Kinetic constant high-throughput rapid determination method for characterizing carbonyl reductase substrate suitability

Abstract

The invention relates to a high-throughput rapid determination method for kinetic constants for characterizing substrate suitability of carbonyl reductase. The method comprises the following steps: determining the protein concentration of target carbonyl reductase in crude enzyme liquid by adopting a high-throughput method; carrying out catalytic reduction on multiple carbonyl compounds in a substrate library by using the crude enzyme liquid, and rapidly determining the catalytic activity of the carbonyl reductase at high flux; according to the measured activity of the carbonyl reductase for catalyzing reduction of substrates with different concentrations and the measured protein concentration of the target carbonyl reductase, kinetic parameters Vmax and KM of the carbonyl reductase are calculated through software fitting; according to the kinetic parameter Vmax of the carbonyl reductase and the molar concentration of the corresponding carbonyl reductase, a quasi-primary kinetic parameter kcat is calculated, and then the catalytic efficiency constant kcat / KM is calculated. Compared with the prior art, the method has the advantages that the catalytic specific activity and kinetic parameters of the carbonyl reductase to a plurality of substrates in a mode substrate library can be quickly represented in a high-throughput manner.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a high-throughput rapid determination method for kinetic constants for characterizing substrate adaptability of carbonyl reductase. BACKGROUND

[0002] Carbonyl reductase is a very important and useful class of oxidoreductases, which catalyzes the reduction of carbonyl compounds (such as aldehydes or ketones) to generate the corresponding primary or secondary alcohols. At the same time, the enzyme also has the ability to catalyze the reverse reaction, that is, to promote the oxidative dehydrogenation process of alcohol to generate the corresponding aldehyde or ketone, and is also known as alcohol dehydrogenase.

[0003] Compared with the limitations of traditional enantiomeric resolution method in preparing single configuration chiral alcohol chemicals, the ketone asymmetric synthesis method catalyzed by carbonyl reductase shows significant advantages, and the theoretical yield is as high as 100%, which has become the first choice for the synthesis of single configuration chiral secondary alcohol. Due to the high chemical selectivity, regioselectivity and stereoselectivity of carbonyl reductase, as well as the wide substrate adaptability, they are increasingly widely used in fine chemical industry, pharmaceutical industry and other fields.

[0004] However, the industrial application of carbonyl reductase still faces challenges, mainly including the scarcity of enzyme resources suitable for industrial substrates, the cumbersome and time-consuming process of screening new enzymes. Although there have been numerous reports on new carbonyl reductases, the mining and screening of these enzymes are often targeted at specific target substrates, and enzymes with certain catalytic activity for a single target substrate are screened through activity determination. Although in the mining literature of these new enzymes, the expansion of substrate spectrum is usually also carried out, due to the difficulty in obtaining substrates and the limitation of experimental workload, the reported catalytic substrate spectrum range of carbonyl reductase is relatively narrow, mainly limited to the same type of structural derivatives of the target substrate, and it is difficult to comprehensively evaluate the multi-substrate adaptability and comprehensive and accurate structure-activity relationship of carbonyl reductase.

[0005] With the rapid development of artificial intelligence technology, by using deep machine learning strategy, analyzing a large number of experimental data of carbonyl reductase sequence-multiple substrate adaptability (such as catalytic efficiency constant k cat / K M ), and deeply mining and analyzing the quantitative structure-activity relationship between enzyme sequence and multiple substrates, it is possible to efficiently predict the catalytic efficiency constant of carbonyl reductase with different sequences for target substrates, which helps to speed up the speed of new enzyme mining and modification. Efficient machine learning, especially deep machine learning, relies on a large amount of substrate spectrum data of carbonyl reductase, the diversity of enzymes and substrates, and the quantity and quality of related data, which is crucial to the effect of machine learning, and is the key to solving the problem of serious lack of data on recognition and catalysis of artificial synthesized unnatural substrates by natural enzymes (survivor bias).

[0006] Therefore, it is urgent to develop a rapid, efficient and economical method for characterizing the substrate adaptability (e.g., specific activity) and kinetic constants of carbonyl reductases, so as to comprehensively and quantitatively analyze the interaction (binding constant K M ) and mutual adaptability (catalytic constant k cat ) of carbonyl reductases with different sequences / structures and carbonyl substrates with different structures, which can provide valuable and high-quality catalytic function experimental data for quantitative structure-activity relationship studies of carbonyl reductases.

[0007] The kinetic parameters of enzymes, such as the maximum rate V max and Michaelis constant K M , are usually obtained by fitting the conversion rates of enzyme catalysis of a series of different concentrations of substrates based on the Michaelis equation in the past. In order to quickly obtain the reaction kinetic parameters of carbonyl reductases catalyzing a series of concentrations of carbonyl substrates, the key is to use a specific automated instrument to obtain the substrate / co-substrate concentration-time curve of the target carbonyl reductase with different substrate sets in parallel, high-throughput and rapidly.

[0008] In view of the presence of a large amount of impurities in the cell-free extract (crude enzyme solution) obtained after centrifugation of microbial cells, in order to accurately measure the protein concentration of the target carbonyl reductase and the normalized intrinsic kinetic parameters (such as the conversion frequency TOF = k cat = V max / [E]0) of the enzyme, the prior art usually needs to separately purify the enzyme for a time-consuming and tedious process, and then use a precise amount of pure enzyme protein to catalyze the substrate conversion reaction to measure the initial reaction rate of the enzyme reaction at a series of different concentrations. The process is usually carried out on a spectrophotometer, usually using a 1cm cuvette, and the reaction liquid volume is 1-3mL, which is time-consuming and laborious and low in efficiency. In addition, for some enzymes, it is difficult to obtain the expected purity of the pure enzyme by using the relatively simple nickel ion affinity chromatography method. The prior art seriously restricts the high-throughput and rapid determination of the kinetic parameters of carbonyl reductases. SUMMARY

[0009] There is a lack of high-throughput and rapid determination method for the kinetic parameters of carbonyl reductases in the prior art, and based on this, the present application provides a high-throughput and rapid determination method for characterizing the kinetic constants of the substrate adaptability of carbonyl reductases.

[0010] Based on the method provided in the present application, the rapid and high-throughput determination of the enzymatic reaction kinetic parameters of carbonyl reductases for a plurality of different substrates can be realized.

[0011] The present application provides a high-throughput rapid determination method for kinetic constants for characterizing the substrate adaptability of carbonyl reductase, and secondly, in order to realize the structural diversity and functional representation of carbonyl reductase substrates, a substrate library including aldehydes, aliphatic ketones, alicyclic ketones, heterocyclic ketones, monocyclic aromatic ketones, double aromatic ring ketones, aliphatic ketone esters, aromatic ketone esters and aryl alicyclic ketones is designed, and a high-throughput determination method for the target enzyme protein concentration in the crude enzyme solution of carbonyl reductase is optimized.

[0012] The object of the present application can be achieved by the following technical solutions:

[0013] The present application provides a high-throughput rapid determination method for kinetic constants for characterizing the substrate adaptability of carbonyl reductase, and secondly, in order to realize the structural diversity and functional representation of carbonyl reductase substrates, a substrate library including aldehydes, aliphatic ketones, alicyclic ketones, heterocyclic ketones, monocyclic aromatic ketones, double aromatic ring ketones, aliphatic ketone esters, aromatic ketone esters and aryl alicyclic ketones is designed, and a high-throughput determination method for the target enzyme protein concentration in the crude enzyme solution of carbonyl reductase is optimized.

[0014] (1) obtaining a crude enzyme solution containing a target carbonyl reductase;

[0015] (2) determining the protein concentration of the target carbonyl reductase in the crude enzyme solution obtained in step (1) by using a high-throughput method;

[0016] (3) using the crude enzyme solution obtained in step (1) to catalytically reduce a plurality of carbonyl compounds in the substrate library, and high-throughput rapidly determining the catalytic activity of the carbonyl reductase;

[0017] (4) according to the activity of the carbonyl reductase catalyzing the reduction of different concentrations of substrates determined in step (3) and the protein concentration of the target carbonyl reductase determined in step (2), fitting and calculating the kinetic parameters V max and K M of the carbonyl reductase by software;

[0018] (5) according to the kinetic parameters V max of the carbonyl reductase and the molar concentration of the corresponding carbonyl reductase, calculating the pseudo-first-order kinetic parameter k cat , and further calculating the catalytic efficiency constant k cat / K M .

[0019] In an embodiment of the present application, the determination of the protein concentration of the target carbonyl reductase in the crude enzyme solution in step (2) comprises the following steps:

[0020] (2.1) taking a certain volume of the crude enzyme solution and performing SDS-polyacrylamide gel electrophoresis;

[0021] (2.2) using the concentration of standard molecular weight protein markers as a standard, and using gray scale analysis strategy to quantitatively calculate the concentration of the target carbonyl reductase in the crude enzyme solution (cell-free extract).

[0022] In an embodiment of the present application, in step (3), the high-throughput rapid determination of the catalytic activity of carbonyl reductase is performed in a 96-well plate on an enzyme label meter, comprising the following steps:

[0023] (3.1) A buffer, a stock solution of a plurality of carbonyl compounds in a substrate library, a stock solution of coenzyme NADH or NADPH, and a concentrated stock solution of the enzyme are sequentially added to the wells of the 96-well plate in different volumes;

[0024] (3.2) After the addition is complete, the 96-well plate is placed on an enzyme label meter, briefly shaken to mix, and the enzyme-catalyzed reaction is performed, and the characteristic absorbance value A of the coenzyme NADH or NADPH at 340 nm is recorded 340 (t), according to the Beer-Lambert law, the relationship between absorbance and concentration is: A 340 (t) = ε × C(t) × l, where ε is the molar absorption coefficient of NADH or NADPH, taken as 6220 cm·L·mol -1 ; C(t) is the coenzyme concentration at a certain time point in the enzyme label plate (mol·L -1 ); l is the depth of the solution in the wells of the enzyme label plate, unit: cm; through data fitting, the coenzyme concentration C(t) as a function of time is obtained, and its change rate is equal to the instantaneous rate of the enzyme-catalyzed reaction of the carbonyl substrate;

[0025] (3.3) According to the change rate of the coenzyme concentration C(t) as a function of time, it is recorded as the enzyme-catalyzed reaction rate V(t), V(t) = -dC(t) / dt = -(1 / ε×l)·dA 340 (t) / dt, the initial reaction rate V 0i value corresponding to different carbonyl substrate concentrations is obtained, and the Michaelis equation is fitted to obtain the corresponding kinetic constants K M (NADH / NADPH) and V max or k cat (k cat = V max / [E]0).

[0026] In an embodiment of the present application, in step (1), the target carbonyl reductase is selected from any one of CR01 to CR30, and the amino acid sequences of CR01 to CR30 are shown in the sequence table SEQ ID No. 1-SEQ ID No. 30.

[0027] In an embodiment of the present application, in step (2), a high-throughput method is used to determine the protein concentration of a plurality of different target carbonyl reductases obtained in step (1), comprising the following steps:

[0028] With E. coli BL21(DE3) as the host and plasmid pET 28a as the vector, different target carbonyl reductases are recombinantly expressed: the nucleic acid sequence of the carbonyl reductase is connected to the multiple cloning site of pET 28a to construct a recombinant expression plasmid, which is transformed into E. coli BL21(DE3) to construct a series of recombinant strains E. coli BL21 / pET 28a-CR;

[0029] The recombinant strain E. coli BL21 / pET 28a-CR is inoculated into a triangular flask containing LB medium and cultured to OD 600 1.0, then transferred into a triangular flask containing fresh LB medium at a ratio of 1% (v / v) and cultured to OD 600 0.6, isopropyl-β-D-thiogalactoside is added, and the shaking is continued, the bacterial cells are harvested by centrifugation, resuspended in potassium phosphate buffer, ultrasonically broken, the broken liquid is centrifuged to remove cell debris, and the broken liquid is stored at 4°C for short-term preservation;

[0030] The total protein concentration of the crude enzyme solution is determined by Bradford protein concentration determination method, three groups of parallel samples are set for each carbonyl reductase, 30 kinds of crude enzyme solutions corresponding to the carbonyl reductases are added to the holes of a 96-hole plate, a total of 90 hole samples, 100 μL of enzyme solution is added to each hole; 6 remaining holes are used as blank controls, 100 μL of deionized water is added to each hole, after the sample addition is completed, 100 μL of coomassie brilliant blue R250 staining solution is added to each hole, 25°C incubation is performed for 5 min, and the enzyme label instrument is used to read the absorbance value at 595 nm;

[0031] The obtained carbonyl reductase crude enzyme solution is subjected to SDS-PAGE protein electrophoresis separation, a standard molecular weight protein is used as a protein standard, a coomassie brilliant blue staining method is used to stain the protein gel sample obtained after electrophoresis, a gel imaging instrument is used to scan the protein gel image, ImageJ software is used to perform gray scale analysis on the protein staining bands, a standard curve is drawn using the known concentration of the protein molecular weight standard, and the protein concentration of the target carbonyl reductase in the crude enzyme solution is quantified.

[0032] In an embodiment of the present application, in step (3), the design and selection of the carbonyl compounds in the substrate library cover one or more of the following principles, with structural diversity and functional representation:

[0033] (1) widely reported in the literature, with wide application;

[0034] (2) has a structurally diverse molecular skeleton;

[0035] (3) covers different types of chemical functional groups, covering a wide range of physical and chemical properties;

[0036] (4) The reduction products of some carbonyl substrates are drug precursors or building blocks, which have practical values;

[0037] (5) Easy to synthesize or commercially available.

[0038] In one embodiment of the present application, in step (3), the carbonyl compounds in the substrate library include aldehydes, aliphatic ketones, alicyclic ketones, heterocyclic ketones, monocyclic aromatic ketones, bis-aromatic ketones, aliphatic ketone esters, aromatic ketone esters and aryl alicyclic ketones.

[0039] The aldehydes include compounds S01-S08, the structures of which are shown as follows:

[0040]

[0041] The aliphatic ketones include compounds S09-S18, the structures of which are shown as follows:

[0042]

[0043] The alicyclic ketones include compounds S19-S25, the structures of which are shown as follows:

[0044]

[0045] The heterocyclic ketones include compounds S26-S34, the structures of which are shown as follows:

[0046]

[0047] The monocyclic aromatic ketones include compounds S35-S60, the structures of which are shown as follows:

[0048] The bis-aromatic ketones include compounds S61-S68, the structures of which are shown as follows:

[0049]

[0050] The aliphatic ketone esters include compounds S69-S77, the structures of which are shown as follows:

[0051]

[0052] The aromatic ketone esters include compounds S78-S83, the structures of which are shown as follows:

[0053]

[0054] The aryl alicyclic ketones include compounds S84-S90, the structures of which are shown as follows:

[0055] In one embodiment of the present application, in step (3), the substrate is added to the 96-well plate in the following order, with 6 holes reserved as blank controls, Blk1, Blk2;

[0056] 1 2 3 4 5 6 7 8 9 10 11 12 A Blk1 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 B S12 S13 Blk2 S14 S15 S16 S17 S18 S19 S20 S21 S22 C S23 S24 S25 S26 Blk1 S27 S28 S29 S30 S31 S32 S33 D S34 S35 S36 S37 S38 S39 Blk2 S40 S41 S42 S43 S44 E S45 S46 S47 S48 S49 S50 S51 S52 Blk1 S53 S54 S55 F S56 S57 S58 S59 S60 S61 S62 S63 S64 S65 Blk2 S66 G S67 S68 S69 S70 S71 S72 S73 S74 S75 S76 S77 S78 H S79 S80 S81 S82 S83 S84 S85 S86 S87 S88 S89 S90

[0057] Blk1 is Control 1: only coenzyme, carbonyl reductase and buffer are added, without adding the carbonyl substrate solution, but replaced by an equal volume of the auxiliary solvent;

[0058] Blk2 is Control 2: only coenzyme (NADH / NADPH) and buffer are added, without adding the carbonyl substrate and carbonyl reductase.

[0059] Since there are some bioreducible natural metabolites in the enzyme solution during the preparation of the enzyme solution, when the enzyme activity of the target substrate is determined, the decrease in the absorbance value at 340 nm caused by these substances must be deducted. Therefore, two groups of blank controls are set.

[0060] In one embodiment of the present application, in step (3.2), the enzyme-catalyzed reaction is carried out in a 96-well plate on an enzyme marker. The volume of each hole reaction solution is 200 μL, containing 140 μL buffer, 20 μL coenzyme stock solution (NADH / NADPH), 20 μL carbonyl reductase solution, 1-20 μL substrate concentrate dissolved in DMSO, and the rest volume is made up with DMSO (19-0 μL). All solutions are pre-incubated at 30°C. After the addition is completed, the 96-well plate is placed on the enzyme marker, briefly shaken to mix, and the enzyme-catalyzed reaction is carried out, and the change in the absorbance value of coenzyme NADH / NADPH at 340 nm during the reaction is tracked in time to calculate the rate of enzyme reaction and the activity of dehydrogenase.

[0061] In one embodiment of the present application, in step (4), the concentration of the carbonyl substrate is selected to be a typical concentration of 1.0 mM to 20.0 mM for determination, and the initial concentration of the coenzyme is 0.1 mM NADH / NADPH; the concentration of the enzyme solution is diluted so that the rate of enzyme reaction is controlled at 0.005-0.02 mM·min -1 .

[0062] In the present application, a large number of kinetic parameters of carbonyl reductase need to be determined. Here, the accurate determination of the concentration of the target enzyme protein is crucial for the quantification of enzyme activity.

[0063] Traditionally, when determining the kinetic parameters of an enzyme, the enzyme needs to be purified, and the enzyme is usually rapidly purified by nickel column affinity chromatography. In the design and construction of a recombinant enzyme protein, a His-tag label of 6 histidines is added to the N-terminus or C-terminus of the enzyme, and the specific chelation between histidine and nickel ions is used to bind the gel resin combined with nickel ions. Then, the impure proteins not combined with the resin are removed by washing. Subsequently, the target protein is eluted using different concentrations of imidazole to obtain a purified enzyme solution. Then, the protein concentration of the purified enzyme solution is determined, and the enzyme activity is calculated by using the purified enzyme to catalyze the reaction.

[0064] However, in practice, it is found that some enzymes are difficult to combine with nickel ions due to structural factors, and therefore, the nickel ion affinity chromatography method cannot be used for rapid purification of enzyme proteins. The traditional resin column chromatography purification method is time-consuming and labor-intensive, and the enzyme activity is greatly lost, which cannot meet the requirements of rapid purification of enzyme proteins.

[0065] Therefore, in the present application, a method for rapidly quantifying target carbonyl reductase protein in a crude enzyme solution is established to solve the above problems.

[0066] E. coli is used as a host for expressing target carbonyl reductase protein, and the collected recombinant E. coli cells are broken to obtain a crude enzyme solution (supernatant of the cell-free extract after centrifugation) containing target carbonyl reductase protein. On this basis, the total protein concentration of the crude enzyme solution is determined by the classic Bradford method. At the same time, the crude enzyme solution is subjected to protein electrophoresis separation and gel imaging. The gray scale of the gel strip after electrophoresis separation of the crude enzyme solution is scanned and analyzed based on the concentration of the molecular weight standard protein to determine the relative content and actual concentration of the target protein, thereby establishing a rapid and standard method for determining the concentration of target enzyme protein, thereby omitting the time-consuming and labor-intensive target enzyme protein affinity chromatography purification process which may fail.

[0067] Meanwhile, in the high-throughput enzyme activity and reaction rate rapid determination method of the present application, the carbonyl reductase catalyzes the reduction reaction of the carbonyl substrate, and at the same time, the co-substrate (coenzyme) NADH / NADPH also undergoes oxidation reaction to generate NAD + / NADP + , accompanied by a significant decrease in the absorbance of the reducing coenzyme at 340 nm. By using this property, the enzyme activity and reaction rate of the carbonyl reductase can be rapidly determined by using an enzyme marker.

[0068] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0069] The application provides a high-throughput rapid determination method for kinetic constants of substrate adaptability of a carbonyl reductase. Specifically, the application constructs a model substrate library of carbonyl reductases with structural diversity and functional representation, establishes a high-throughput determination method for the catalytic activity of the carbonyl reductases on multiple substrates based on the substrate library and a multi-well plate enzyme marker, and a high-throughput detection method for directly determining the concentration of target carbonyl reductase protein in cell lysate expressed by recombination. Based on this, the catalytic specific activity of the carbonyl reductase on the model substrate library and the kinetic parameters (including Michaelis constant K M , pseudo-first-order speed constant k cat , and catalytic efficiency constant k cat / K M ) of the substrates can be rapidly and high-throughput characterized. The technology disclosed in the application is of great significance for breaking the speed bottleneck of obtaining big data of the adaptability characteristics and structure-activity relationship of different sequences of carbonyl reductases / alcohol dehydrogenases on different substrates, and is helpful to accelerate the development of machine learning, functional prediction and intelligent design technologies and industrial application of industrial enzymes. DETAILED DESCRIPTION

[0070] The application will be described in detail below in combination with specific embodiments.

[0071] Example 1: A substrate library of carbonyl reductases with structural diversity and functional typicality

[0072] Carbonyl reductases often have a relatively wide substrate spectrum. In this embodiment, the active substrates involved are summarized, and the carbonyl reductase catalytic substrates are simplified according to a series of principles to design and construct a substrate library containing 90 carbonyl reductases with structural diversity and functional typicality.

[0073] The principles are as follows:

[0074] (1) The molecular skeleton has structural diversity. It includes aldehydes, ketones (aliphatic ketones, aromatic ketones, heterocyclic ketones, ketone esters, etc.) with different structures; and straight chains, branched chains, aromatic / heterocyclic, bridged rings, and polyrings with different structures.

[0075] (2) A wide range of chemical functional groups are covered. It includes aliphatic groups, alkenyl groups, hydroxyl groups, cyano groups, halogenated groups, etc.

[0076] (3) A wide range of physical and chemical properties are covered. Different polar substituents are introduced to adjust the hydrophobicity parameter (such as logP) of the substrate.

[0077] (4) The application is widely used. The compounds are selected according to the number of literature reports of enzymatic reduction in the literature.

[0078] (5) Certain practicality. The reduction products of some carbonyl substrates are drug precursors or building blocks.

[0079] (6) Easy to obtain. The substrates need to be easily synthesized or commercially available to ensure the feasibility of the experiment.

[0080] The diversity of the substrate structures provided by the present embodiment is a key element to ensure the quality of the database, mainly embodied in the following aspects:

[0081] 1) Structural adaptability: the diversity of the substrate molecular skeleton, the types of substituent groups, steric hindrance and physicochemical properties directly affect the degree of stereomatching and the level of binding energy between them and the active pocket of the enzyme (including the binding pocket and the catalytic site);

[0082] 2) Interaction mechanism: polar groups (such as hydroxyl, amino, etc.) may be involved in forming a hydrogen bond network, while non-polar groups interact with hydrophobic cavities through van der Waals interactions; adjacent counterions may form salt bridges; benzene rings may form π-π stacking interactions with adjacent aromatic groups.

[0083] 3) Functional expandability: diverse substrate structures help to explore the potential application value of enzymes in the synthesis of unnatural products and chiral drugs.

[0084] It is of great significance to construct a pattern substrate library with strong diversity, including but not limited to:

[0085] 1) Systematic analysis of enzyme catalytic flexibility and substrate broadness (adaptation map);

[0086] 2) Improve the prediction width and generalization ability of the structure-activity relationship model, and reduce the survivor bias caused by natural evolution;

[0087] 3) Provide high-quality, comprehensive and diverse big data support for the rational design and industrial application of enzymes.

[0088] Through the integration of molecular docking, quantum chemical calculations and machine learning, diverse substrate adaptability data can be used to establish a complete structure-activity relationship from molecular interactions to macroscopic catalytic performance.

[0089] Specifically, the substrate library of the carbonyl reductase provided in the present embodiment includes aldehyde compounds, aliphatic ketones, alicyclic ketones, heterocyclic ketones, monocyclic aromatic ketones, bis-aromatic ketones, aliphatic ketone esters, aromatic ketone esters and aryl alicyclic ketones.

[0090] Among them, the aldehyde compounds include compounds S01-S08, the structures of which are as follows:

[0091]

[0092] Aliphatic ketones include compounds S09-S18, the structures of which are shown below:

[0093]

[0094] Alicyclic ketones include compounds S19-S25, the structures of which are shown below:

[0095]

[0096] Heterocyclic ketones include compounds S26-S34, the structures of which are shown below:

[0097]

[0098] Monocyclic aromatic ketones include compounds S35-S60, the structures of which are shown below:

[0099] Bicyclic aromatic ketones include compounds S61-S68, the structures of which are shown below:

[0100]

[0101] Aliphatic ketoesters include compounds S69-S77, the structures of which are shown below:

[0102]

[0103] Aromatic ketoesters include compounds S78-S83, the structures of which are shown below:

[0104]

[0105] Arylalicyclic ketones include compounds S84-S90, the structures of which are shown below:

[0106]

[0107] Example 2

[0108] In this example, a rapid determination method for the total protein concentration in a crude enzyme solution of a carbonyl reductase is provided

[0109] In this example, 30 carbonyl reductases (CR01-CR30) were selected as examples, and the amino acid sequences thereof are shown in the sequence listing SEQ ID No. 1-SEQ ID No. 30. Using E. coli BL21 (DE3) as the host and plasmid pET 28a as the vector, the 30 carbonyl reductases (CR01-CR30) were recombinantly expressed.

[0110] The nucleic acid sequences of the carbonyl reductases CR01-CR30 were respectively connected to the multiple cloning site of pET 28a to construct recombinant expression plasmids, which were transformed into E. coli BL21(DE3) to construct a series of recombinant strains E. coli BL21 / pET 28a-CR.

[0111] The recombinant strain E. coli BL21 / pET 28a-CR was inoculated into a 250 mL triangular flask containing 50 mL of LB medium and cultured at 37°C and 200 rpm until the OD 600 was 1.0, and then transferred at a ratio of 1% (v / v) into a 500 mL triangular flask containing 100 mL of fresh LB medium and cultured at 37°C and 200 rpm until the OD 600 was 0.6, and isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2 mM, and the shaking was continued at 16°C for 24 h, and the bacterial cells were harvested by centrifugation at 10,000 x g, resuspended in 10 mL of potassium phosphate buffer (100 mM, pH 7.0), broken by ultrasonication, the broken solution was centrifuged at 15000 x g to remove cell debris, and stored at 4°C for short-term use.

[0112] Each carbonyl reductase was set in triplicate, and 10 μL of the crude enzyme solution of each of the 30 carbonyl reductases was added to the holes of a 96-well plate, for a total of 90 sample wells; the remaining 6 holes were used as blank controls, and 100 μL of deionized water was added to each hole. After the addition, 200 μL of Coomassie Brilliant Blue R250 staining solution was added to each hole, and the plate was incubated at 25°C for 5 min, and then placed on an enzyme marker to read the absorbance value at 595 nm. The protein concentration of the crude enzyme solution was calculated based on the standard curve prepared in advance.

[0113] Table 1: Sample addition setting table for total protein concentration determination of crude enzyme solution

[0114] 1 2 3 4 5 6 7 8 9 10 11 12 1 Blank CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 2 CR12 CR13 Blank CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 3 CR23 CR24 CR25 CR26 Blank CR27 CR28 CR29 CR30 CR1 CR2 CR3 4 CR4 CR5 CR6 CR7 CR8 CR9 Blank CR10 CR11 CR12 CR13 CR14 5 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 Blank CR23 CR24 CR25 6 CR26 CR27 CR28 CR29 CR30 CR1 CR2 CR3 CR4 CR5 Blank CR6 7 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 8 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30

[0115] It was determined that the total protein concentrations of the crude enzyme solutions of the carbonyl reductases CR01-30 were as shown in Table 2.

[0116] Table 2: Total protein concentrations of crude enzyme solutions of carbonyl reductases CR01-CR30

[0117]

[0118]

[0119] Example 3: Quantification of target carbonyl reductase protein concentration

[0120] The obtained carbonyl reductase crude enzyme solution was subjected to SDS-PAGE protein electrophoresis separation, and the standard molecular weight (14.4-116 kD) protein of Biyun Tian Biotechnology was used as a protein standard. The protein gel obtained after electrophoresis was stained by using a traditional coomassie brilliant blue staining method. The gel imaging instrument was used to scan the protein gel image, and the ImageJ software was used to analyze the gray scale of the protein staining band. The protein concentration of the target carbonyl reductase in the crude enzyme solution was quantified by using the known concentration of the protein molecular weight standard as a standard curve, and the results are shown in Table 3.

[0121] Table 3 Protein concentration of target enzyme in carbonyl reductase CR01-CR30 crude enzyme solution

[0122]

[0123] Example 4 High-throughput determination of the activity of carbonyl reductase CR01 on different substrates in a 96-well plate

[0124] The enzymatic reaction was carried out in a 96-well plate on an enzyme marker. The total volume of the reaction solution was 200 μL, which contained 140 μL of potassium phosphate buffer (50 mM, pH 6.5), 20 μL of coenzyme NADH / NADPH solution (1.00 mM, final concentration 0.10 mM), 20 μL of carbonyl reductase CR01 crude enzyme diluent (x10), and 20 μL of each substrate DMSO solution. Each substrate was previously dissolved in DMSO to prepare a stock solution with a concentration of 200 mM, and 20 μL was added to the reaction system, with a final substrate concentration of 20.0 mM (the final concentration of DMSO was 10%, v / v). All solutions were pre-incubated at 30°C, and the samples were added in the positions listed in Table 4 using a row gun. A total of 90 substrates (S1-S90 as disclosed in Example 1), plus 2 groups of 6 blank controls (Blk1*3+Blk2*3). Three parallel experiments were performed, and the average values were taken.

[0125] Control 1 (Blk1): only add coenzyme, carbonyl reductase and buffer, do not add substrate / DMSO solution (only replace with equal volume of DMSO);

[0126] Control 2 (Blk2): only add coenzyme and buffer solution, do not add carbonyl reductase (replace with equal volume of buffer), and do not add substrate / DMSO solution (only replace with equal volume of DMSO).

[0127] Table 4 Substrates for enzymatic reaction and blank control settings

[0128] 1 2 3 4 5 6 7 8 9 10 11 12 A Blk1 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 B S12 S13 Blk2 S14 S15 S16 S17 S18 S19 S20 S21 S22 C S23 S24 S25 S26 Blk1 S27 S28 S29 S30 S31 S32 S33 D S34 S35 S36 S37 S38 S39 Blk2 S40 S41 S42 S43 S44 E S45 S46 S47 S48 S49 S50 S51 S52 Blk1 S53 S54 S55 F S56 S57 S58 S59 S60 S61 S62 S63 S64 S65 Blk2 S66 G S67 S68 S69 S70 S71 S72 S73 S74 S75 S76 S77 S78 H S79 S80 S81 S82 S83 S84 S85 S86 S87 S88 S89 S90

[0129] After the addition of the substrate, the 96-well plate was placed on the microplate reader, briefly shaken to mix, and the enzymatic reaction was performed. The microplate reader was set to analyze at a wavelength of 340 nm, and data was read every 5 seconds.

[0130] The recorded data was analyzed, and the linear portion was extracted to calculate the rate of change in absorbance at 340 nm, ΔA / Δt, of coenzyme NADH / NADPH during the reaction.

[0131] During the enzymatic reaction, the change in absorbance A per minute should not be higher than 30% of the absorbance value A2 of the blank control group 2. If the determination of ΔA per minute exceeds 30% of A2, the enzyme solution needs to be appropriately diluted and measured again according to the above method.

[0132] The calculation formula of the unit volume enzymatic reaction rate v for the target substrate is:

[0133] v = (ΔA / Δt - ΔA0 / Δt) / 6220 / 0.7 × 1000

[0134] Where the unit of the unit volume enzymatic reaction rate v is μmol·min -1 ·mL -1 ;

[0135] ΔA / Δt is the rate of decrease in absorbance at 340 nm during the reaction of the target substrate (taking the average of three experiments), with the unit of min -1 ;

[0136] ΔA0 / Δt is the average of the absorbance decrease rate at 340 nm of three blank controls 1 (Blk1*3), with the unit of min -1 ;

[0137] 6220 is the molar absorption coefficient (ε) of coenzyme NADH / NADPH, with the unit of L·mol -1 ·cm -1 ;

[0138] 0.7 is the optical path (l) of the reaction solution in the 96-well plate, with the unit of cm;

[0139] 1000 is the conversion coefficient between different volumes and concentrations.

[0140] According to the data recorded by the microplate reader, the carbonyl reductase CR01 is an NADPH-dependent reductase. When the substrate concentration is 20.0 mM, the unit volume enzymatic reaction rate v of the carbonyl reductase CR01 for the model substrate is calculated, and the results are shown in Table 5. Among them, the carbonyl reductase CR01 has activity for some model substrates, but also does not show activity for other model substrates, indicating that the carbonyl reductase has a certain degree of substrate broadness. Only some of the model substrates with detectable activity are listed in Table 5, and the activity of other substrates is not detected.

[0141] Table 5 Enzymatic reaction rate v (pmol min-1 mL-1) of carbonyl reductase CR01 for some model substrates -1 ·mL -1 ).

[0142] S11 18.2±0.4 S12 7.16±0.31 S13 5.39±0.13 S26 1.75±0.08 S27 2.02±0.12 S28 2.19±0.22 S32 2.27±0.22 S35 12.7±0.4 S36 14.4±0.23 S37 4.54±0.24 S40 6.22±0.41 S42 22.4±1.4 S44 3.87±0.22 S50 0.45±0.07 S52 4.09±0.37 S53 10.22±0.15 S57 4.43±0.46 S58 9.38±0.13 S69 3.51±0.28 S70 14.9±0.9 S71 10.5±0.8 S72 5.84±0.32 S74 8.17±0.16 S76 3.26±0.17 S78 0.91±0.12 S79 1.91±0.09

[0143] Example 5 High-throughput determination of reaction rate of carbonyl reductase CR01 for different concentrations of substrates in 96-well plates

[0144] According to the results of Example 4, when the substrate concentration is 20 mM, the carbonyl reductase CR01 does not have activity for some model substrates. Further, only for the active substrates, the kinetic parameter determination analysis is carried out, and the final substrate concentration is set to 1, 2, 4, 8 mM. The loading position is still set as in Table 4, but the substrates without catalytic activity are removed. The concentration of the substrate stock solution is still 200 mM, and as the final concentration of the substrate changes, the volume of the substrate sample is changed, and the insufficient volume is supplemented with DMSO, so that the content of DMSO in the reaction system remains unchanged (10%, v / v). The determination results are shown in Table 6.

[0145] Table 6 Unit volume enzymatic reaction rate v (pmol min-1 mL-1) of carbonyl reductase CR01 for different concentrations of substrates -1 ·mL -1 )

[0146]

[0147]

[0148] Example 6 Calculation of catalytic kinetic parameters of carbonyl reductase CR01 for different substrates

[0149] According to the concentration C of the target protein in the crude enzyme solution of the carbonyl reductase CR01 in Example 3 酶 and the unit volume enzymatic reaction rate v of the carbonyl reductase CR01 for different concentrations of various model substrates in Examples 4 and 5, the catalytic reaction rate V (pmol min-1 mg-1) of the carbonyl reductase CR01 for different concentrations of substrates is calculated. -1 ·mg-1 )。

[0150] wherein the calculation formula of V is:

[0151] V = v / C 酶

[0152] wherein C 酶 is the average value of the target carbonyl reductase protein concentration in the reaction hole, in mg·mL -1 .

[0153] According to the Michaelis equation, the carbonyl reductase CR01 catalytic reaction rate V determined at different substrate concentrations was fitted by Origin software, to obtain the maximum reaction rate V max and Michaelis constant K M of the carbonyl reductase catalytic reaction; according to V max and the molecular weight of the enzyme, the turnover number k cat of the enzyme was calculated, and then the catalytic efficiency constant k cat / K M of the enzyme could be calculated. Among them, the molecular weight of the carbonyl reductase CR01 is 30.1 kDa.

[0154] According to the fitting results, the kinetic parameters of the carbonyl reductase CR01 for a series of model substrates were calculated, as shown in Table 7.

[0155] Table 7 Kinetic constants of carbonyl reductase CR01 for model substrates

[0156]

[0157]

[0158] Example 7 High-throughput determination of reaction rates of carbonyl reductase CR09 catalysis of different substrates in 96-well plates

[0159] The enzymatic reaction was carried out in a 96-well plate on an enzyme marker. The total volume of the reaction solution was 200 μL, which contained 140 μL of potassium phosphate buffer (50 mM, pH 6.5), 20 μL of coenzyme NADH / NADPH solution (final concentration 0.1 mM) with a concentration of 1 mM, 20 μL of crude enzyme dilution of carbonyl reductase CR09 (x20), and 20 μL of each substrate DMSO solution. The substrate was previously dissolved in DMSO to prepare a stock solution with a concentration of 200 mM, which was added to the reaction system with a final concentration of 20 mM. All solutions were pre-incubated at 30°C, and the addition was carried out according to the positions listed in Table 4. A total of 90 substrates, and 2 groups of 6 blank controls were set. A total of 3 groups of parallel experiments were carried out.

[0160] After the addition of reagents, the 96-well plate was placed on the microplate reader, briefly shaken to mix, and the enzymatic reaction was performed. The microplate reader was set to analyze at a wavelength of 340 nm, and data was read every 5 seconds.

[0161] The recorded data was analyzed, and the linear portion was extracted to calculate the rate of change in absorbance value of coenzyme NADH / NADPH at 340 nm, ΔA / Δt.

[0162] In the enzymatic reaction, the change in absorbance value A per minute should not be higher than 30% of the absorbance value A2 of the blank control group. If ΔA per minute exceeds 30% of A2, the enzyme solution needs to be appropriately diluted and measured again according to the above method.

[0163] According to the data from the microplate reader, carbonyl reductase CR09 is an NADH-dependent reductase. The unit volume enzymatic reaction rate v of carbonyl reductase CR09 for the model substrate was calculated when the substrate concentration was 20 mM, and the results are shown in Table 8.

[0164] Table 8 Enzymatic reaction rate v (μmol·min -1 ·mL -1 )

[0165] S01 12.4±0.6 S02 11.3±2.6 S04 10.4±1.5 S09 10.8±0.8 S10 11.5±1.4 S11 9.36±2.40 S16 2.81±0.14 S21 8.77±2.31 S23 11.1±1.7 S30 6.48±0.32 S32 7.07±1.32 S38 7.92±1.05 S39 7.80±1.42 S40 2.93±0.36 S45 9.28±0.89 S46 5.52±0.56 S50 7.76±0.47 S53 6.52±0.35 S55 4.67±0.14 S58 4.85±0.44 S60 1.19±0.12 S61 5.48±0.53 S64 4.66±0.74 S69 4.30±0.51 S71 3.93±0.41 S72 7.91±1.40 S73 3.51±0.47 S75 4.76±0.35 S76 0.35±0.06 S78 1.88±0.21 S79 0.79±0.12 S82 2.04±0.33 S84 0.41±0.06 S88 1.62±0.22 S89 2.21±0.29

[0166] Example 8 High-throughput determination of catalytic constants of carbonyl reductase CR09 for different concentrations of substrates in a 96-well plate

[0167] According to the results of Example 7, further, only for active substrates, the determination and analysis of kinetic parameters were set, and the initial concentration of the substrate was set to 1, 2, 4, and 8 mM. The addition position was still set as in Table 4, but the substrates without catalytic activity were removed. The concentration of the substrate stock solution was still 200 mM, and as the concentration of the substrate changed, the volume of the substrate was changed, and the insufficient volume was supplemented with DMSO. The results are shown in Table 9.

[0168] Table 9 Enzymatic reaction rate v (μmol·min -1 ·mL -1 )

[0169]

[0170]

[0171] Example 9 Calculation of kinetic parameters of carbonyl reductase CR09 for different substrates

[0172] According to the concentration C of the target protein in the crude enzyme solution of carbonyl reductase CR09 in Example 3酶 and the unit volume enzymatic reaction rate v of carbonyl reductase CR09 on various model substrates with different concentrations in Examples 7 and 8, the catalytic reaction rate V (pmol min -1 ·mg -1 ) of carbonyl reductase CR09 on different concentrations of substrates was calculated. According to the Michaelis equation, the catalytic reaction rate V of carbonyl reductase CR09 measured at different substrate concentrations was fitted by Origin software to obtain the maximum reaction rate V max and Michaelis constant K M of carbonyl reductase catalytic reaction; according to V max and the molecular weight of the enzyme, the turnover frequency TOF = k cat＝Vmax / [E] of the enzyme was calculated, and then the catalytic efficiency constant k cat / K M of the enzyme can be calculated. The molecular weight of carbonyl reductase CR09 is 35.7 kDa.

[0173] According to the fitting results, the kinetic parameters of carbonyl reductase CR09 on a series of model substrates were calculated, as shown in Table 10.

[0174] Table 10 Kinetic constants of carbonyl reductase CR09 for model substrates

[0175]

[0176]

[0177] In the present application, the sequence of the carbonyl reductase involved is as follows:

[0178] CR01

[0179] VDLGIQGKLAVVTAGSKGLGFASALELARNGARLLLFSRNREKLEAAASRIASLVSGAQVDIVAGDIREPGDIDRLFEKARDLGGADILVYSRGGPRPGRFMELGVEDWDESYRLLARSAVWVGRRAAEQMVEKGWGRMVYIGSVTLLRPWQDLALSNIMRLPVIGVVRTLALELAPHGVTVNAVLPSLILTDRVRSLGEERARRSGITVEEALKSMASRIPMGTVGKPEELASVVAFLASEKASFITGAVIPVDGGAHI

[0180] CR02

[0181] MENVNMVKSKAALCKKFSEPLSIEDVNIPEPQGEEVLIRIGGAGVCRTDLRVWKGVEAKQ GFRLPIILGHENAGTIVEVGELAKVKRGDNVVVYATWGDLTCRYCREGKFNICKNQIIPGQ TTNGGFSEYMLVKSTRWLVKLNSLSPVEAAPLADAGTTSMGAIRQALPFISKFAEPVVIVNG IGGLAVYTIQILKALMKNITIVGISRSKKHRDFALVLGADYVSEMKDAESLINKLTDGLGAS IAIDLVGTEETTYNLGKLLAQEGAIILVGMEGKRVSLEAFDTAVWNKKLLGSNYGSLNDLE DVVRLSESGKIKPYIIKVPLNDINKAFTNLDEGRVDGRQVITP

[0182] CR03

[0183] MANPTVIKAQDGNVMPQLGLGVWQASNEEVITAIQKALEVGYRSIDTAAAYKNEEGVGKAL KNASVNREELFITTKLWNDDHKRPREAGLDSLKKLQLDYIDLYLMHWPVPAIDHYVEAWKG MIELQKEGLIKSIGVCNFQIHHLQRLIDETGVTPVINQIELHPLMQQRQLHAWNATHKIQTE SWSPLAQGGKGVFDQKVCRDLADKYGKTPAQIVIRWHLDSGLVVIPKSVTPSRIAENFDW DFRLDKDELGEIAKLDQGKRLGPDPDQFGG

[0184] CR04

[0185] MVWLANPERYGQMVYRYCGKSGLRLPALSLGLWHNFGHVNALESQRAILRKAFDLGITHFDLANNYGPPPGSAEENFGRLLREDFAAYRDELIISTKAGYDMWPGPYGSGGSRKYLLASLDQSLKRMGLEYVDIFYSHRVDENTPMEETASALATAVQSGKALYVGISSYSPERTQKMVELLREWKIPLLIHQPSYNLGNRWVDKSGLLDTLQNNGVGCIAFTPLAQGLLTGKYLNGIPQDSRMHREGNKVRGLTPKMLTEANLNSLRLLNEMAQQRGQSMAQMALTWLLKDDRVTSVLIGASRAEQLEENVQALNNLTFSTKELAQIDQHIADGELNLWQASSDK

[0186] CR05

[0187] MSYQSLKNKCVIVTGAGSGIGRAIAKKFALNDSIVVAVELLEDRLNQIVQELRGMGKEVLGVKADVSKKKDVEEFVRRTFETYSRIDVLCDNAGIMDGVTPVAEVSDELWERVLAVNLYSAFYSSRAVIPIMLKQGKGVIVNTASIAGIRGGFARAPYTVAKHGLIGLTRSIAAHYGDQGIRAVAVLPGTVKTNIGLGSSKPSELGMRTLTKLMSLTSRLAEPEDIANVIVFLASDEASFVNGDAVVVDGGLTVL

[0188] CR06

[0189] MKVAVITGASRGIGEAIARALARDGYALALGARSVDRLEKIAHELMQEQGVEVFYHHLDVSKAESVEEFSKKVLERFGDADVVVANAGLGYFKRLEELSEEEFHEMIEVNLLGVWRTLKAFLDSLKRTGGLALVTTSDVSARLIPYGGGYVSTKWAARLLVRTFQIENPDVRFFELRPGAVDTYFGGSKPGKPKEKGYLKPNEIAEAVRCLLKLPKDVRVEELMLRSVYQRPEY

[0190] CR07

[0191] MKAIAVKRGEDRLVVIEKPRPEPESGEALVRTLRVGVDGTDHEVIAGGHGGFPEGEDHLVLGHEAVGVVVDPNDTELEEGDIVVPTVRRPPASGTVEYFERDQPDMAPDGMYFERGIVGAHGYMSEFFTSPEKYLVRIPRSQAELGFLIEPISITEKALEHAYASRSAFDWDPSSAFVLGNGSLGLLTLAMLKVDDKGYENLYCLGRRDRPDPTIDIIEELDATYVDTRQTPVEDVPDVYEQMDFIYEATGFPKHAIQSVQALAPNGVGALLGVPSDWAFEVDAGAFHREMVLHNKALVGSVNSHVEHFEAATVTFTKLPRWFLEDLVTGVHPLSEFEAAFDDDDTTIKTAIEFSTV

[0192] CR08

[0193] MSSTLFITGATSGFGEACLRRFAEAGWSLVLTGRREERLQALAGELSAKTRVLPLTLDVRDRAAMSAAVDNLPEEFATLRPLINNAGLALGTDPAQSCDLDDWDTMVDTNIKGLLYSTRLLLPRLIAHGAGASIVNLGSVAGKWPYPGSHVYGGTKAFVEQFSLNLRCDLQGTGVRVTNLEPGLCESEFSLVRFGGDQARYDKTYAGAHPIQPENIAETIFWIMNQPAHLNINSLEIMPVSQSWAGFAIHRES

[0194] CR09

[0195] MVFQHDIYAGQQVLVTGGSSGIGAAIAMQFAELGADVVALGLDADGVHAPRHPRIRREELDITDSQRLQRLFEALPRLDVLVNNAGISRDREEYDLASFERVLRLNLSAAMLASQLARPLLAQRGGSILNIASMYSTFGSADRPAYSISKGAIVQLTRSLACEYAAERIRVNAIAPGWIDTPLGAGLKADVEATRRIMQRTPLARWGEAPEVASAAAFLCGPGASFVTGAVLAVDGGYLCA

[0196] CR10

[0197] MKPYDLSEAVAVFTGGSSGIGLATVELLLEAGAAVAFCARDGERLRAAESALRQRFPGARLFASVCDVLDALQVRAFAEACERTLGCASILVNNAGQGRVSTFAETTDEAWSEELQLKFFSVIHPVRAFLPQLESRADAALVCVNSLLASQPEPHMVATSAARAGVKNLVRSMGFEFAPKGVRVNGILIGLVESGQWRRRFEAREERELDWAQWTAQLARNKQIPLGRLGKPLEAARAILFLASPLSAYTTGSHIDVSGGLSRHA

[0198] CR11

[0199] MIRDTLHDLHRPIGDTGLAVSPLGLGTVKFGRDQGVKYPSGFTIPDDREAADLLALARDLGINLIDTAPAYGRSEERLGPLLRGQREHWVIVSKTGEEFVDGQSVFDFSAAHTRRSVERSLKRLDTDRIELVLVHSDGNDLDILENSEVYPTLAALKREGLIGAYGLSGKTVEGGLRALREGDCAMVTYNLNERAERPVIEYAAAHAKGILVKRALASGHACLGAGQDPVRASFELVFDQPGVAAAIVGTINPLHLAHNVAMAAQALKKA

[0200] CR12

[0201] MTSQINRQYQLAQRPSGLPGRDTFSWVETPLGEPAEGQILVKNEYLSLDPAMRGWMNDARSYIPPVGIGEVMRALGVGKVLVSKHPGFQAGDYVNGALGVQDYFIGEPKGFYKVDPSRAPLPRYLSALGMTGMTAYFALLDVGNPKNGETVVISGAAGAVGSVAGQIARLKGCRVVGIAGGAEKCRFLVEELGFDGAIDYKNEDLAAGLKRECPKGIDVFFDNVGGEILDTVLTRIAFKARIVLCGAISQYNNKDAVRGPANYLSLLVNRARMEGMVVMDYAQRFPEGLKEMATWLAEGKLQSKEDIVEGLETFPETLLKLFSGENFGKLVLKV

[0202] CR13

[0203] MTRSALVTGITGQEGAYLAKLLLEKGYRVHGLVARRSSDTRWRLRELGIEGDIQYEDGDMADACSVQRAVIKAQPQEVYNLAAQSFVGASWNQPVTTGVVDGLGVTHLLEAIRQFSPETRFYQASTSEMFGLIQAERQDELTPFYPRSPYGVAKLYGHWITVNYRESFGLHASSGILFNHESPLRGIEFVTRKVTDGVARIKLGKQQELRLGNVDAKRDWGFAGDYVEAMWLMLQQDKADDYVVATGVTTTVRDMCQIAFEHVGLDYRDFLKIDPAFFRPAEVDVLLGNPAKAQRVLGWKPRTSLDELIRMMVEADLKRVSRE

[0204] CR14

[0205] MTGLLDGKRIIVSGIITDSSIAFHIARVAQEQGAQLVLTGFDRLRLIQRITDRLPAKAPLLELDVQNEEHLASLAGRVTEAIGAGNKLDGVVHSIGFMPQTGMGINPFFDAPYADVSKGIHISAYSYASMAKALLPIMNPGGSIVGMDFDPTRAMPAYNWMTVAKSALESVNRFVAREAGKYGVRSNLVAAGPIRTLAMSAIVGGALGEEAGANIQLLEEGWDQRAPIGWNMKDATPVAKTVCALLSDWLPATTGDIIYADGGAHTQLL

[0206] CR15

[0207] MRVGIPTETKNNEFRVAITPAGVAELTRRGHEVLIQAGAGEGSAITDADFKAAGAQLVGTADQVWADADLLLKVKEPIAAEYGYLRHGQILFTFLHLAASRACTDALLDSGTTSIAYETVQTADGALPLLAPMSEVAGRLAAQVGAYHLMRTQGGRGVLMGGVPGVEPADVKVIGAGTAGYNAARIANGMGATVTVLDINIDKLRQLDAEFCGRIHTRYSSAYELEGAVKRADLVIGAVLVPGAKAPKLVSNSLVAHGKPGAVLVDIAIDQGGCFEGSRPTTYDHPTFAVHDTLFYCVANMPASVPKTSTYALTNATMPYVLELADHGWRAACRSNPALAKGLSTHEGALLSERVATDLGVPFTEPASVLA

[0208] CR16

[0209] MKKRQLMTSDLHVSELGFGCMSLGTDETKARRIMDEVLELGINYLDTADLYNQGLNEQFVGKALKGRRQDIILATKVGNRFEQGKEGWWWDPSKAYIKEAVKDSLRRLQTDYIDLYQLHGGTIDDPIDETIEAFEELKQEGVIRYYGLSSIRPNVIKEYLKRSNIVSIMMQYSILDRRPEEWFPLIQEHGVSVVVRGPVARGLLSRRPLPEGEGYLNYRYDELKLLRESLPTDRPLHELALQYCLAHDVVATVAAGASSLDQVKANVQAVEATPLTAEERQHIQKLAKAAVYEQHRE

[0210] CR17

[0211] VKEVFDLRGRVALVTGGSRGLGFGIAQGLAEAGCSVVVASRNLEEASEAAQKLTEKYGVETMAFRCDVTNYEEVKKLLEAVKEKFGKLDTVVNAAGINRRHPAEDFPLDEFRQVIEVNLFGTYYVCREAFSLLRESENPSIINIGSLTVEEVTMPNISAYAASKGGVASLTKALAKEWGRYGIRVNVIAPGWYRTKMTEAVFSDPEKLDWMLKRIPLGRTGVPEDLKGVAVFLASEEAKYVTGQIIFVDGGWTAN

[0212] CR18

[0213] MQFTGKNVLISGASKGIGAEIAKTLASMGLKVWINYRSNAEVADALKNELEEKGYKAAVIKFDAASESDFIEAIQTIVQSDGGLTYLVNNAGVVRDKLAIKMKTEDFHHVIDNNLTSAFIGCREALKVMSKSRFGSVVNVASIIGDRGNMGQTNYSASKGGMIAMSKSFAYEGALRNIRFNSVTPGFIETDMNANLKDELKADYVKNIPLNRLGSAKEVAEAVAFKLSDHSSYITGETLKVNGGLYM

[0214] CR19

[0215] MSFFHASNRDALNQSLAEVQGQINVSFEFFPPRTSEMEQTLWNSIDRLSSLKPKFVSVTYGANSGERDRTHSIIKGIKDRTGLEAAPHLTCIDATPDELRTIARDYWNNGIRHIVALRGDLPPGSGKPEMYASDLVTLLKEVADFDISVAAYPEYHPEAKSAQADLLNLKRKVDAGANRAITQFFFDVESYLRFRDRCVSAGIDVEIIPGILPVSNFKQAKKFADMTNVRIPAWMAQMFDGLDDDAETRKLVGANIAMDMVKILTREGVKDFHFYTLNRAEMSYAICHTLGVRPGL

[0216] CR20

[0217] LNHPDLVGKVAIVTGAGAGIGLAVARRLADEGCHVLCADIDGDAADAAATKIGCGAAACRVDVSDEQQIIAMVDACVAAFGGVDKLVANAGVVHAASLIDTTVEDFDRVIAINLRGAWLCTKHAAPRMIERGGGAIVNLSSLAGQVAVGGTGAYGMSKAGIIQLSRITAAEIRSSGIRSNTLLPAFVDTPMQQTAMAMFDGALGAGGARSMIARLNGRMAAPEEMAGIVVFLLSDDASMITGTTQIADGGTIAALW

[0218] CR21

[0219] VPTSLKDTVKLHNGVEMPWFGLGVFKVENGNEATESVKAAIKNGYRSIDTAAIYKNEEGVGIGIKESGVAREELFITSKVWNEDQGYETTLAAFEKSLERLQLDYLDLYLIHWPGKDKYKDTWRALEKDYKDGKIRAIGVSNFQVHHLEELLKDAEIKPMVIQVEFHPRLTQKELRDYCKGQGIQLEAWSPLMQGQLLDNEVLTQIAEKHNKSVAQVILRWDLQHGVVTIPKSIKEHRIIENADIFDFEISQEDMDKIDALNKDERVGPNPDELLF

[0220] CR22

[0221] LSKRTAFVMGLSQGIGKAIALKLADQHFSLVINSRNLDNIESVKEDILAKHPEASVIVLAGDMSDQHTRAGIFQKIESQCGRLDVLINNIPGGAPDTFDNCNIEDMTATFTQKTVAYIDAIKRASSLMKQNEFGRIINIVGNLWKEPGANMFTNSMMNADLINASKNISIQLAPHNITVNCLNPGFIATDRYHQFVENVMKKASISKQKAEEQIASGIPMKRVGSAEETAALAAFLASEEASYITGQQISADGGSMKSI

[0222] CR23

[0223] MDFSGKNVWVTGAGKGIGYATALAFVEAGAKVTGFDQAFTQEQYPFATEVMDVADAAQVAQVCQRLLAETERKDALVNAAGILRMGATDQLSKEDWQQTFAVNVGGAFNLFQQTMNQFRRQRGGAIVTVASDAAHTPRIGMSAYGASKAALKSLALSVGLELAGSGVYCNVVSPGSTDTDMQRTLWVSDDAEEDRIRGFGEQFKLGIPLGKIARPQEIANTILFLASDLASHITLQDIVVDGGSTLGA

[0224] CR24

[0225] MRVLVTGGSGYIGSMTCVQLLQNGHDVIILDNLCNSKRSVLPVIERLGGKHPTFVEGDIRNEALMTEILHDHAIDTVIHFAGLKAVGESVQKPLEYYDNNVNGTLRLISAMRAANVKNFIFSSSATVYGDQPKIPYVESFPTGTPQSPYGKSKLMVEQILTDLQKAQPDWSIALLRYFNPVGAHPSGDMGEDPQGIPNNLMPYIAQVAVGRRDSLAIFGNDYPTEHGTGVRDYIHVMDLADGHVVAMEKLANKPTVHIYNLGAGVGNSVLDVVNAFSKACGKPVNYHFAPRREGDLPAYWADASKADRELNWRVTRTLDEMAQDTWHWQSRHPQGYPD

[0226] CR25

[0227] MTLQGKVAVVYGGSRGIGRDIAINLAKEGANIFFNYNGSPEAAEETAKLVAEHGVEVEAMKANVAIAEDVDAFFKQAIERFGRVDILVNNAGITRDNLLMRMKEDEWDDFININLKGTFLCTKAVSRTMMKQRAGKIINMASVVGLIGNAGQANYVASKAGVIGLTKTTARELAPRGINVNAVAPGFITTDMTDKLDRKTKEAMLAQIPLGAYGTTEDIANAVLFLASDASKYITGQTLSVDGGMVM

[0228] CR26

[0229] MIIVTGGAGFIGSGIVKALNDKGITDILVVDNLKDGTKFVNLVDLNIADYMDKEDFLIQIMAGEEFGDVEAIFHEGACSSTTEWDGKYMMDNNYQYSKELLHYCLEREIPFLYASSAATYGGRTSDFIESREYEKPLNVYGYSKFLFDEYTRQILPEANSQIVGFRYFNVYGPREGHKGSMASVAFHLNTQLNNGESPKLFEGSENFKRDFVYVGDVADVNLWFLENGVSGIFNLGTGRAESFQAVADATLAYHKKGQIMYIPFPDKLKGRYQAFTQADLTNLRAAGYDKPFKTVAEGVTEYMAWLNRDA

[0230] CR27

[0231] MSKQRVFIAGHRGMVGSAIRRQLEQRGDVELVLRTRDELNLLDSRAVHDFFASERIDQVYLAAAKVGGIVANTTYPADFIYQNMMIESNIIHAAHQNDVNKLLFLGSSCIYPKLAKQPMAESELLQGTLEPTNEPYAIAKIAGIKLCESYNRQYGRDYRSVMPTNLAGPHDNFHPSNSHVIPALLRRFHEATAQNAPDVVVWGSGTPMREFLHVDDMAAASIHVMELAHEVWLENTQPMLSHINVGTGVDCTIRELGQTIAKVVGYKGRVVFDASKPDGTPRKLLDVTRLHQLGWYHEISLEAGLASTYQWFLENQDRFRG

[0232] CR28

[0233] MVQRITIAPQGPEFSRFVMGYWRLMDWNMSARQLVSFIEEHLDLGVTTVDHADIYGGYQ CEAAFGEALKLAPHLYERMEIVSKCGIATTAREENVIGHYITDRDHIIKSAEQSLINLATDH LDLLLIHRPDPLMDADEVADAFKHLHQSGKVRHFGVSNFTPAQFALLQSRLPFTLATNQ VEISPVHQPLLLDGTLCQLQQLRVRPMAWSCLGGGRLFNDDYFQPLRDELAVVAEELN AGSIEQVVYAWVLRLPSQPLPIIGSGKIERVRAAVEAETLKMTRQQYFRIRKAALGYD VP

[0234] CR29

[0235] MQYHRIPHSSLEVSTLGLGTMTFGEQNSEADAHAQLDYAVAQGINLIDVAEMYPVPPRPET QGLTETYVGNWLAKHGSREKLIIASKVSGPSRNNDKGIRPDQALDRKNIREALHDSLK RLQTDYLDLYQVHWSQRPTNCFGKLGYSWTDSAPAVSLLDTLDALAEYQRAGKIRYIG VSNETAFGVMRYLHLADKHDLPRIVTIQNPYSLLNRSFEVGLAEVSQYEKVELLAYS CLGFGTLTGKYLNGAKPALARNTLFSRFTRYSGEQTQKAVAAYVDIARRHGLDPAQMA LAFVRRQPFVASTLLGATTMDQLKTNIESLHLELSEDVLAEIEAVHQVYTYPAP

[0236] CR30

[0237] MTVRVAINGFGRIGRNVVRALYESGRRAEITVVAINELADAAGMAHLLKYDTSHGRFAWEVRQERDQLFYGDDAIRVLHERSLQSLPWRELGVDVVLDCTGVYGSREHGEAHIAAGAKKVLFSHPGSNDLDATVVYGVNQDQLRAEHRIVSNASCTTNCIIPVIKLLDDAYGIESGTVTTIHSAMHDQQVIDAYHPDLRRTRAASQSIIPTDTKLAAGITRFFPQFNDRFEAIAVRVPTINVTAIDLSVTVKKPVKANEVNLLLQKAAQGAFHGIVDYTELPLVSQDFNHDPHSAIVDGTQTRVSGAHLIKTLVWCDNEWGFANRMLDTTLAMATVAFR

[0238] The foregoing description of the examples has been presented for the purposes of conduction and enabling those of ordinary skill in the art to make and use the application. Modifications to, and variations of, the examples described herein will be apparent to those of ordinary skill in the art and can be made without departing from the scope or spirit of the application. The application is not limited to the examples described above, but is amenable to modification and alteration unattaining the scope of the application. Accordingly, this application is intended to embrace all such alterations, modifications, and variations that fall within the scope of the application.

Claims

1. A method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases, characterized in that, A high-throughput rapid determination of kinetic constants of a plurality of carbonyl compounds in a substrate library using a target carbonyl reductase, comprising the following steps: (1) obtaining a crude enzyme solution containing the target carbonyl reductase; (2) determining the protein concentration of the target carbonyl reductase in the crude enzyme solution obtained in step (1) using a high-throughput method; (3) using the crude enzyme solution obtained in step (1) to catalytically reduce a plurality of carbonyl compounds in a substrate library, and high-throughput rapid determination of the catalytic activity of the carbonyl reductase; (4) According to the activity of the carbonyl reductase catalyzing the reduction of different concentrations of substrates determined in step (3) and the protein concentration of the target carbonyl reductase determined in step (2), the kinetic parameters V max and K M of the carbonyl reductase are calculated by software fitting. (5) The catalytic efficiency constant kcat / Km was calculated from the kinetic parameters Vmax and Km of the carbonyl reductase max and the molar concentration of the respective carbonyl reductase. cat and the molar concentration of the respective carbonyl reductase. cat M .​ 2. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 1, characterized in that, In step (2), the determination of the protein concentration of the target carbonyl reductase in the crude enzyme solution includes the following steps: (2.1) Take a certain volume of crude enzyme solution and perform SDS-polyacrylamide gel electrophoresis; (2.2) Using the concentration of standard molecular weight protein markers as a standard, the concentration of the target carbonyl reductase in the crude enzyme solution (cell-free extract) is quantitatively calculated using gray scale analysis strategy.

3. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 1, characterized in that, In step (3), the high-throughput rapid determination of the catalytic activity of the carbonyl reductase is carried out in a 96-well plate on an enzyme marker instrument, including the following steps: (3.1) Add buffer, stock solution of a plurality of carbonyl compounds in a substrate library, coenzyme NADH or NADPH stock solution, and enzyme concentrated stock solution into the holes of the 96-well plate in different volumes; (3.2) After the addition of reagents, the 96-well plate was placed on the microplate reader, briefly shaken to mix, and the enzymatic reaction was carried out. The characteristic absorbance value A of the cofactor NADH or NADPH at 340 nm was recorded 340 (t) = ε x C(t) x l, where ε is the molar absorption coefficient of NADH or NADPH, taken as 6220 cm-1L-1mol-1 340 ; l is the depth of the solution in the hole of the enzyme plate, unit: cm; through data fitting, the time function C(t) at a certain time point is obtained -1 ; (3.3) The rate of change of coenzyme concentration as a function of time C(t) is determined, which is equal to the instantaneous rate of the enzymatic reaction of the carbonyl substrate, denoted as the initial reaction rate V(t), V(t) = -dC(t) / dt = -[1 / (εxl)]-dA 340 (t) / dt, the initial reaction rates V(0) corresponding to different carbonyl substrate concentrations are determined i , and the corresponding kinetic constants K M are fitted according to the Michaelis equation. max , and the corresponding kinetic constants K cat , and the corresponding kinetic constants K cat = V max / [E]0, wherein [E]0is the molar concentration of the carbonyl reductase corresponding to the determination of the kinetic constants.

4. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 3, characterized in that, In step (3.2), the enzymatic reaction is carried out in the 96-well plate on the enzyme marker instrument; the reaction volume of each hole is 200 μL, containing 140 μL buffer, 20 μL coenzyme stock solution NADH or NADPH, 20 μL carbonyl reductase solution, 1-20 μL substrate concentrated solution dissolved in DMSO, and the rest volume is made up with DMSO; all solutions are pre-incubated at 30°C, after adding the materials, the 96-well plate is placed on the enzyme marker instrument, shaken briefly to mix, and the enzymatic reaction is carried out, and the change of absorbance value of coenzyme NADH or NADPH at 340 nm during the reaction is tracked in time to calculate the rate of enzyme reaction.

5. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 1, characterized in that, In step (2), the protein concentration of a plurality of different target carbonyl reductases obtained in step (1) is determined using a high-throughput method, including the following steps: Using E. coli BL21(DE3) as the host and plasmid pET 28a as the vector, different target carbonyl reductases are recombinantly expressed: the nucleic acid sequence of the carbonyl reductase is linked to the multiple cloning site of pET 28a to construct a recombinant expression plasmid, which is transformed into E. coli BL21(DE3) to construct a series of recombinant strains E. coli BL21 / pET 28a-CR; The recombinant strain E. coli BL21 / pET 28a-CR was inoculated into a triangular flask containing LB medium and cultured to OD 600 1.0, then subcultured into a triangular flask containing fresh LB medium at a ratio of 1% (v / v) and cultured to OD 600 0.6, isopropyl-β-D-thiogalactoside was added, shaking was continued, the bacterial cells were harvested by centrifugation, resuspended in potassium phosphate buffer, broken by ultrasonic, the broken solution was centrifuged to remove cell debris, and stored at 4°C for short-term use. The total protein concentration of the crude enzyme solution is determined using the Bradford protein concentration determination method, and three groups of parallel samples are set for each carbonyl reductase. The crude enzyme solution corresponding to 30 kinds of carbonyl reductases is added to the holes of the 96-well plate, a total of 90 sample holes, and 100 μL of enzyme solution is added to each hole. The remaining 6 holes are used as blank controls, and 100 μL of deionized water is added to each hole. After adding the sample, 100 μL of Coomassie Brilliant Blue R250 staining solution is added to each hole, incubated at 25°C for 5 min, and then placed on the enzyme marker instrument to read the absorbance value at 595 nm. The obtained carbonyl reductase crude enzyme solution is subjected to SDS-PAGE protein electrophoresis separation, a standard molecular weight protein is used as a protein standard, a Coomassie brilliant blue staining method is used to stain the protein gel obtained after electrophoresis, a gel imaging instrument is used to scan the protein gel image, ImageJ software is used to analyze the gray scale of the protein staining bands, a known concentration of protein molecular weight standard is used as a standard curve, and the protein concentration of the target carbonyl reductase in the crude enzyme solution is quantified.

6. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 1, characterized in that, In step (3), the carbonyl compounds in the substrate library include aldehyde compounds, aliphatic ketones, alicyclic ketones, heterocyclic ketones, monocyclic aromatic ketones, bis-aromatic ketones, aliphatic ketone esters, aromatic ketone esters, and aryl alicyclic ketones.

7. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 6, characterized in that, The aldehyde compounds include compounds S01-S08, the structures of which are as follows: The aliphatic ketones include compounds S09-S18, the structures of which are as follows: The alicyclic ketones include compounds S19-S25, the structures of which are as follows: The heterocyclic ketones include compounds S26-S34, the structures of which are as follows: The monocyclic aromatic ketones include compounds S35-S60, the structures of which are as follows: The bis-aromatic ketones include compounds S61-S68, the structures of which are as follows: The aliphatic ketone esters include compounds S69-S77, the structures of which are as follows: The aromatic ketone esters include compounds S78-S83, the structures of which are as follows: The aryl alicyclic ketones include compounds S84-S90, the structures of which are as follows:

8. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 7, characterized in that, In the step (3), the substrates are added to the 96-well plate in the following order, with 6 holes reserved as blank controls, Blk1, Blk2; Blk1 is Control 1: only coenzyme, carbonyl reductase and buffer are added, without adding carbonyl substrate solution, but its equal volume of auxiliary solvent is replaced; Blk2 is Control 2: only coenzyme (NADH / NADPH) and buffer are added, without adding carbonyl substrate and carbonyl reductase.

9. The method for high-throughput rapid determination of kinetic constants for characterizing substrate complementarity of carbonyl reductases according to claim 1, characterized in that, In step (4), the concentration of the carbonyl substrate was selected to be a typical concentration of 1.0 mM to 20.0 mM, and the initial concentration of the coenzyme was 0.1 mM of NADH or NADPH; the concentration of the enzyme solution was diluted so that the rate of the enzyme reaction was controlled to be 0.005 to 0.02 mM min -1 .

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