exploratory stage.
  • D- ⁇ -hydroxy- ⁇ -amino acid synthetase such as D-threonine aldolase
  • the D configuration diastereomer derivative does not It is biologically active and needs to be retransformed by a complex chemical method to obtain the L configuration product. Therefore, this still cannot achieve the goal of mild conditions, environmentally friendly and simple.
  • the highly selective D- ⁇ -hydroxy- ⁇ -amino acid synthetase still fails to achieve the object of the present invention.
  • step (a) of the method of the present invention it was unexpectedly found that under the conditions described in the present invention, as the reaction proceeds, L-erythro-p-methylsulfone phenylserine can be precipitated from the reaction system, and L-threo-p-methylsulfone phenylserine can remain dissolved.
  • L-erythro-p-methylsulfone phenylserine it is preferable to add L-erythro-p-methylsulfone phenylserine seed crystals.
  • L-erythro-p-methylsulfone phenylserine seed crystals it is not necessary to add L-erythro-p-methylsulfone phenylserine seed crystals.
  • L-erythro-p-methylsulfone phenylserine seed crystals are added to the reaction, the time of addition is not particularly limited. They can be added to the reaction system with the reactants, or they can be added after the reaction starts, for example Any time within 0-72 hours during the reaction, for example, 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours after the start of the reaction, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 33 hours, 35 hours, 38 hours, 40 hours, 42 hours, 45 hours, 48 hours, 50 hours, 52 hours 55 hours, 58 hours, 60 hours, 62 hours, 65 hours, 68 hours, 70 hours or 72 hours.
  • step (a) as the reaction proceeds, L-erythro-p-sulfone phenylserine is continuously precipitated, and L-threo-p-sulfone phenylserine is continuously accumulated in the liquid phase.
  • the equilibrium state is reached, the L-erythro-p-methylsulfone phenylserine content in the liquid phase is very small, and most of the L-erythro-p-sulfone phenylserine is precipitated.
  • the reaction system is subjected to solid-liquid separation to obtain a liquid phase containing L-threo-p-sulfone phenylserine (step (b)).
  • the solid-liquid separation operation may use conventional solid-liquid separation means, such as filtration, centrifugation, or suction filtration. However, it can be understood that the solid-liquid separation operation may not be performed until the reaction reaches an equilibrium state, and the solid-liquid separation operation may be performed before the equilibrium state is reached.
  • step (c) the temperature of the liquid phase containing L-threo-p-sulfone phenylserine is lowered to 10 ° C or lower, for example, 9 ° C or lower, 8 ° C or lower, 7 ° C or lower, 6 ° C or lower, or 5 ° C or lower. , 4 ° C or lower, 3 ° C or lower, 2 ° C or lower, 1 ° C or lower, 0 ° C or lower; however, considering actual operating conditions, the temperature is generally lowered to -5 ° C or higher, such as -4 ° C or higher, -3 ° C or higher,- Above 2 ° C and above -1 ° C.
  • L-threo-p-sulfone phenylserine was precipitated from the liquid phase. Theoretically, it is believed that the precipitation may include a mixture of crystallization and ordinary precipitation. In order to speed up the precipitation process, it is preferable to add L-threo-p-sulfone phenylserine seed crystals, but from the perspective of reducing production costs, L-threo-p-sulfone phenylserine seed crystals may not be added.
  • the time used for precipitation in step (c) is not specifically limited, as long as at least a part (preferably most) of L-threo-p-methylsulfonephenylserine in the liquid phase is precipitated out. It can be comprehensively determined according to the cooling speed, cooling temperature, and cooling equipment used. Generally, the time taken for the precipitation to cool down is in the range of 0.5-36 hours, such as 1-25 hours, 1-15 hours, and 1-5 hours.
  • step (d) the precipitated L-threo-p-methylsulfone phenylserine is obtained by solid-liquid separation.
  • the solid-liquid separation operation may use conventional solid-liquid separation means, such as filtration, centrifugation, or suction filtration.
  • the second aspect of the present invention relates to a method for preparing L-threo-p-methylsulfone phenylserine by using the waste produced by the aforementioned method. Specifically, it relates to a method for enzymatically preparing L-threo-p-sulfone phenylserine, which method includes step (e) in addition to steps (a) to (d) of the method of the first aspect described above. : The liquid phase produced in step (d) is used for the reaction in step (a). That is, the liquid phase after separation of L-threo-p-methylsulfone phenylserine is reused for the reaction of step (a).
  • the third aspect of the present invention relates to a method for enzymatically preparing L-erythro-p-methylsulfone phenylserine, wherein L-erythro-p-methylsulfone phenylserine has the following structure:
  • the method includes the following steps:
  • step (a) In the method for preparing L-erythro-p-methylsulfone phenylserine, the conditions of step (a) are basically the same as those in the method for preparing L-threo-p-methylsulfone phenylserine. Conditions can be used for the preparation of L-erythro-p-methylsulfone phenylserine.
  • step (a) of the method for preparing L-erythro-p-methylsulfone phenylserine of the present invention it was unexpectedly found that under the conditions described in the present invention, as the reaction proceeds, L-erythro-p-methyl Sulfonylphenylserine can be precipitated from the reaction system. In order to accelerate this precipitation process, it is preferable to add L-erythro-p-methylsulfone phenylserine seeds. However, from the perspective of reducing production costs, it is not necessary to add L-erythro-p-methylsulfone phenylserine seed crystals.
  • L-erythro-p-methylsulfone phenylserine seed crystals are added to the reaction, the time of addition is not particularly limited. They can be added to the reaction system with the reactants, or they can be added after the reaction starts, for example Any time within 0-72 hours during the reaction, for example, 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours after the start of the reaction, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 33 hours, 35 hours, 38 hours, 40 hours, 42 hours, 45 hours, 48 hours, 50 hours, 52 hours 55 hours, 58 hours, 60 hours, 62 hours, 65 hours, 68 hours, 70 hours or 72 hours.
  • step (a) as the reaction proceeds, L-erythro-p-sulfone phenylserine is continuously precipitated, and as L-erythro-p-sulfone phenylserine is precipitated, the equilibrium of the above reaction is continuously maintained It is broken, so that the reaction is continuously carried out in the direction of generating L-erythro-p-methylsulfone phenylserine.
  • the enzyme catalyzes the reaction of glycine and p-methylsulfonylbenzaldehyde to form L-erythro-p-methylsulfone phenylserine and L-threo-p-sulfone phenylserine is a reversible reaction
  • the unprecipitated L-threo -P-methylsulfone phenylserine is catalyzed to re-decompose into glycine and p-methylsulfone benzaldehyde. Therefore, in theory, all reactants added to the reaction system may be all converted into L-erythro-p-methylsulfone phenylserine. However, it can be understood that in actual operation, it is not necessary to wait until all the reactants are converted to L-erythro-p-methylsulfone phenylserine.
  • the reaction time of step (a) can be selected within a wide range according to the amount of enzyme used and reaction conditions such as reaction temperature, for example, the reaction can be performed for 2-150 hours, such as 5-80 hours, 10 -75 hours.
  • the reaction system was subjected to solid-liquid separation to obtain a solid phase containing L-erythro-p-methylsulfonephenylserine (step (b)).
  • the solid-liquid separation operation may use conventional solid-liquid separation means, such as filtration, centrifugation, or suction filtration. However, it can be understood that the separation operation can be performed at any time point after the precipitation of L-erythro-p-methylsulfonephenylserine.
  • the solid phase comprising L-erythro-p-methylsulfonylphenylserine is isolated, the solid phase is washed, for example, using water and / or an organic solvent, which may be acetone , Acetonitrile, DMSO, DMF, etc .; Deionized water and acetone are preferred. Washing can be performed multiple times, for example 2-5 times.
  • an organic solvent which may be acetone , Acetonitrile, DMSO, DMF, etc .; Deionized water and acetone are preferred. Washing can be performed multiple times, for example 2-5 times.
  • L-erythro-p-methylsulfone phenylserine be easily and environmentally friendly prepared by the method of the present invention, but also L-erythro-p-sulfone phenylserine can be obtained with high purity by simple separation.
  • the fourth aspect of the present invention relates to a preparation system used in the method of the present invention.
  • the preparation system includes: 1) a reaction device for making the Glycine and p-methylsulfonylosaldehyde are reacted in the presence of a co-solvent in an aqueous solution system in the presence of L- ⁇ -hydroxy- ⁇ -amino acid synthetase; 2) a solid-liquid separation device; and 3) a cooling device.
  • the preparation system is composed of the above-mentioned devices 1) to 3).
  • the preparation system only includes: 1) a reaction device for reacting glycine and p-methylsulfone benzaldehyde at L- ⁇ -hydroxy- ⁇ -Reaction in the presence of an amino acid synthetase in an aqueous solution system containing a co-solvent; 2) a solid-liquid separation device.
  • the preparation system is composed of the above-mentioned devices 1) and 2).
  • the reaction device is not particularly limited, and may be a commonly used reactor for biocatalytic reaction, for example, a kettle type reactor, a mechanically stirred type reactor, a bubble type reactor, and the like, as long as it can be performed in the reactor Reaction of Glycine and p-Methionylbenzaldehyde in the presence of L- ⁇ -hydroxy- ⁇ -amino acid synthetase in an aqueous solution system containing a cosolvent.
  • a commonly used reactor for biocatalytic reaction for example, a kettle type reactor, a mechanically stirred type reactor, a bubble type reactor, and the like, as long as it can be performed in the reactor Reaction of Glycine and p-Methionylbenzaldehyde in the presence of L- ⁇ -hydroxy- ⁇ -amino acid synthetase in an aqueous solution system containing a cosolvent.
  • the solid-liquid separation device is also not particularly limited, and may be a separation device commonly used for solid-liquid separation, such as a filter, a suction filter, a centrifuge, etc., as long as the solid-liquid separation in the method of the present invention can be achieved.
  • the cooling device is not particularly limited as long as the cooling of the liquid phase in the method of the present invention can be achieved, such as a jacketed water circulation cooling device, a cooling heat exchanger, and the like.
  • the system of the present invention is unique in that it uses a combination of these several devices. That is, corresponding to the combination of the respective steps of the method of the present invention, the various devices described above are used in combination accordingly. However, in the prior art, it has not been found that a combination of these devices is used for the preparation of L-threo / erythro-p-sulfone phenylserine.
  • a fifth aspect of the present invention relates to L- ⁇ -hydroxy- ⁇ -amino acid synthetase that can be used in the method of the present invention, including L- ⁇ -hydroxy- ⁇ -amino acid synthetase cyclized with SpyTag-SpyCatcher.
  • L- ⁇ -hydroxy- ⁇ -amino acid synthetases capable of catalyzing the above-mentioned reactions can be used in the method of the present invention.
  • the method having SEQ ID No. 1 or SEQ ID No. 1 is used in the method of the present invention.
  • an enzyme having the amino acid sequence of SEQ ID No. 1 is referred to as enzyme 24-1 (the enzyme is L-phenylserine aldolase, which belongs to the L- ⁇ -hydroxy- ⁇ -amino acid synthetase) and will have The enzyme with the amino acid sequence of SEQ ID No. 2 is called KT2440 (the enzyme is L-threonine aldolase and belongs to L- ⁇ -hydroxy- ⁇ -amino acid synthetase).
  • KT2440 the enzyme is L-threonine aldolase and belongs to L- ⁇ -hydroxy- ⁇ -amino acid synthetase.
  • SpyTag-SpyCatcher cyclized enzyme 24-1 (the enzyme is called SR-24-1) or SpyTag-SpyCatcher cyclized enzyme KT2440 (the enzyme is called the enzyme SR-KT2440).
  • SpyTag-SpyCatcher cyclase 24-1 refers to the enzyme obtained by cyclizing enzyme 24-1 using the SpyTag-SpyCatcher technology
  • SpyTag-SpyCatcher cyclizing enzyme KT2440 refers to the cyclization of enzyme 2440 using SpyTag-SpyCatcher technology The enzyme.
  • Enzyme 24-1 can be obtained by recombining a DNA sequence capable of translating the amino acid sequence of SEQ ID No. 1 into pET-28a plasmid, obtaining a recombinant plasmid pET28a-24-1, and recombining the recombinant plasmid pET28a-24 -1 was transferred into E. coli BL21 (DE3) to obtain recombinant strain BL21 (DE3) / pET28a-24-1, and the recombinant strain was cultured to express enzyme 24-1.
  • the method for culturing the recombinant strain BL21 (DE3) / pET28a-24-1 can be as follows: a single colony of the recombinant strain BL21 (DE3) / pET28a-24-1 is inoculated into LB medium at 35-40 Cultivate with shaking for 5-18h at °C, transfer the culture solution after culture and shaking to the lactose medium with an inoculum of 0.5-5%, and shake culture at 20-37 ° C for 12-40h. Collect cells by centrifugation after shaking culture Deionized water was added to the collected cells to resuspend the cells, and the cells were homogenized and crushed with a high-pressure homogenizer. The crushed solution was centrifuged and the supernatant was collected to obtain the enzyme 24-1.
  • the enzyme KT2440 can be obtained by recombining a DNA sequence capable of translating the amino acid sequence of SEQ ID No. 2 into a pET28a plasmid, obtaining a recombinant plasmid pET28a-KT2440, and transferring the recombinant plasmid pET28a-KT2440 into E. coli E.
  • a recombinant strain BL21 (DE3) / pET28a-KT2440 was obtained from coli BL21 (DE3), and the recombinant strain was cultured to express the enzyme KT2440.
  • the method for culturing the recombinant strain BL21 (DE3) / pET28a-KT2440 can be as follows: a single colony of the recombinant strain BL21 (DE3) / pET28a-KT2440 is inoculated into an LB medium, and cultured with shaking at 35-40 ° C 5-18h, transfer the culture solution after culture and shaking to the lactose medium with 0.5-5% inoculum, and shake culture at 20-37 ° C for 12-40h. After the shaking culture is complete, collect cells by centrifugation. Deionized water was added to the cells to resuspend the cells, and the cells were homogenized and crushed with a high-pressure homogenizer. The crushed solution was centrifuged and the supernatant was collected to obtain the enzyme KT2440.
  • the present invention utilizes the method of full plasmid MegaWHOP (Miyazaki, K., & Takenouchi, M. (2002). , 33 (5), 1033-4.),
  • the target gene containing SpyTag and SpyCatcher linker was obtained by constructing a synthetic lap primer, and then performing a mixed PCR with the synthetic primer and the target L- ⁇ -hydroxy- ⁇ -amino acid synthetase gene.
  • the cells were collected by centrifugation, and the cells were added to deionized water to resuspend the cells, and the cells were homogenized and crushed in a high-pressure homogenizer.
  • the broken solution was centrifuged and the supernatant was collected to obtain the enzyme KT2440.
  • the enzyme activity of the enzyme solution was measured to be 13.6 U / mL.
  • the enzyme SR-24-1 of this embodiment has the amino acid sequence structure described in SEQ ID No. 5, and the enzyme SR-24-1 is obtained by the following method:
  • the ⁇ -Lactamase gene in the SpyTag- ⁇ -Lactamase-SpyCatcher plasmid is replaced with a gene having the DNA sequence structure described in SEQ ID No. 3 to obtain the ability to translate SEQ.
  • the gene with the amino acid sequence structure described in ID No. 5 and the DNA sequence structure described in SEQ ID No. 8 is amplified by DpnI and transformed into E. coli BL21 (DE3).
  • the cyclase SR-KT2440 in this embodiment has the amino acid sequence structure described in SEQ ID No. 9, and the enzyme SR-KT2440 is obtained by the following method:
  • the ⁇ -Lactamase gene in the SpyTag- ⁇ -Lactamase-SpyCatcher plasmid was replaced with a gene having the DNA sequence structure described in SEQ ID No. 12 to obtain the translation of SEQ ID No. 9
  • the amino acid sequence structure of the gene having the DNA sequence structure described in SEQ ID No. 12 was amplified by DpnI and transformed into E. coli BL21 (DE3) to obtain the recombinant strain BL21 ( DE3) / pET28a-SR-KT2440.
  • the cyclized cephalosporin C acylase SR-CCA of this comparative example has the amino acid sequence structure described in SEQ ID No. 13, and the cyclized cephalosporin C acylase SR-CCA is obtained by the following method:
  • the ⁇ -lactamase ( ⁇ -Lactamase) gene in the SpyTag- ⁇ -Lactamase-SpyCatcher plasmid was replaced with a gene having the DNA sequence structure described in SEQ ID No. 15 to obtain a sequence capable of translating SEQ ID ID No.
  • the gene having the amino acid sequence structure of 13 having the DNA sequence structure of SEQ ID No. 18, the product obtained after amplification is digested with DpnI and transformed into E. coli BL21 (DE3) to obtain the recombinant strain.
  • BL21 (DE3) / pET28a-SR-CCA The gene having the amino acid sequence structure of 13 having the DNA sequence structure of SEQ ID No. 18, the product obtained after amplification is digested with DpnI and transformed into E. coli BL21 (DE3) to obtain the recombinant strain.
  • the medium after shaking culture was transferred to a lactose medium at 2.5% inoculation amount (the components and the aforementioned lactose medium) The same), shaking culture at 28 ° C for 24 hours; after the shaking culture, the cells were collected by centrifugation, deionized water was added to the collected cells to resuspend the cells, and the cells were homogenized and crushed in a high-pressure homogenizer, and the crushed solution was centrifuged Then, the supernatant was collected to obtain the cyclized cephalosporin C acylase SR-CCA.
  • the enzyme activity of the enzyme solution was 5.66 U / mL.
  • the recombinant strain BL21 (DE3) / pET28a-CCA is cultured in the same manner as in (3) to express cephalosporin C acylase to obtain cephalosporin C acylase CCA.
  • the enzyme activity of the enzyme solution was 6.96 U / mL.
  • Example 1 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 30% DMF
  • Example 2 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme KT2440 in 20% DMSO
  • Example 3 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 30% DMF
  • Example 4 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 30% ethanol
  • Example 5 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 40% DMSO
  • the main component of the filter cake was L-threo-p-methyl A sulfone phenylserine, wherein the ratio of L-threo-p-sulfone phenylserine to L-erythro-p-sulfone phenylserine in the filter cake is 82.1: 17.9.
  • Example 6 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 40% ethanol
  • Example 7 Preparation of L-threo-p-methylsulfone phenylserine by cyclase SR-24-1 in 30% ethanol
  • the nuclear cake and high pressure liquid chromatography determined that the main components of the filter cake were L-threo-p-methylsulfone phenylserine, and the L-threo- The ratio of p-methylsulfone phenylserine to L-erythro-p-methylsulfone phenylserine was 83.5: 16.5.
  • Example 8 Catalytic preparation of L-threo-p-methylsulfone phenylserine by enzyme 24-1 in 30% DMSO
  • the main components of the filter cake were L-threo-p-methylsulfone phenylserine, in which the L-threo-p-sulfone phenylserine and L-erythro-p-methyl
  • the ratio of sulfone phenylserine was 92.6: 7.4.
  • p-methanesulfonylbenzaldehyde and glycine form L-threo-p-methanesulfonylphenylserine and L-erythro-p-methanesulfonylphenylserine, which are precipitated and filtered to isolate the product L-threonine -After p-methylsulfanylphenylserine, add glycine and p-methylsulfanylbenzaldehyde to the filtrate after suction filtration, and repeat the above-mentioned catalytic reaction process and separation process.
  • the application of the filtrate can be performed multiple times before the enzyme is inactivated.
  • the main component of the filter cake was L-threo-p-methylsulfone phenylserine, in which the L-threo-p-methylsulfone phenylserine and L
  • the ratio of -erythro-p-methylsulfonephenylserine was 91.7: 8.3.
  • a second application was performed on the filtrate from which L-threo-p-methylsulfone phenylserine was removed by suction filtration, and 0.56 g of glycine was added to the filtrate, and the filter cake (containing unreacted Methanesulfonyl benzaldehyde), and the above-mentioned catalytic reaction process (reaction 36h) and separation process were repeated.
  • a filter cake containing a ratio of L-threo-p-methylsulfone phenylserine to L-erythro-p-sulfone phenylserine of 94.3: 5.7 can be obtained.
  • Example 10 Enzyme KT2440 catalyzes 200 mM p-methylsulfonylbenzaldehyde in 30% DMSO
  • Example 11 The enzyme KT2440 catalyzes 400 mM p-methylsulfonylbenzaldehyde in 30% DMSO
  • Example 12 Enzyme KT2440 catalyzes 400 mM p-methylsulfonylbenzaldehyde in 15% DMSO
  • Example 13 Enzyme 24-1 catalyzes 400 mM p-methylsulfonylbenzaldehyde in 30% DMSO
  • Example 14 The enzyme KT2440 catalyzes 300 mM p-methylsulfonylbenzaldehyde in 30% DMSO
  • Example 15 Enzyme 24-1 catalyzes 400 mM p-methylsulfonylbenzaldehyde in 30% DMF
  • Example 16 Enzyme KT2440 catalyzes 100 mM p-methylsulfonylbenzaldehyde in 30% DMSO
  • the enzyme solutions of Enzyme Preparation Example 1 and Enzyme Preparation Example 3 were respectively treated at a temperature of 60 ° C for 60 minutes. After shaking and shaking uniformly, a certain amount of enzyme solution was taken to determine the enzyme activity, and the enzyme activity data was compared with the initial value without any treatment. Enzyme activity was compared to calculate the percentage of residual enzyme activity.
  • the residual enzyme activity ratio of enzyme 24-1 in Enzyme Preparation Example 1 was 42.7%
  • the residual enzyme activity ratio of cyclase SR-24-1 in Enzyme Preparation Example 3 was 91.8%.
  • Example 19 Evaluation of the thermal stability of the enzyme KT2440 and the cyclase SR-KT2440
  • the enzyme solutions of Enzyme Preparation Example 2 and Enzyme Preparation Example 4 were respectively treated at a temperature of 50 ° C for 60 minutes. After shaking and shaking uniformly, a certain amount of enzyme solution was taken to determine the enzyme activity, and the enzyme activity data was compared with the initial value without any treatment. Enzyme activity was compared to calculate the percentage of residual enzyme activity.
  • the residual enzyme activity ratio of the enzyme KT2440 in the enzyme preparation example 2 was 18.4%
  • the residual enzyme activity ratio of the cyclase SR-KT2440 in the enzyme preparation example 4 was 32.4%.
  • Example 20 Evaluation of denaturant tolerance of enzyme 24-1 and cyclase SR-24-1
  • the enzyme solutions of Enzyme Preparation Example 1 and Enzyme Preparation Example 3 were added to a 4M guanidine hydrochloride denaturant, and after incubating at room temperature for 30 minutes, the enzyme activity was measured.
  • the enzyme activity without any treatment (denaturant concentration in the solution is 0M) is 100%, and the residual enzyme activity percentage at the denaturant concentration is calculated.
  • the residual enzyme activity ratio of enzyme 24-1 in Enzyme Preparation Example 1 was 3.6%
  • the residual enzyme activity ratio of cyclase SR-24-1 in Enzyme Preparation Example 3 was 38.6%.
  • Example 21 Evaluation of DMSO tolerance of enzyme 24-1 and cyclase SR-24-1
  • Example 22 Evaluation of ethanol tolerance of enzyme 24-1 and cyclase SR-24-1
  • Example 23 Evaluation of methanol tolerance of enzyme 24-1 and cyclase SR-24-1
  • the main component of the solid phase was L-erythro Formula-p-methylsulfone phenylserine, the liquid phase was cooled to 4 ° C., stirred for 1 h, and then filtered by suction to obtain a filter cake, which was determined by nuclear magnetic resonance and high pressure liquid chromatography.
  • the main component of the filter cake was L-threo-p-methyl A sulfone phenylserine, wherein the ratio of L-threo-p-methylsulfone phenylserine to L-erythro-p-sulfone phenylserine in the filter cake is 83.5: 16.5.
  • Example 25 Enzyme SR-2440 catalyzes the preparation of L-threo-p-methylsulfone phenylserine in DMSO
  • the collected filter cake was washed with deionized water, and the filter cake was washed three times with acetone, and the unreacted substrate p-methylsulfanyl benzaldehyde and glycine were washed away. Finally, the filter cake was dried in a vacuum drying box, and the obtained That is L-erythro-p-methylsulfone phenylserine. After analysis by high performance liquid chromatography, the purity is 94.5%, and the yield is 62%.
  • the cephalosporin C acylase CCA and the cyclized cephalosporin C acylase SR-CCA enzyme solution of Comparative Enzyme Preparation Example 1 were respectively treated at a temperature of 55 ° C for 40 minutes. After shaking and shaking evenly, a certain amount of enzyme solution was taken. The enzyme activity was determined by the reaction, and the enzyme activity data was compared with the initial enzyme activity without any treatment to calculate the percentage of residual enzyme activity.
  • the residual enzyme activity ratio of cephalosporin C acylase was 11.7%, and the residual enzyme activity ratio of cyclized cephalosporin C acylase SR-CCA was 9.9%.

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Abstract

提供一种酶催化制备L-苏式/赤式-对甲砜基苯丝氨酸的方法，特别是涉及利用L-β-羟基-α-氨基酸合成酶催化甘氨酸和对甲砜基苯丝氨酸制备L-苏式/赤式-对甲砜基苯丝氨酸的方法。提供用于上述方法的制备系统以及L-β-羟基-α-氨基酸合成酶，包括用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶。

Description

制备L-苏式/赤式-对甲砜基苯丝氨酸的方法以及用于该方法的酶 技术领域
本发明涉及酶催化制备L-苏式/赤式-对甲砜基苯丝氨酸的方法，特别是涉及利用L-β-羟基-α-氨基酸合成酶催化甘氨酸和对甲砜基苯丝氨酸制备L-苏式/赤式-对甲砜基苯丝氨酸的方法。本发明还涉及用于本发明方法的制备系统以及L-β-羟基-α-氨基酸合成酶，包括用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶。
背景技术
L-苏式-对甲砜基苯丝氨酸是生产甲砜霉素及其氟化衍生物氟甲砜霉素(氟苯尼考)的原料L-苏式-对甲砜基苯丝氨酸乙酯的前体物质。甲砜霉素和氟苯尼考均是拥有广泛用途的广谱抗生素，近年来，人们对他们的需求量正在逐年增加。
目前，生产L-苏式-对甲砜基苯丝氨酸主要通过化学法合成(例如，参见韩玉英等.对甲砜基苯丝氨酸铜合成的研究.化学工业与工程，2011，28(2):29-34；以及韩玉英等.DL-苏式-对甲砜基苯丝氨酸乙酯的合成及拆分.精细化工，2011，28(6):599-602,619)，但化学法存在反应步骤较长，反应条件苛刻，生产成本高，对环境不友好等特点。
鉴于目前化学法生产L-苏式-对甲砜基苯丝氨酸存在诸多问题，亟待开发出能够克服一种或多种上述缺点的制备L-苏式-对甲砜基苯丝氨酸的方法。
L-赤式-对甲砜基苯丝氨酸是L-苏式-对甲砜基苯丝氨酸的非对映异构体。L-苏式-对甲砜基苯丝氨酸是制备甲砜霉素和及其氟化衍生物氟甲砜霉素(氟苯尼考)的原料L-苏式-对甲砜基苯丝氨酸乙酯的前体物质。虽然L-苏式-对甲砜基苯丝氨酸乙酯直接决定了氟苯尼考的质量和成本，但是，由于L-赤式-对甲砜基苯丝氨酸可以经过化学转化获得L-苏式-对甲砜基苯丝氨酸，如果能够以非常简单且低成本地制备L-赤式-对甲砜基苯丝氨酸，其也将具有十分重要的市场开发价值。
使用L-β-羟基-α-氨基酸合成酶(包括L-苏氨酸醛缩酶，L-苯丝氨酸醛缩酶，丝氨酸羟甲基转移酶等)通过催化甘氨酸和相应的醛类化合物可一步合成L-β-羟基-α-氨基酸，且反应条件较为温和。
在L-β-羟基-α-氨基酸合成酶催化甘氨酸与对甲砜基苯甲醛的反应过程中，可能需要在反应体系加入有机溶剂，这些有机溶剂的存在对酶的稳定性造成了不利的影响。因此，对酶分子进行改造使其稳定性(尤其是热稳定性和有机溶剂耐受性)提高就有重要意义。
发明内容
本发明的第一方面涉及酶催化制备L-苏式-对甲砜基苯丝氨酸的方法，通过该方法能够以温和且环境友好的反应条件制备并通过简单分离就可得到L-苏式-对甲砜基苯丝氨酸。
本发明的第二方面涉及利用第一方面的方法产生的废料制备L-苏式-对甲砜基苯丝氨酸的方法中。换句话说，该制备方法实现了反应体系的重复使用。
本发明的第三方面涉及酶催化制备L-赤式-对甲砜基苯丝氨酸的方法，通过该方法能够以温和且环境友好的反应条件制备并通过简单分离就可得到L-赤式-对甲砜基苯丝氨酸。
本发明的第四方面涉及用于上述第一方面至第三方面的方法的制备系统。
本发明的第五方面涉及可以用于上述第一方面至第三方面的方法的L-β-羟基-α-氨基酸合成酶，包括用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶。
具体实施方式
如上所述，本发明的第一方面涉及酶催化制备L-苏式-对甲砜基苯丝氨酸的方法，并通过简单分离制备L-苏式-对甲砜基苯丝氨酸的方法。所述L-苏式-对甲砜基苯丝氨酸结构式如下：
Figure PCTCN2019102718-appb-000001
具体来说，本发明提供酶催化制备L-苏式-对甲砜基苯丝氨酸的方法，所述方法包括以下步骤：
(a)使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应，
(b)固液分离得到包含L-苏式-对甲砜基苯丝氨酸的液相，
(c)将步骤(b)中得到的液相降温以使L-苏式-对甲砜基苯丝氨酸沉淀析出，和
(d)固液分离得到沉淀析出的L-苏式-对甲砜基苯丝氨酸。
在步骤(a)的反应体系中，甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下进行如下反应：
Figure PCTCN2019102718-appb-000002
上述反应中生成L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸，其中L-赤式-对甲砜基苯丝氨酸不能直接用于制备具有生物活性的产品。
在本发明中，术语“L-β-羟基-α-氨基酸合成酶”泛指能够催化醛基(-CH(＝O))与氨基酸中的氨基(-NH 2)发生缩合反应生成L-β-羟基-α-氨基酸的酶，特别是指能够催化醛基(-CH(＝O))与甘氨酸中的氨基(-NH 2)发生缩合反应生成L-β-羟基-α-氨基酸的酶，尤其是指能够催化甘氨酸和对甲砜基苯甲醛发生缩合反应生成L-苏式/赤式-对甲砜基苯丝氨酸的酶，例如，L-β-羟基-α-氨基酸合成酶包括L-苏氨酸醛缩酶、L-苯丝氨酸醛缩酶、丝氨酸羟甲基转移酶等等。
在本文中，“L-苏式/赤式-对甲砜基苯丝氨酸”是指代L-苏式-对甲砜基苯丝氨酸和/或L-赤式-对甲砜基苯丝氨酸。
在本发明中，应当理解所给出的数值点包括适当的偏差，例如所给出值±10％，优选±5％，更优选±3％，更优选±1％内的偏差。
在本发明中，L-β-羟基-α-氨基酸合成酶在反应中的用量没有特别的要求。用量少，反应进行的慢，所需反应时间长；用量多，反应进行的快，所需反应时间短。可以根据反应需要选择和调节L-β-羟基-α-氨基酸合成酶的用量。
在本发明中，L-β-羟基-α-氨基酸合成酶的活性以其催化分解L-苯丝氨酸生成苯甲醛和甘氨酸的能力进行衡量。在本发明中，L-β-羟基-α-氨基酸合成酶的活性定义为：在约30℃、pH为约8.5和L-苯丝氨酸浓度为约10mmol/L的条件下，每分钟催化L-苯丝氨酸生成1μmol苯甲醛所需的酶量为1个活力单位(U)。
在本发明中，L-β-羟基-α-氨基酸合成酶的活性测定方式为：
(1)底物溶液的配制：称取DL-苯丝氨酸(以其中的L-苯丝氨酸为酶的底物)于去离子水中，超声使其完全溶解，再加入5-磷酸吡哆醛(PLP)，调节pH＝8.5，使得最终溶液中包含40mmol/L DL-苯丝氨酸、40μmol/L PLP。
(2)反应条件：取10μl酶液、190μl去离子水、200μl底物溶液于1.5ml离心管中混匀，于30℃下计时反应，反应10min时立即加入400μl 1.7％磷酸终止液混匀终止反应。取终止后的反应液测量其290nm处的吸光度，与苯甲醛浓度—290nm吸光度标准曲线进行比较，得到反应液中的苯甲醛浓度，进而计算得到L-β-羟基-α-氨基酸合成酶的活力单位。
需要说明的是，在本发明的方法中，甘氨酸与对甲砜基苯甲醛的反应需要使用L-β-羟基-α-氨基酸合成酶进行催化，而不能使用D-β-羟基-α-氨基酸合成酶(例如D-苏氨酸醛缩酶)进行催化，因为D-β-羟基-α-氨基酸合成酶催化甘氨酸与对甲砜基苯甲醛的反应生成上述两个产物的手性对映体D-苏式-对甲砜基苯丝氨酸和D-赤式-对甲砜基苯丝氨酸。由于生产甲砜霉素及其氟化衍生物的活性前体是L-苏式-对甲砜基苯丝氨酸，其D构型对映体不具有生物活性，需要通过复杂的化学法再转化获得L构型的产物。因此，使用D-β-羟基-α-氨基酸合成酶在生产中不能通过简单分离获得L-苏式/赤式-对甲砜基苯丝 氨酸，即，使用D-β-羟基-α-氨基酸合成酶催化甘氨酸与对甲砜基苯甲醛的反应不能实现本发明的目的。
作为本发明的反应底物，甘氨酸易溶于水，而对甲砜基苯甲醛在水中的溶解度非常低，需要加入助溶剂来在提高对甲砜基苯甲醛在水中的溶解度。优选地，对甲砜基苯甲醛和甘氨酸均以饱和状态溶解在反应体系中。优选地，对甲砜基苯甲醛以远大于其溶解度的量加入反应体系中。更优选加入到反应体系中的甘氨酸的摩尔量大于对甲砜基苯甲醛的摩尔量。例如，甘氨酸以0.5-2mol/L，例如0.6-1.5mol/L，0.8-1.2mol/L的浓度加入到反应体系中，对甲砜基苯甲醛以0.1-0.8mol/L，例如0.2-0.6mol/L，0.2-0.5mol/L的浓度加入到反应体系中。
在本发明中，可以使用的助溶剂选自以下中的一种或多种：甲醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、仲丁醇、叔丁醇、1-戊醇、2-戊醇、3-戊醇、2-甲基-1-丁醇、3-甲基-1-丁醇、2-甲基-2-丁醇、3-甲基-2-丁醇、2,2-二甲基丙醇、乙二醇、丙三醇、巯基乙醇、乙酸乙酯、乙酸丁酯、聚乙二醇6000(PEG 6000)、聚乙二醇辛基苯醚(Triton X-100)、乙腈、丙酮、二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)、二甲基乙醇胺(DMAE)、乙二醇二甲醚(DME)、甲基叔丁基醚、四丁基溴化铵、三乙胺、咪唑、吡啶、二甲基四氢呋喃、十二烷基磺酸钠(SDS)、3-吗啉丙磺酸(MOPS)、β-环糊精和亚硫酸氢钠。优选选自DMSO、DMF、乙醇和丙酮。
优选地，助溶剂在水溶液中的体积浓度不超过60％，优选地，不超过55％，不超过50％，不超过45％，不超过40％，不超过35％。优选地，助溶剂的体积浓度为至少5％，至少10％，至少12％，至少15％，至少20％，至少22％，至少25％，至少30％。
可以在步骤(a)中的反应体系中加入5-磷酸吡哆醛(PLP)，5-磷酸吡哆醛作为L-β-羟基-α-氨基酸合成酶的辅酶可以提高酶的活性。但是，5-磷酸吡哆醛在本发明的方法中不是必须的。在加入5-磷酸吡哆醛的情况下，5-磷酸吡哆醛的加入量可以为不超过200μmol/L，例如不超过100μmol/L，不超出80μmol/L，不超过60μmol/L，不超过50μmol/L。
步骤(a)的反应可以在宽范围的温度条件下进行，例如，4-50℃的温度范围内，10-30℃的温度范围内，15-28℃的温度范围内。一般而言，温度越高，反应速度越快，但是，有可能造成L-赤式-对甲砜基苯丝氨酸生成过多；温度越低，反应速度越慢，但是，可能有利于L-苏式-对甲砜基苯丝氨酸的形成。因此，根据酶的催化反应特点以及所需的最终所需产物选择所需反应温度，但是通常在4-50℃温度范围内。
步骤(a)的反应体系的pH可以在5-10的范围内，例如，6-9的范围内，6-8的范围内，6-7的范围内。可以在反应体系中使用缓冲液，也可以不使用缓冲液。从简化工艺的角度来说，优选不使用缓冲液。
步骤(a)的反应优选在搅拌下进行，搅拌可以连续进行，也可以间断进行。搅拌速度没有特别的限制，只要使得反应体系处于大致的混合均匀状态即可。在不同的反应容器中，可以根据需要调节适合的搅拌速度。
优选地，进行步骤(a)的反应直到反应体系的反应达到平衡状态。但是，也不是必须达到反应平衡状态，可以在达到平衡状态前终止反应。根据使用的酶的量以及反应温度等反应条件，反应时间可以在宽的范围内选择，例如，反应可以进行2-75个小时，例如5-70个小时，10-68个小时，10-45个小时，10-40个小时。
在酶催化制备L-苏式-对甲砜基苯丝氨酸的方法中，重要的是将L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸进行分离。二者作为非对映体结构，根据常规的分离方法难以进行分离。提高酶的选择性是一种方法，也就是说，使得L-β-羟基-α-氨基酸合成酶仅选择性催化合成L-苏式-对甲砜基苯丝氨酸而不催化合成或者尽可能少地催化合成L-赤式-对甲砜基苯丝氨酸。但是，这种方法尚处于探索阶段。另外，即使D-β-羟基-α-氨基酸合成酶(例如D-苏氨酸醛缩酶)能够实现高的选择性，但是，如前所述，D构型非对映体的衍生物不具有生物活性，需要通过复杂的化学法再转化获得L构型的产物，因此，这依然不能实现条件温和且环境友好且简单的目标。换句话说，高选择性的D-β-羟基-α-氨基酸合成酶依然不能实现本发明的目标。
在本发明方法的步骤(a)中，出人意料的发现，在本发明中描述的条件下，随着反应的进行，L-赤式-对甲砜基苯丝氨酸能够从反应体系中沉淀出来，而L-苏式-对甲砜基苯丝氨酸却可以保持溶解状态。为了加速L-赤式-对甲砜基苯丝氨酸这一沉淀进程，优选加入L-赤式-对甲砜基苯丝氨酸晶种。但是，从降低生产成本的角度，也可以不加入L-赤式-对甲砜基苯丝氨酸晶种。这一发现使得对酶的选择性没有高的要求，只要能够催化甘氨酸和对甲砜基苯甲醛进行上述反应的L-β-羟基-α-氨基酸合成酶都可以用于本发明的方法中。但是，仍然优选将高选择性的L-β-羟基-α-氨基酸合成酶用于本发明的方法中。
如果在反应中加入L-赤式-对甲砜基苯丝氨酸晶种，其加入时间点没有特别的限制，可以随反应物一同加入到反应体系，也可以在反应开始之后反应结束之前加入，例如在反应过程中0-72小时内的任意时间内，例如，在反应开始后0小时，1小时，2小时，3小时，4小时，6小时，8小时，10小时，12小时，14小时，16小时，18小时，20小时，22小时，24小时，26小时，28小时，30小时，33小时，35小时，38小时，40小时，42小时，45小时，48小时，50小时，52小时，55小时，58小时，60小时，62小时，65小时，68小时，70小时或72小时。
在步骤(a)中，随着反应的不断进行，L-赤式-对甲砜基苯丝氨酸不断沉淀，而L-苏式-对甲砜基苯丝氨酸在液相中不断的积累。在达到平衡状态时，液相中的L-赤式-对甲砜基苯丝氨酸含量很少，大部分的L-赤式-对甲砜基苯丝氨酸沉淀出。优选地，在反应达到平衡状态后，将反应体系固液分离得到包含L-苏式-对甲砜基苯丝氨酸的液相(步骤(b))。所述固液分离操作可以采用常规的固液分离手段，例如过滤、离心或抽滤等。但是，可以理解的是，不是必须等到反应达到平衡状态才可以进行上述固液分离操作，也可以在达到平衡状态前进行上述固液分离操作。
在步骤(c)中，将包含L-苏式-对甲砜基苯丝氨酸的液相降温至10℃以下，例如，9℃ 以下、8℃以下、7℃以下、6℃以下、5℃以下、4℃以下、3℃以下、2℃以下、1℃以下，0℃以下；但是，考虑到实际操作条件限制，一般降温到-5℃以上，例如-4℃以上，-3℃以上、-2℃以上、-1℃以上。L-苏式-对甲砜基苯丝氨酸从液相中沉淀析出。理论上初步认为，该沉淀析出可能包括了结晶过程和普通析出过程的混合。为了加快这一沉淀析出进程，优选加入L-苏式-对甲砜基苯丝氨酸晶种，但是从降低生产成本的角度，也可以不加入L-苏式-对甲砜基苯丝氨酸晶种。
步骤(c)中的沉淀析出所用时间没有具体的限制，只要液相中的至少一部分(优选大部分)L-苏式-对甲砜基苯丝氨酸沉淀析出出来即可。可以根据降温速度、降温温度和所用降温设备等条件进行综合确定。一般情况下，降温沉淀析出所用时间在0.5-36小时范围内，例如1-25小时范围内，1-15小时范围内，1-5小时范围内。
在沉淀析出完成后，在步骤(d)中，通过固液分离得到沉淀析出的L-苏式-对甲砜基苯丝氨酸。所述固液分离操作可以采用常规的固液分离手段，例如过滤、离心或抽滤等。
本发明的第二方面涉及利用前述的方法产生的废料制备L-苏式-对甲砜基苯丝氨酸的方法。具体来说，涉及酶催化制备L-苏式-对甲砜基苯丝氨酸的方法，该方法除了包括前述第一方面的方法的步骤(a)至(d)之外，还包括步骤(e)：将步骤(d)产生的液相用于步骤(a)的反应。也就是说，将分离L-苏式-对甲砜基苯丝氨酸之后的液相重新用于步骤(a)的反应。在该分离后的液相中加入对甲砜基苯甲醛和甘氨酸，不加或仅少量补加L-β-羟基-α-氨基酸合成酶就可以直接重新用于反应，这可以大大降低生产成本，实现反应体系的重复套用。
本发明的第三方面涉及酶催化制备L-赤式-对甲砜基苯丝氨酸的方法，其中，L-赤式-对甲砜基苯丝氨酸具有下述结构：
Figure PCTCN2019102718-appb-000003
该方法包括以下步骤：
(a)使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应，
(b)固液分离得到包含L-赤式-对甲砜基苯丝氨酸的固相。
在制备L-赤式-对甲砜基苯丝氨酸的方法中，步骤(a)的条件与前述制备L-苏式-对甲砜基苯丝氨酸的方法基本上相同，前述的步骤(a)的条件可以用于L-赤式-对甲砜基苯丝氨酸的制备。
在酶催化制备L-赤式-对甲砜基苯丝氨酸的方法中，重要的是将L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸进行分离。二者作为非对映体结构，根据常规的分 离方法难以进行分离。提高酶的选择性是一种方法，也就是说，使得L-β-羟基-α-氨基酸合成酶仅选择性催化合成L-赤式-对甲砜基苯丝氨酸。但是，这种方法尚处于探索阶段。另外，即使D-β-羟基-α-氨基酸合成酶(例如D-苏氨酸醛缩酶)能够实现高的选择性，但是，如前所述，D构型对映体不具有生物活性，需要通过复杂的化学法再转化获得L构型的产物，因此，这依然不能实现条件温和且环境友好且简单的目标。换句话说，高选择性的D-β-羟基-α-氨基酸合成酶依然不能实现本发明的目标。
在本发明制备L-赤式-对甲砜基苯丝氨酸的方法的步骤(a)中，出人意料的发现，在本发明中描述的条件下，随着反应的进行，L-赤式-对甲砜基苯丝氨酸能够从反应体系中沉淀出来。为了加速这一沉淀进程，优选加入L-赤式-对甲砜基苯丝氨酸晶种。但是，从降低生产成本的角度，也可以不加入L-赤式-对甲砜基苯丝氨酸晶种。这一发现使得对酶的选择性没有高的要求，只要能够催化甘氨酸和对甲砜基苯甲醛进行上述反应的L-β-羟基-α-氨基酸合成酶都可以用于本发明的方法中。但是，仍然优选将高选择性的L-β-羟基-α-氨基酸合成酶用于本发明的方法中。
如果在反应中加入L-赤式-对甲砜基苯丝氨酸晶种，其加入时间点没有特别的限制，可以随反应物一同加入到反应体系，也可以在反应开始之后反应结束之前加入，例如在反应过程中0-72小时内的任意时间内，例如，在反应开始后0小时，1小时，2小时，3小时，4小时，6小时，8小时，10小时，12小时，14小时，16小时，18小时，20小时，22小时，24小时，26小时，28小时，30小时，33小时，35小时，38小时，40小时，42小时，45小时，48小时，50小时，52小时，55小时，58小时，60小时，62小时，65小时，68小时，70小时或72小时。
在步骤(a)中，随着反应的不断进行，L-赤式-对甲砜基苯丝氨酸不断沉淀，而随着L-赤式-对甲砜基苯丝氨酸的沉淀，上述反应的平衡不断被打破，使得反应不断朝着生成L-赤式-对甲砜基苯丝氨酸的方向进行。另外，由于酶催化甘氨酸和对甲砜基苯甲醛生成L-赤式-对甲砜基苯丝氨酸和L-苏式-对甲砜基苯丝氨酸的反应是可逆反应，未沉淀的L-苏式-对甲砜基苯丝氨酸会被催化重新分解为甘氨酸和对甲砜基苯甲醛。因此，理论上，加入到反应体系中的所有反应物可能全部被转变为L-赤式-对甲砜基苯丝氨酸。但是，可以理解的是，在实际操作过程中，不需要等到所有的反应物都转变为L-赤式-对甲砜基苯丝氨酸。
优选地，根据使用的酶的量以及反应温度等反应条件，步骤(a)的反应时间可以在宽的范围内选择，例如，反应可以进行2-150个小时，例如5-80个小时，10-75个小时。
在停止反应后，将反应体系固液分离得到包含L-赤式-对甲砜基苯丝氨酸的固相(步骤(b))。所述固液分离操作可以采用常规的固液分离手段，例如过滤、离心或抽滤等。但是，可以理解的是，可以在L-赤式-对甲砜基苯丝氨酸发生沉淀后的任何时间点进行分离操作。
任选地，在分离获得包含L-赤式-对甲砜基苯丝氨酸的固相后，对该固相进行洗涤， 例如，使用水和/或有机溶剂进行洗涤，所述有机溶剂可以为丙酮、乙腈、DMSO、DMF等；优选去离子水和丙酮。洗涤可以进行多次，例如2-5次。
通过本发明的方法不仅可以容易地且环境友好地制备L-赤式-对甲砜基苯丝氨酸，而且可以通过简单分离获得高纯度的L-赤式-对甲砜基苯丝氨酸。
本发明的第四方面涉及用于本发明的方法的制备系统，在制备L-苏式-对甲砜基苯丝氨酸的方法中，该制备系统包括：1)反应装置，该反应装置用于使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应；2)固液分离装置；3)降温装置。或者，该制备系统由上述装置1)至3)组成。在制备L-赤式-对甲砜基苯丝氨酸的方法中，该制备系统仅包括：1)反应装置，该反应装置用于使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应；2)固液分离装置。或者，该制备系统由上述装置1)和2)组成。
所述反应装置没有特别的限制，可以是用于生物催化反应的常用反应器，例如，釜式反应器，机械搅拌式反应器，鼓泡式反应器等等，只要在该反应器中能够进行甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中的反应。
所述固液分离装置也没有特别的限制，可以是固液分离常用的分离装置，例如过滤器、抽滤器、离心机等等，只要能够实现本发明的方法中的固液分离即可。
所述降温装置也没有特别的限制，只要能实现本发明的方法中液相的降温即可，例如夹套水循环降温装置，冷却换热器等等。
本发明的系统的特别之处是将这几种装置的组合使用。也就是说，对应于本发明的方法的各个步骤的组合，相应地组合使用上述各种装置。而在现有技术中，没有发现将这几种装置进行组合用于L-苏式/赤式-对甲砜基苯丝氨酸的制备。
本发明的第五方面涉及可以用于本发明的方法的L-β-羟基-α-氨基酸合成酶，包括用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶。如上所述，能够催化上述反应的L-β-羟基-α-氨基酸合成酶都可以用于本发明的方法，优选地，在本发明的方法中使用具有SEQ ID No.1或SEQ ID No.2所示的氨基酸序列的酶。
在本文中，将具有SEQ ID No.1氨基酸序列的酶称为酶24-1(该酶为L-苯基丝氨酸醛缩酶，属于L-β-羟基-α-氨基酸合成酶)，将具有SEQ ID No.2氨基酸序列的酶称为KT2440(该酶为L-苏氨酸醛缩酶，属于L-β-羟基-α-氨基酸合成酶)。本领域技术人员可以理解，在保持L-β-羟基-α-氨基酸合成酶的酶活性的前提下，可以对SEQ ID No.1或SEQ ID No.2所示的氨基酸序列进行少量氨基酸残基(例如1-10个或1-5个或1-3个氨基酸残基)的替换、增加或缺失。
可选地，在本发明的方法中也可以使用SpyTag-SpyCatcher环化的酶24-1(该酶称为酶SR-24-1)或SpyTag-SpyCatcher环化的酶KT2440(该酶称为酶SR-KT2440)。SpyTag-SpyCatcher环化酶24-1是指用SpyTag-SpyCatcher技术将酶24-1进行环化获得的 酶，SpyTag-SpyCatcher环化的酶KT2440是指用SpyTag-SpyCatcher技术将酶2440进行环化获得的酶。
酶24-1可以通过下述方法得到：将能够翻译出SEQ ID No.1氨基酸序列的DNA序列重组到pET-28a质粒上，得到重组质粒pET28a-24-1，将所述重组质粒pET28a-24-1转入大肠杆菌E.coli BL21(DE3)中获得重组菌株BL21(DE3)/pET28a-24-1，培养所述重组菌株使其表达酶24-1。
优选地，培养重组菌株BL21(DE3)/pET28a-24-1的方法可以如下：将所述重组菌株BL21(DE3)/pET28a-24-1的单菌落接种到LB培养基中，在35-40℃下振荡培养5-18h，取培养振荡后的培养液以0.5-5％的接种量转接于乳糖培养基中，在20-37℃下振荡培养12-40h，振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其用高压匀浆机匀浆破碎，将破碎液离心，收集上清液，即得所述酶24-1。
酶KT2440可以通过下述方法得到：将能够翻译出SEQ ID No.2氨基酸序列的DNA序列重组到pET28a质粒上，得到重组质粒pET28a-KT2440，将所述重组质粒pET28a-KT2440转入大肠杆菌E.coli BL21(DE3)中获得重组菌株BL21(DE3)/pET28a-KT2440，培养所述重组菌株使其表达酶KT2440。
优选地，培养重组菌株BL21(DE3)/pET28a-KT2440的方法可以如下：将所述重组菌株BL21(DE3)/pET28a-KT2440的单菌落接种到LB培养基中，在35-40℃下振荡培养5-18h，取培养振荡后的培养液以0.5-5％的接种量转接于乳糖培养基中，在20-37℃下振荡培养12-40h，振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其用高压匀浆机匀浆破碎，将破碎液离心，收集上清液，即得所述酶KT2440。
为了构建环化的酶24-1或环化的酶KT2440，本发明利用全质粒MegaWHOP的方法(Miyazaki,K.,&Takenouchi,M.(2002).Creating random mutagenesis libraries using megaprimer PCR of whole plasmid.Biotechniques,33(5),1033-4.)，通过构建合成搭接引物，再将合成的引物和目的L-β-羟基-α-氨基酸合成酶基因进行混合PCR获得含有SpyTag和SpyCatcher接头的目的基因片段，然后再将这一片段和SpyTag-β-Lactamase-SpyCatcher质粒(从Addgene购得，质粒编号52656)混合，进行全质粒MegaWHOP PCR以获得含有SpyTag-24-1-SpyCatcher或SpyTag-2440-SpyCatcher的SpyTag/SpyCatcher环化酶的质粒DNA。接着，将MegaWHOP PCR产物用DpnI进行消化然后将其转化入BL21(DE3)中，液体LB培养后涂抗性平板进行筛选，经过核酸测序确定SpyTag/SpyCatcher环化的酶SR-24-1或SpyTag/SpyCatcher环化的酶SR-KT2440是否构建成功。
以下通过实施例示例性地说明本发明，但是可以理解的是，本发明的范围不限于这些实施例。
实施例
酶制备例1
具有SEQ No.1的氨基酸序列的酶24-1的制备
(1)合成pET28a-24-1质粒，将能够翻译出SEQ ID No.1氨基酸序列的DNA序列SEQ ID No.3基因进行合成，并插入到pET28a质粒的BamHI-HindIII之间，即得重组质粒pET28a-24-1，将所述重组质粒pET28a-24-1转化至大肠杆菌E.coli BL21(DE3)中，即得所述重组菌株BL21(DE3)/pET28a-24-1。
(2)培养所述重组菌株BL21(DE3)/pET28a-24-1使其表达所述酶24-1，具体包括以下步骤：将所述重组菌株BL21(DE3)/pET28a-24-1的单菌落接种到LB培养基中，在37℃下振荡培养12h，取振荡培养后的培养基以2.5％的接种量转接于乳糖培养基(蛋白胨10g/L，酵母粉5g/L，Na 2HPO 4·12H 2O 8.95g/L，KH 2PO 43.4g/L，NH 4Cl 2.67g/L，Na 2SO 40.7g/L，MgSO 40.24g/L，甘油5g/L，葡萄糖0.5g/L，乳糖2g/L)中，在28℃下振荡培养24h；振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其置于高压匀浆机中匀浆破碎，将破碎液离心，收集上清液，即得所述酶24-1。通过前述活性测定方法，测得酶液的酶活为33U/mL。
酶制备例2
具有SEQ No.2的氨基酸序列的酶KT2440的制备
(1)合成pET28a-KT2440质粒，将能够翻译出SEQ ID No.2氨基酸序列的DNA序列SEQ ID No.4基因进行合成，并插入pET28a质粒的BamHI-HindIII之间，即得重组质粒pET28a-KT2440，将所述重组质粒pET28a-KT2440转化至大肠杆菌E.coli BL21(DE3)中，即得所述重组菌株BL21(DE3)/pET28a-KT2440。
(2)培养所述重组菌株BL21(DE3)/pET28a-KT2440使其表达所述酶KT2440，具体包括以下步骤：将所述重组菌株BL21(DE3)/pET28a-KT2440的单菌落接种到LB培养基中，在37℃下振荡培养12h，取振荡培养后的培养基以2.5％的接种量转接于乳糖培养基(成分与酶制备例1中相同)中，在28℃下振荡培养24h；振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其置于高压匀浆机中匀浆破碎，将破碎液离心，收集上清液，即得所述酶KT2440。通过前述活性测定方法，测得酶液的酶活为13.6U/mL。
酶制备例3
SpyTag-SpyCatcher环化的酶SR-24-1的制备
本实施例的酶SR-24-1具有SEQ ID No.5所述的氨基酸序列结构，所述酶SR-24-1通过以下方法得到：
(1)设计、合成具有SEQ ID No.6所述DNA序列结构的上游引物和具有SEQ ID No.7所述DNA序列结构的下游引物，用所述两引物以酶制备例1中所述pET28a-24-1质粒为 模板进行PCR扩增，所得到的产物为MegaWHOP扩增所需的长引物，用所述长引物以SpyTag-β-Lactamase-SpyCatcher质粒(Addgene编号#52656)为模板进行MegaWHOP扩增，经过扩增后SpyTag-β-Lactamase-SpyCatcher质粒中的β-内酰胺酶(β-Lactamase)基因被替换为具有SEQ ID No.3所述DNA序列结构的基因，得到能够翻译出SEQ ID No.5所述氨基酸序列结构的具有SEQ ID No.8所述DNA序列结构的基因，扩增所得到的产物经DpnI消化后转化至大肠杆菌E.coli BL21(DE3)中，即得所述重组菌株BL21(DE3)/pET28a-SR-24-1。
(2)培养所述重组菌株BL21(DE3)/pET28a-SR-24-1使其表达所述酶SR-24-1，具体包括以下步骤：将所述重组菌株BL21(DE3)/pET28a-SR-24-1的单菌落接种到LB培养基中，在37℃下振荡培养12h，取振荡培养后的培养基以2.5％的接种量转接于乳糖培养基(成分与酶制备例1中相同)中，在28℃下振荡培养24h；振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其置于高压匀浆机中匀浆破碎，将破碎液离心，收集上清液，即得所述酶SR-24-1。通过前述活性测定方法，测得酶液的酶活为13U/mL。
酶制备例4
SpyTag-SpyCatcher环化的酶SR-KT2440的制备
本实施例的环化酶SR-KT2440具有SEQ ID No.9所述的氨基酸序列结构，所述酶SR-KT2440通过以下方法得到：
(1)设计、合成具有SEQ ID No.10所述DNA序列结构的上游引物和具有SEQ ID No.11所述DNA序列结构的下游引物，用该两引物以酶制备例2中pET28a-KT2440质粒为模板进行PCR扩增，所得到的产物为MegaWHOP扩增所需的长引物，用所述长引物以SpyTag-β-Lactamase-SpyCatcher质粒(Addgene编号#52656)为模板进行MegaWHOP扩增，经过扩增后SpyTag-β-Lactamase-SpyCatcher质粒中的β-内酰胺酶(β-Lactamase)基因被替换为具有SEQ ID No.12所述DNA序列结构的基因，得到能够翻译出SEQ ID No.9所述氨基酸序列结构的具有SEQ ID No.12所述DNA序列结构的基因，扩增所得到的产物经DpnI消化后转化至大肠杆菌E.coli BL21(DE3)中，即得所述重组菌株BL21(DE3)/pET28a-SR-KT2440。
(2)培养所述重组菌株BL21(DE3)/pET28a-SR-KT2440使其表达所述环化酶SR-KT2440，具体包括以下步骤：将所述重组菌株BL21(DE3)/pET28a-SR-KT2440的单菌落接种到LB培养基中，在37℃下振荡培养12h，取振荡培养后的培养基以2.5％的接种量转接于乳糖培养基(成分与前述乳糖培养基相同)中，在28℃下振荡培养24h；振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其置于高压匀浆机中匀浆破碎，将破碎液离心，收集上清液，即得所述环化酶SR-KT2440。酶液的酶活为10.5U/mL。
对比酶制备例1
环化的头孢菌素C酰化酶SR-CCA的制备
本对比例的环化头孢菌素C酰化酶SR-CCA具有SEQ ID No.13所述的氨基酸序列结构，所述环化头孢菌素C酰化酶SR-CCA通过以下方法得到：
(1)pET28a-CCA质粒，按文献(Wang,Y.,Yu,H.,Song,W.,An,M.,Zhang,J.,Luo,H.,&Shen,Z.(2012).Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel.Journal of bioscience and bioengineering,113(1),36-41.)制备，将能够翻译出具有SEQ ID No.14氨基酸序列的头孢菌素C酰化酶CCA的DNA序列SEQ ID No.15插入pET-28a质粒BamHI-HindIII之间，即得重组质粒pET28a-CCA。
(2)设计、合成具有SEQ ID No.16所述DNA序列结构的上游引物和具有SEQ ID No.17所述DNA序列结构的下游引物，用所述两引物以(1)中所述pET28a-CCA质粒为模板进行PCR扩增，所得到的产物为MegaWHOP扩增所需的长引物，用所述长引物以SpyTag-β-Lactamase-SpyCatcher质粒(Addgene编号#52656)为模板进行MegaWHOP扩增，经过扩增后SpyTag-β-Lactamase-SpyCatcher质粒中的β-内酰胺酶(β-Lactamase)基因被替换为具有SEQ ID No.15所述DNA序列结构的基因，得到能够翻译出SEQ ID No.13所述氨基酸序列结构的具有SEQ ID No.18所述DNA序列结构的基因，扩增所得到的产物经DpnI消化后转化至大肠杆菌E.coli BL21(DE3)中，即得所述重组菌株BL21(DE3)/pET28a-SR-CCA。
(3)培养所述重组菌株BL21(DE3)/pET28a-SR-CCA使其表达所述环化头孢菌素C酰化酶SR-CCA，具体包括以下步骤：将所述重组菌株BL21(DE3)/pET28a-SR-CCA的单菌落接种到LB培养基中，在37℃下振荡培养12h，取振荡培养后的培养基以2.5％的接种量转接于乳糖培养基(成分与前述乳糖培养基相同)中，在28℃下振荡培养24h；振荡培养结束后离心收集细胞，向收集的细胞中加入去离子水重悬细胞，将其置于高压匀浆机中匀浆破碎，将破碎液离心，收集上清液，即得所述环化头孢菌素C酰化酶SR-CCA。酶液的酶活为5.66U/mL。
(4)采用与(3)同样的方法培养所述重组菌株BL21(DE3)/pET28a-CCA使其表达头孢菌素C酰化酶，得到头孢菌素C酰化酶CCA。酶液的酶活为6.96U/mL。
实施例1：酶24-1在30％DMF中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入22.5ml DMF(占总体积的约30体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，15℃摇床200rpm振荡反应，反应61h后抽滤，上清液降温到4℃，搅拌1.5h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜 基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为90.6:9.4。
实施例2：酶KT2440在20％DMSO中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水35ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例2中制备得到的酶KT2440酶液，混匀后加入15ml DMSO(占总体积的约20体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入2.77g对甲砜基苯甲醛，37℃摇床200rpm振荡反应，反应36h后抽滤，将上清液降温到-4℃，搅拌1.5h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中的L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为65.6:34.4。
实施例3：酶24-1在30％DMF中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水25ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入22.5ml DMF(占总体积的约30体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应38h加入10mg L-赤式-对甲砜基苯丝氨酸晶种，反应62h后抽滤，降温到4℃，搅拌1.5h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为88.9:11.1。
实施例4：酶24-1在30％乙醇中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入22.5ml乙醇(占总体积的约30体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应62h后抽滤，降温到4℃，搅拌2.5h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为93.4:6.6。
实施例5：酶24-1在40％DMSO中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水20ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入30ml DMSO(占总体积的约40体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应19.5h时加入10mg L-赤式-对甲砜基苯丝氨酸晶种，反应62h后抽滤，降温到4℃，搅拌1h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为82.1：17.9。
实施例6：酶24-1在40％乙醇中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水20ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入30ml乙醇(占总体积的约40体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应19.5h时加入10mg L-赤式-对甲砜基苯丝氨酸晶种，反应62h后抽滤，降温到4℃，搅拌0.33h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为90.4:9.6。
实施例7：环化酶SR-24-1在30％乙醇中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例3中制备得到的环化酶SR-24-1酶液，混匀后加入22.5ml乙醇(占总体积的约30体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应39h后抽滤，降温到4℃，搅拌1h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为83.5:16.5。
实施例8：酶24-1在30％DMSO中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入22.5ml DMSO(占总体积的30体积％)，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应72h后抽滤，上清液降温到4℃，搅拌1.5h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为92.6:7.4。
实施例9：酶催化体系的重复套用
在酶催化体系中，对甲砜基苯甲醛与甘氨酸生成L-苏式-对甲砜基苯丝氨酸和L-赤式-对甲砜基苯丝氨酸，在降温沉淀并过滤分离产物L-苏式-对甲砜基苯丝氨酸之后，在抽滤后的滤液中加入甘氨酸和对甲砜基苯甲醛，重复上述催化反应过程以及分离过程。在酶未失活之前，该滤液的套用操作可以进行多次。
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例1中制备得到的酶24-1酶液，混匀后加入22.5ml乙醇，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，15℃摇床200rpm振荡反应，反应61h后抽滤，将上清液降温到4℃，搅拌0.58h，抽滤获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成 分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中的L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为91.7:8.3。
将抽滤除去L-苏式-对甲砜基苯丝氨酸的滤液进行第一次套用，向该滤液中加入1.69g甘氨酸和4.14g对甲砜基苯甲醛，重复上述催化反应过程(反应36h)以及分离过程。可以得到含L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为88.7:11.3的滤饼。
将抽滤除去L-苏式-对甲砜基苯丝氨酸的滤液进行第二次套用，向该滤液中加入0.56g甘氨酸及第一次滤液套用时催化后的滤饼(含有未反应完的对甲砜基苯甲醛)，重复上述催化反应过程(反应36h)以及分离过程。可以得到含L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为94.3:5.7的滤饼。
实施例10：酶KT2440在30％DMSO中催化200mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63甘氨酸，加入25ml的上述酶制备例2的KT2440酶酶液，及0.994mg的辅酶因子PLP，再添加22.5ml(占总体积的约30体积％)的有机溶剂DMSO，加入2.76g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中振荡反应72h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为98.5％，得率65％。
对比例1：酶KT2440无助溶剂催化200mM对甲砜基苯甲醛
(1)在50ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例2的酶KT2440酶液，及0.994mg的辅酶因子PLP，加入2.76g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中振荡反应36h，反应结束后取样进行高效液相色谱分析，没有发现L-赤式-对甲砜基苯丝氨酸沉淀。
实施例11：酶KT2440在30％DMSO中催化400mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例2的酶KT2440酶液，及0.994mg的辅酶因子PLP，再添加22.5ml(占总体积的约30体积％)有机溶剂DMSO，加入5.53g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于15℃的200rpm的摇床中振荡反应72h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为94.9％，得率68.5％。
实施例12：酶KT2440在15％DMSO中催化400mM对甲砜基苯甲醛
(1)在38.75ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例2的酶KT2440酶液，及0.994mg的辅酶因子PLP，再添加11.25ml(占总体积的约15体积％)有机溶剂DMSO，加入5.53g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于15℃和200rpm的摇床中振荡反应72h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为91.2％，得率56％。
实施例13：酶24-1在30％DMSO中催化400mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例1的酶24-1酶液，及0.994mg的辅酶因子PLP，再添加22.5ml(占总体积的约30体积％)有机溶剂DMSO，加入5.53g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于15℃和200rpm的摇床中振荡反应72h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为85.7％，得率56％。
实施例14：酶KT2440在30％DMSO中催化300mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例2的酶KT2440酶液，再添加22.5ml(占总体积的约30体积％)有机溶剂DMSO，加入5.53g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中震荡反应72h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为94.5％，得率58.7％
对比例2：酶24-1无助溶剂催化200mM对甲砜基苯甲醛
(1)在50ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例1的酶24-1酶液，及0.994mg的辅酶因子PLP，加入2.76g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中振荡反应72h，反应结束后取样进行高效液相色谱分析，没有发现L-赤式-对甲砜基苯丝氨酸沉淀。
实施例15：酶24-1在30％DMF中催化400mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例1的酶24-1酶液，及0.994mg的辅酶因子PLP，再添加22.5ml(占总体积的约30体积％)有机溶剂DMF，加入5.53g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中振荡反应108h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，所得沉淀中L-赤式-对甲砜基苯丝氨酸纯度为92.5％，得率为44.3％。
实施例16：酶KT2440在30％DMSO中催化100mM对甲砜基苯甲醛
(1)在27.5ml去离子水中，先超声溶解底物5.63g甘氨酸，加入25ml的上述酶制备例2的酶KT2440酶液，及0.994mg的辅酶因子PLP，再添加22.5ml(占总体积的约30体积％)有机溶剂DMSO，加入1.38g对甲砜基苯甲醛，溶液pH值为约6.5。
(2)将整个催化反应体系，置于28℃和200rpm的摇床中振荡反应36h，反应结束后进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，沉淀中L-赤式-对甲砜基苯丝氨酸含量为96.2％，得率为56.5％。
实施例17：环化酶SR-24-1在DMSO体系中进行催化
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例3中制备得到的环化酶SR-24-1酶液，混匀后加入22.5ml DMSO(占总体积的约30体积％)混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应144h后抽滤获得滤饼，用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，沉淀中L-赤式-对甲砜基苯丝氨酸含量为95.7％，得率为53.5％。
实施例18：酶24-1和环化的酶SR-24-1的热稳定性评价
将酶制备例1和酶制备例3的酶液分别放置到60℃温度下处理60min，振荡摇晃均匀后取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。酶制备例1中的酶24-1的残留酶活比例为42.7％，酶制备例3的环化酶SR-24-1的残留酶活比例为91.8％。
实施例19：酶KT2440和环化酶SR-KT2440的热稳定性评价
将酶制备例2和酶制备例4的酶液分别放置到50℃温度下处理60min，振荡摇晃均匀后取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。酶制备例2中的酶KT2440的残留酶活比例为18.4％，酶制备例4的环化酶SR-KT2440的残留酶活比例为32.4％。
实施例20：酶24-1和环化酶SR-24-1的变性剂耐受能力的评价
将酶制备例1和酶制备例3的酶液加入到4M的盐酸胍变性剂中，室温孵育30min后，取出测定酶活。以未经任何处理的酶液(溶液中变性剂浓度为0M)酶活为100％，计算得到变性剂浓度下残余酶活百分比。酶制备例1中的酶24-1的残留酶活比例为3.6％，酶制备例3的环化酶SR-24-1的残留酶活比例为38.6％。
实施例21：酶24-1和环化酶SR-24-1的DMSO耐受能力的评价
在酶制备例1和酶制备例3的酶液中，加入终浓度为30％浓度的DMSO，常温处理一段时间后，取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。酶制备例1中的酶24-1的残留酶活比例为68.2％，酶制备例3的环酶SR-24-1的残留酶活比例为100.7％。
实施例22：酶24-1和环化酶SR-24-1的乙醇耐受能力的评价
在酶制备例1和酶制备例3的酶液中，加入终浓度为30％浓度的乙醇，常温处理36h后，取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。酶制备例1的酶24-1的残留酶活比例为55.9％，酶制备例3的环化酶SR-24-1的残留酶活比例为96.8％。
实施例23:酶24-1和环化酶SR-24-1的甲醇耐受能力的评价
在酶制备例1和酶制备例3的酶液中，加入终浓度为50％浓度的甲醇，常温处理一段时间后，取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。酶制备例1中的酶24-1的残留酶活比例为14.1％，酶制备例3的环化酶SR-24-1的残留酶活比例为74.8％。
实施例24:环化酶SR-24-1在30％乙醇中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例3中制备得到的环化酶SR-24-1酶液，混匀后加入22.5ml乙醇(占总体积的约30体积％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应39h后抽滤获得包含L-赤式-对甲砜基苯丝氨酸的固相和包含L-苏式-对甲 砜基苯丝氨酸的液相，经核磁和高压液相色谱法确定，固相主要成分为L-赤式-对甲砜基苯丝氨酸，将液相降温到4℃，搅拌1h，再抽滤，获得滤饼，经核磁和高压液相色谱法确定，滤饼主要成分为L-苏式-对甲砜基苯丝氨酸，其中滤饼中L-苏式-对甲砜基苯丝氨酸与L-赤式-对甲砜基苯丝氨酸的比例为83.5:16.5。
实施例25:酶SR-2440在DMSO中催化制备L-苏式-对甲砜基苯丝氨酸
取去离子水27.5ml于250ml锥形瓶中，加入5.63g甘氨酸，混匀后加入25ml酶制备例3中制备得到的酶SR-24-1酶液，混匀后加入22.5ml DMSO(占总体积的30％)，混匀后加入0.994mg 5-磷酸吡哆醛，混匀后加入5.53g对甲砜基苯甲醛，28℃摇床200rpm振荡反应，反应72h后，进行抽滤获得滤饼。用去离子水清洗收集的滤饼，再用丙酮清洗3次滤饼，洗去未反应完全的底物对甲砜基苯甲醛和甘氨酸，最后将滤饼置于真空干燥箱中烘干，所得即为L-赤式-对甲砜基苯丝氨酸，经高效液相色谱分析，纯度为94.5％，得率62％。
对比例3:头孢菌素C酰化酶SR-CCA的热稳定性评价
将对比酶制备例1的头孢菌素C酰化酶CCA和环化的头孢菌素C酰化酶SR-CCA酶液分别放置到55℃温度下处理40min，振荡摇晃均匀后取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。头孢菌素C酰化酶的残留酶活比例为11.7％，环化的头孢菌素C酰化酶SR-CCA的残留酶活比例为9.9％。
对比例4:头孢菌素C酰化酶SR-CCA的DMSO耐受能力评价
在对比酶制备例1的头孢菌素C酰化酶CCA和环化的头孢菌素C酰化酶SR-CCA酶液中，加入终浓度为30％浓度的DMSO，常温处理40h后，取一定量酶液反应测定酶活，并将该酶活数据与不进行任何处理的初始酶活进行对比，计算残余酶活百分比。头孢菌素C酰化酶的残留酶活比例为80.3％，环化的头孢菌素C酰化酶的残留酶活比例为70.8％。
最后所应说明的是：以上实施例仅用以说明而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应该理解：依然可以对本发明进行修改或者等同替换；而不脱离本发明的精神和范围的任何修改或局部替换，均应涵盖在本发明的范围内。

Claims (18)

  1. 酶催化制备L-苏式-对甲砜基苯丝氨酸的方法，包括以下步骤：
    (a)使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应，
    (b)固液分离得到包含L-苏式-对甲砜基苯丝氨酸的液相，
    (c)将步骤(b)中得到的液相降温以使L-苏式-对甲砜基苯丝氨酸沉淀析出，和
    (d)固液分离得到沉淀析出的L-苏式-对甲砜基苯丝氨酸。
  2. 权利要求1所述的方法，还包括步骤(e)：将步骤(d)产生的液相用于步骤(a)的反应。
  3. 权利要求1或2所述的方法，其中，步骤(c)的降温温度在-5℃至25℃之间，例如0-10℃之间，0-4℃之间。
  4. 权利要求1-3任一项所述的方法，其中，在步骤(a)中加入L-赤式-对甲砜基苯丝氨酸晶种，优选在搅拌下加入；和/或在步骤(c)的降温期间加入L-苏式-对甲砜基苯丝氨酸晶种，优选在搅拌下加入。
  5. 酶催化制备L-赤式-对甲砜基苯丝氨酸的方法，包括以下步骤：
    (a)使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应；
    (b)固液分离得到包含L-赤式-对甲砜基苯丝氨酸的固相。
  6. 权利要求5所述的方法，其中，在步骤(a)中加入L-赤式-对甲砜基苯丝氨酸晶种，优选在搅拌下加入。
  7. 权利要求1-6任一项所述的方法，其中，步骤(a)的反应温度在4-50℃之间，例如10-30℃之间，15-28℃之间。
  8. 权利要求1-7任一项所述的方法，其中，在步骤(a)中加入5-磷酸吡哆醛。
  9. 权利要求1-8任一项所述的方法，其中，所述助溶剂选自以下物质：甲醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、仲丁醇、叔丁醇、1-戊醇、2-戊醇、3-戊醇、2-甲基-1-丁醇、3-甲基-1-丁醇、2-甲基-2-丁醇、3-甲基-2-丁醇、2,2-二甲基丙醇、乙二醇、丙三醇、巯基乙醇、乙酸乙酯、乙酸丁酯、聚乙二醇6000、聚乙二醇辛基苯醚、乙 腈、丙酮、二甲基亚砜、二甲基甲酰胺、二甲基乙醇胺、乙二醇二甲醚、甲基叔丁基醚、四丁基溴化铵、三乙胺、咪唑、吡啶、二甲基四氢呋喃、十二烷基磺酸钠、3-吗啉丙磺酸、β-环糊精和亚硫酸氢钠；优选选自二甲基亚砜、二甲基甲酰胺、乙醇和丙酮。
  10. 权利要求1-9任一项所述的方法，其中，助溶剂在水溶液体系中的体积浓度为5-60％，例如10-50％，或20-40％。
  11. 权利要求1-10任一项所述的方法，其中，所述L-β-羟基-α-氨基酸合成酶具有SEQ ID No.1或SEQ ID No.2所示的氨基酸序列，或者具有在SEQ ID No.1或SEQ ID No.2所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸序列。
  12. 权利要求1-10任一项所述的方法，其中，所述L-β-羟基-α-氨基酸合成酶是用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶，
    其中，所述L-β-羟基-α-氨基酸合成酶具有SEQ No.1或SEQ No.2的氨基酸序列，或者具有在SEQ ID No.1或SEQ ID No.2所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸序列；或者
    所述用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶具有SEQ ID No.5或SEQ ID No.9的氨基酸序列，或者具有在SEQ ID No.5或SEQ ID No.9所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸序列。
  13. 用于权利要求1所述方法的制备系统，该制备系统包括：1)反应装置，该反应装置用于使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应；2)固液分离装置；3)降温装置。
  14. 用于权利要求5所述方法的制备系统，该制备系统包括：1)反应装置，该反应装置用于使甘氨酸和对甲砜基苯甲醛在L-β-羟基-α-氨基酸合成酶存在下在含助溶剂的水溶液体系中反应；和2)固液分离装置。
  15. L-β-羟基-α-氨基酸合成酶，具有SEQ ID No.1或SEQ ID No.2所示的氨基酸序列，或者具有在SEQ ID No.1或SEQ ID No.2所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸 序列。
  16. 用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶，其中，所述L-β-羟基-α-氨基酸合成酶具有SEQ No.1或SEQ No.2的氨基酸序列，或者具有在SEQ ID No.1或SEQ ID No.2所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸序列。
  17. 权利要求16所述的用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶，具有SEQ ID No.5或SEQ ID No.9的氨基酸序列，或者具有在SEQ ID No.5或SEQ ID No.9所示的氨基酸序列基础上进行1-10个或1-5个或1-3个氨基酸残基替换、增加或缺失但具有L-β-羟基-α-氨基酸合成酶活性的氨基酸序列。
  18. 权利要求15所述的L-β-羟基-α-氨基酸合成酶或权利要求16或17所述的用SpyTag-SpyCatcher环化的L-β-羟基-α-氨基酸合成酶在制备L-苏式-对甲砜基苯丝氨酸和/或L-赤式-对甲砜基苯丝氨酸中的用途。
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