Carboxylesterase, coding gene, recombinant vector, recombinant strain, enzyme preparation and application of carboxylesterase and coding gene, recombinant vector, recombinant strain and enzyme preparation

Abstract

The invention relates to the technical field of enzyme engineering and biology, and discloses carboxylesterase as well as a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application thereof. The carboxylesterase (GutestA) has an amino acid sequence as shown in SEQ ID NO. 1 (sequence identifier number 1). When the GutestA takes an acid ester compound as a substrate, the GutestA is high in specific enzyme activity, high in thermal stability, wide in pH application range and high in catalytic efficiency, and has a good industrial application prospect in the fields of drug synthesis, industrial catalysis, chiral compound resolution, grease processing, environmental governance and the like.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering and biotechnology, and in particular to a carboxylesterase and its encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation and applications thereof. Background Art

[0002] As a complex microbial community that colonizes the gastrointestinal tract of animals, the intestinal flora is closely related to the health of the host. Its core functions cover several key aspects: at the nutritional metabolism level, it can decompose and metabolize complex nutrients, helping the host to absorb and utilize various nutrients; in terms of the synthesis of bioactive substances, it can produce short-chain fatty acids (SCFAs), neurotransmitter precursors, etc., which play an important role in regulating the host's metabolism and improving thinking ability; in the field of physiological regulation, with the help of multi-organ interaction networks such as the gut-liver axis and the gut-brain axis, it can accurately regulate the host's energy metabolism and immune homeostasis, and is a key factor in maintaining intestinal barrier function and immune tolerance. For this reason, the intestinal flora is regarded as a "second genome" co-evolved with the host, which contains a huge number of genetic resources with diverse functions. However, current research on the intestinal flora still has major limitations. Among the sequenced intestinal bacteria, only about 21% (400 / 1952 species) can be achieved in pure culture in vitro. More than 80% of the bacterial species are still in the state of "microbial dark matter" that has not been fully studied due to limitations in culture technology and other reasons. The large number of functional genes carried by these unknown bacterial species also need to be analyzed.

[0003] Carboxylesterases play an indispensable role in numerous fields. They specifically catalyze the hydrolysis of carboxylate ester bonds, breaking down carboxylates into carboxylic acids and alcohols in the presence of water molecules. In the biopharmaceutical field, carboxylesterases can be used to activate prodrugs, converting inactive or low-activity prodrugs into therapeutically effective drugs, thereby enhancing their efficacy. In the fine chemical industry, they play a key role in the synthesis of chiral compounds, enabling the preparation of compounds with specific chiral configurations through precise catalytic reactions. In environmental remediation, they can effectively degrade pollutants such as organophosphorus pesticides, making them of great significance for environmental protection.

[0004] Currently, carboxylesterases are primarily derived from plants, animals, and microorganisms. However, these carboxylesterases suffer from low expression levels and difficulty in purification, limiting their large-scale industrial application. In contrast, microbial carboxylesterases offer significant advantages, such as high genetic plasticity and robust stress tolerance, making them a key research target for industrial development. The challenge is to identify carboxylesterases that possess excellent heat resistance and pH stability, enabling them to stably catalyze under the complex conditions of high temperature and varying pH levels encountered in chemical production, while also possessing long service life and good immobilization potential to meet the demands of industrial applications. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of low expression level and great purification difficulty of animal and plant-derived carboxylesterases in the prior art, as well as the difficulty of simultaneously meeting the requirements of industrial application in terms of thermal stability, pH stability and substrate specificity of the discovered microbial-derived carboxylesterases. The present invention provides a new carboxylesterase derived from mouse intestinal flora and its encoding gene, recombinant vector, recombinant strain, enzyme preparation and their applications. The carboxylesterase has good heat resistance and pH stability, exerts a stable catalytic effect under complex conditions of high temperature and different pH values, has a long service life, and also has good immobilization potential. It has good industrial application prospects in the fields of pharmaceutical synthesis, industrial catalysis, chiral compound separation, oil processing, environmental management, and degradation of ester compounds.

[0006] In order to achieve the above object, the present invention provides a carboxylesterase in one aspect, wherein the carboxylesterase is the enzyme described in (a) to (c): (a) an enzyme having an amino acid sequence as shown in SEQ ID NO. 1; (b) an enzyme represented by an amino acid sequence having 70% or more homology to the amino acid sequence represented by SEQ ID NO. 1 and having carboxylesterase activity; (c) A derivative enzyme obtained by modifying the enzyme having the amino acid sequence shown in SEQ ID NO. 1 and having the same activity as the enzyme before modification.

[0007] Preferably, the carboxylesterase is an enzyme represented by an amino acid sequence having 80% or more, preferably 95% or more, further preferably 98% or more, and most preferably 99% or more homology with the amino acid sequence represented by SEQ ID NO. 1, and having carboxylesterase activity.

[0008] Preferably, the transformation includes: i. at least one of substitution, deletion and addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1; and / or ii. A tag for facilitating purification is connected to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 1; and / or iii. A signal peptide sequence that is beneficial to the secretory expression of the enzyme is connected to the amino terminus of the amino acid sequence shown in SEQ ID NO. 1.

[0009] Preferably, the tag that facilitates purification is at least one selected from Poly-Arg, Poly-His, FLAG, Strep-tag II and c-myc, more preferably Poly-His.

[0010] The second aspect of the present invention provides a gene encoding carboxylesterase, wherein the gene has a nucleotide sequence encoding the carboxylesterase as described above.

[0011] Preferably, the gene has a homology of 70% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.

[0012] Preferably, the gene is the nucleotide sequence shown in SEQ ID NO. 2.

[0013] The third aspect of the present invention provides a recombinant vector comprising the gene as described above.

[0014] Preferably, the expression vector of the recombinant vector is a PBAD vector.

[0015] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0016] Preferably, the expression strain of the recombinant strain is Escherichia coli BL21.

[0017] A fifth aspect of the present invention provides a method for preparing carboxylesterase, which comprises: inoculating the recombinant strain described above into a fermentation medium containing arabinose for fermentation culture.

[0018] In a sixth aspect, the present invention provides an enzyme preparation, which comprises the carboxylesterase prepared by the method described above.

[0019] In a seventh aspect, the present invention provides the use of at least one of the aforementioned carboxylesterase, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, and the carboxylesterase prepared by the aforementioned method in at least one of pharmaceutical synthesis, industrial catalysis, chiral compound resolution, oil and fat processing, environmental remediation, and degradation of ester compounds.

[0020] Preferably, the ester compound is selected from at least one of ethyl lactate, ethyl acetate, ethyl butyrate, ethyl valerate, amyl acetate, ethyl hexanoate and ethyl heptanoate.

[0021] Preferably, the ester compound is at least one of ethyl butyrate, ethyl valerate, pentyl acetate, ethyl hexanoate and ethyl heptanoate.

[0022] Preferably, the degradation conditions include: temperature of 60-95° C. and pH of 8.5-10.

[0023] Through the above-mentioned technical solution, the present invention discovered a novel, previously undiscovered carboxylesterase (GutestA) in the mouse intestinal microbiome through metagenomic sequencing and functional gene mining. This carboxylesterase, through annotation and molecular docking analysis, demonstrated activity against carboxyl esters. A recombinant strain capable of heterologously expressing GutestA was constructed through genetic engineering, and GutestA expression was purified by Ni-NTA affinity chromatography. In vitro experiments demonstrated that this carboxylesterase exhibited excellent heat resistance and pH stability, exhibiting stable catalytic activity under complex conditions of high temperature and varying pH levels, with a long service life. Furthermore, it possessed excellent immobilization potential, suggesting promising industrial applications in pharmaceutical synthesis, industrial catalysis, chiral compound resolution, oil and fat processing, environmental remediation, and ester degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the gene map of the recombinant vector PBAD-GutestA in Example 1; Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified carboxylesterase (GutestA) in Example 2, wherein lane M is a protein marker of 11 KDa-245 KDa, and lane 1 is the purified GutestA protein, which has a size of 35.4 KDa. Figure 3 is a linear relationship diagram of absorbance and the concentration of p-nitrophenol in the system in Example 3; Figure 4 is a graph showing the relationship between temperature and GutestA enzyme activity in the enzymatic property study in Example 4; Figure 5 is a graph showing the relationship between pH and GutestA enzyme activity in the enzymatic property study in Example 4; Figure 6 : is a histogram of enzyme substrate preferences in Example 5; the abscissa is different ester substrates (ethyl lactate, ethyl acetate, ethyl butyrate, ethyl valerate, amyl acetate, ethyl hexanoate, and ethyl heptanoate); the ordinate is the degradation rate, which is used to indicate the enzyme's ability to degrade these substrates; Figure 7 is a graph showing the thermal stability performance of carboxylesterase (GutestA) in Example 6; Figure 8 This is a graph showing the pH stability performance of carboxylesterase (GutestA) in Example 6. DETAILED DESCRIPTION

[0025] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0026] The first aspect of the present invention provides a carboxylesterase, which is the enzyme described in (a) to (c): (a) an enzyme having an amino acid sequence as shown in SEQ ID NO. 1; (b) an enzyme represented by an amino acid sequence having 70% or more homology to the amino acid sequence represented by SEQ ID NO. 1 and having carboxylesterase activity; (c) A derivative enzyme obtained by modifying the enzyme having the amino acid sequence shown in SEQ ID NO. 1 and having the same activity as the enzyme before modification.

[0027] The enzyme provided by the present invention can be a derivative enzyme obtained by modifying the above enzyme in any manner available in the art while maintaining the same activity. There is no particular limitation on the modification method and the specific sequence and properties of the modified derivative enzyme.

[0028] In the present invention, the enzyme may be an enzyme represented by an amino acid sequence having 70% or greater homology to the amino acid sequence represented by SEQ ID NO. 1 and having carboxylesterase activity. Preferably, the carboxylesterase is an enzyme represented by an amino acid sequence having 80% or greater homology, more preferably 95% or greater homology, even more preferably 98% or greater homology, and most preferably 99% or greater homology to the amino acid sequence represented by SEQ ID NO. 1 and having carboxylesterase activity.

[0029] According to a preferred embodiment of the present invention, the transformation may include: i. at least one of substitution, deletion and addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1; and / or ii. A tag for facilitating purification is connected to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 1; and / or iii. A signal peptide sequence that is beneficial to the secretory expression of the enzyme is connected to the amino terminus of the amino acid sequence shown in SEQ ID NO. 1.

[0030] The enzyme provided by the present invention can be obtained by subjecting the amino acid sequence shown in SEQ ID NO. 1 to any one or a combination of the above-mentioned modification methods i, ii and iii, as long as the modified derivative enzyme has the same activity as the amino acid sequence shown in SEQ ID NO. 1.

[0031] Any existing tag in the art that facilitates enzyme purification can be used with the carboxylesterase (GutestA) provided herein. Preferably, the tag facilitating purification is selected from at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc. The specific amino acid sequence of the tag is not particularly limited, and for example, may be the sequence shown in Table 1 below.

[0032] Table 1 Tag sequences for easy purification

[0033] *Poly-Arg can be composed of 5-6 arginine residues. Table 1 only lists the commonly used Poly-Arg tags composed of 5 arginine residues, but Poly-Arg tags composed of 6 arginine residues are also applicable to the present invention.

[0034] **Poly-His can be composed of 2-10 histidine residues. Table 1 only lists the commonly used Poly-His tags composed of 6 histidine residues, but Poly-His tags composed of 2-10 histidine residues are all applicable to the present invention.

[0035] Any signal peptide sequence that is available in the art and can facilitate enzyme secretion expression can be applied to the GutestA provided by the present invention. Preferably, the signal enzyme sequence is a basic amino terminal, an intermediate hydrophobic sequence, or a longer negatively charged C terminal.

[0036] This study, using metagenomic sequencing of mouse intestinal flora and a functional gene mining strategy, identified and cloned a novel carboxylesterase gene (named GutestA) and successfully cloned and expressed the enzyme through heterologous expression. Annotation and molecular docking analysis revealed that GutestA exhibited excellent catalytic activity in the hydrolysis of ester substrates and exhibited resistance to high temperatures and alkaline environments, suggesting promising industrial prospects.

[0037] The enzyme of the present invention is obtained using methods known in the art. It can be chemically synthesized using an automated enzyme synthesizer, or by deriving the nucleotide sequence from the GutestA amino acid sequence and then cloning it into a vector for biosynthesis, or by large-scale extraction and purification from existing organisms. The synthesis of GutestA of the present invention utilizes genetic engineering recombinant expression.

[0038] In the present invention, the separation and purification can be carried out by conventional enzyme separation methods in the art. kromasil C18-5 was purified and separated multiple times.

[0039] The second aspect of the present invention provides a gene encoding an enzyme, which has a nucleotide sequence encoding GutestA as described above.

[0040] According to the present invention, preferably, the gene has a homology of more than 70%, preferably more than 95%, more preferably more than 98%, and most preferably more than 99% with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.

[0041] According to the present invention, preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2. In the present invention, the gene may comprise only the sequence shown in SEQ ID NO. 2, or may be a DNA molecule comprising the sequence shown in SEQ ID NO. 2 as the coding region and additionally comprising other components. These additional components may include any components known in the art for artificially synthesizing gene sequences and expressing them via expression vectors, such as promoters, enhancers, Kozak sequences, and the like.

[0042] The nucleotide sequences provided herein can generally be obtained using polymerase chain reaction (PCR) amplification, recombinant methods, or synthetic methods. Once the nucleotide sequences are obtained, the amino acid sequences can be obtained in large quantities using recombinant methods. Furthermore, the nucleotide sequences can also be synthesized using known synthetic methods. The nucleotide sequence shown in SEQ ID NO. 2 described herein was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0043] A third aspect of the present invention provides a recombinant vector comprising the gene described above. In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as commercially available vectors, cosmids, phages, and retroviruses. The expression vector of the present invention is preferably a PBAD vector, resulting in the recombinant vector PBAD-GutestA.

[0044] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0045] The host cell can be a prokaryotic cell or a eukaryotic cell. According to the present invention, preferably, the expression strain of the recombinant strain is Escherichia coli. According to the present invention, the Escherichia coli is preferably Escherichia coli BL21 and Escherichia coli DE3, and more preferably Escherichia coli BL21.

[0046] In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation using the calcium chloride method or high-voltage electroporation transformation.

[0047] A fifth aspect of the present invention provides a method for preparing carboxylesterase, which comprises: inoculating the aforementioned recombinant strain into a fermentation medium containing arabinose for fermentation culture.

[0048] In the present invention, there is no particular limitation on the conditions for the fermentation of the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Exemplarily, the fermentation process includes: first randomly picking a single colony of the recombinant strain and inoculating it into an activation culture medium (the activation culture medium preferably contains: yeast extract 4-6 g / L, trypsin 8-12 g / L, NaCl 8-12 g / L), and shaking and culturing it at room temperature at a speed of 150-250 rpm for 8-12 h for activation culture; inoculating the strain after activation culture into a seed culture medium (the seed culture medium preferably contains: yeast extract 4-6 g / L, trypsin 8-12 g / L, NaCl 8-12 g / L) for culture, and when the bacterial liquid grows to OD 600 When the pH is 0.6, a fermentation medium containing arabinose (preferably containing 4-6 g / L yeast extract, 8-12 g / L tryptone, and 8-12 g / L NaCl) is added for induction. The culture is shaken at 150-250 rpm for 8-12 hours at room temperature. After incubation, the culture is centrifuged at 5000 rpm for 15 minutes at 2-6°C, and the supernatant is discarded to collect the cells. Prokaryotic cell lysis buffer (ACE) is added at a ratio of 1 mL / g of cell mass. Mix thoroughly by pipetting, and then let stand at 2-6°C for 4-6 minutes to completely lyse the recombinant strain. The lysate is then centrifuged at 12,000 × g for 0.8-1.2 hours. The supernatant is collected to obtain a liquid containing carboxylesterase (GutestA). The inventors have found that this fermentation process is beneficial for increasing the expression level of GutestA.

[0049] The GutestA provided by the present invention has good heat resistance and pH stability.

[0050] In a sixth aspect, the present invention provides an enzyme preparation, which comprises the carboxylesterase prepared by the method described above.

[0051] In the present invention, the GutestA can be made into a corresponding enzyme preparation. Specifically, the enzyme preparation can exist in a solid, semi-solid or liquid form. The enzyme preparation can contain auxiliary materials or additives for preparing the enzyme preparation. Those skilled in the art can select according to needs and will not be described in detail here.

[0052] In the present invention, the fermentation broth obtained by the above-mentioned preparation method can be used directly as an enzyme preparation, or the fermentation broth can be separated and purified to obtain carboxylesterase GutestA as an enzyme preparation, and the separation and purification can adopt the conventional protein separation method in this field. That is to say, carboxylesterase GutestA can be used in the form of whole cells of a recombinant strain, or it can be used in the form of a crude protein preparation or a purified protein preparation separated from recombinant strain cells and not purified. In the present invention, the 6×his tag can form a coordination bond with nickel ions and has a high affinity principle to achieve specific purification of GutestA. The whole process of purification is carried out on ice to prevent excessively high temperatures from denaturing the protein. After the nickel ion affinity medium is loaded into the column, the purified GutestA is obtained through steps such as protein binding, impurity protein washing, target protein elution, dialysis desalting, etc. The inventors have found that the enzyme preparation prepared in the above manner has a higher purity.

[0053] Based on the fact that the GutestA provided by the present invention has good heat resistance and pH stability, exerts a stable catalytic effect under complex conditions of high temperature and different pH values, has a long service life, and also has good immobilization potential, the seventh aspect of the present invention provides the use of at least one of the aforementioned GutestA, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the GutestA prepared by the aforementioned method, and the aforementioned enzyme preparation in at least one of pharmaceutical synthesis, industrial catalysis, chiral compound separation, oil processing, environmental governance, and degradation of ester compounds.

[0054] According to the present invention, preferably, the ester compound is selected from at least one of ethyl lactate, ethyl acetate, ethyl butyrate, ethyl valerate, amyl acetate, ethyl hexanoate, and ethyl heptanoate. Further preferably, the ester compound is at least one of ethyl butyrate, ethyl valerate, amyl acetate, ethyl hexanoate, and ethyl heptanoate.

[0055] According to the present invention, preferably, the degradation conditions include: temperature of 60-95° C. and pH of 8.5-10.

[0056] The present invention will be described in detail below through examples.

[0057] In the following examples, the gel recovery kit was purchased from Corning Life Sciences, Inc.; Escherichia coli BL21 was purchased from Beijing Qingke Biotechnology Co., Ltd.; the vector PBAD was purchased from Beijing Qingke Biotechnology Co., Ltd.; the prokaryotic cell lysate was purchased from Changzhou Boyi Biotechnology Co., Ltd.; the BCA (Keygen) protein quantification kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd.; and the remaining reagents and raw materials were conventional commercially available products.

[0058] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjust the pH to 7.0, steam sterilize under high pressure for 21 min, and set aside.

[0059] LB agar plate medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, adjust the pH to 7.0, steam sterilize under high pressure for 21 min, and set aside.

[0060] Example 1 Recombinant heterologous expression of GutestA The amino acid sequence of GutestA (shown in SEQ ID NO. 1) and the nucleotide sequence encoding the amino acid sequence (shown in SEQ ID NO. 2) were synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized DNA sequence was cloned into the pBAD vector. The pBAD vector was first double-digested with EcoRI and HindIII restriction sites. The components listed in Table 2 were added to 50 μL of the system and digested at 37°C for 4 hours.

[0061] Table 2

[0062] Prepare a 1% by weight agarose gel and run the digested product through gel electrophoresis. Single, bright bands should be present, with the correct size and position. Carefully excise the desired band from the gel using a blue light exciter. Use the Axygen® AxyPrep DNA Gel Extraction Kit to extract the product. After checking the purity of the product using a nanodrop, store it in a refrigerator at 4°C.

[0063] Using PCR, an overlap region matching the pBAD vector was added to the GutestA gene fragment. The GutestA gene fragment was amplified using the primers shown in Table 3 and the PCR system shown in Table 4.

[0064] Table 3

[0065] Table 4

[0066] PCR reactions were performed according to the standard protocol using PrimeSTAR DNA Polymerase (Takara) as specified in the manufacturer's instructions. After completion of the reaction, agarose gel electrophoresis was performed and the gel was excised and recovered. Gibson assembly of the GutestA gene fragment and the digested pBAD vector was performed using the ClonExpress Ultra One Step Cloning Kit V3 (Vazyme). The assembly system was as shown in Table 5, and the reaction was performed at 50°C for 40 min.

[0067] Table 5

[0068] The assembled recombinant vector was transformed into competent E. coli DH5α using the chemical transformation method. The plates were then coated with ampicillin. After overnight culture, transformants were picked and verified by PCR. Positive colonies were inoculated into liquid LB medium supplemented with 100 μg / mL ampicillin, cultured, and the recombinant vector was extracted. The recombinant fragments on the recombinant vector were verified by Sanger sequencing. At this point, the recombinant vector PBAD-GutestA was constructed (see Figure 1 ).

[0069] Example 2 Transformation of recombinant vectors The recombinant vector PBAD-GutestA constructed in Example 1 was chemically transformed into E. coli BL21. After verifying the positive colonies, a single colony was picked and cultured overnight in liquid LB medium supplemented with 100 μg / mL ampicillin. The next day, 1 mL of overnight culture seed solution was inoculated into 100 mL of liquid LB medium supplemented with ampicillin. When the bacterial solution grew to OD 600 When the kinetics of the expression of arabinose were 0.6, arabinose was added to a final concentration of 0.002 wt % for induction at 20°C and 220 rpm for 8 h.

[0070] After induction, the cells were collected by centrifugation at 5000 rpm for 15 min at 4°C. Prokaryotic cell lysis buffer (ACE) was added at a ratio of 1 mL / g of cell mass, mixed by pipetting, and allowed to stand at 4°C for 5 min to completely lyse the E. coli. The cells were centrifuged at 12000 × g for 1 h at 4°C, and the supernatant was collected for subsequent purification steps. In this study, the specific purification of GutestA was achieved by utilizing the principle that the 6×his tag can form a coordination bond with nickel ions and has high affinity. The entire purification process was performed on ice to prevent high temperatures from denaturing the protein. After loading the nickel ion affinity medium onto the column, the purified carboxylesterase was obtained through protein binding, washing of impurities, elution of the target protein, and dialysis for desalting.

[0071] The purity of the purified carboxylesterase was verified by SDS-PAGE electrophoresis. Figure 2 As shown. Figure 2 It can be seen that the purity of carboxylesterase is 85%.

[0072] The concentration of the purified enzyme protein was determined by BCA protein quantification kit (Kagi) and was 0.13 ng / μL.

[0073] This shows that the experimental method can effectively express and purify the target GutestA, providing a reliable material basis for the research and application of subsequent examples.

[0074] Example 3 Enzyme activity assay of GutestA Enzyme activity definition: Under the optimal reaction conditions, the amount of enzyme required to release 1 μM p-nitrophenol from 1 mM p-nitrophenyl butyl ester solution in 1 mL of reaction system within 1 min is one enzyme activity unit (U).

[0075] First, a standard curve was drawn with the concentration of p-nitrophenol as the horizontal axis and the absorbance of the system at 405 nm as the vertical axis to reflect the linear relationship between the absorbance of the system at 405 nm and the concentration of p-nitrophenol in the system (see Figure 3). Then, 50 μL of 20 mM butyl 4-nitrophenyl ester acetonitrile solution, 30 μL of purified GutestA (prepared in Example 2), and 920 μL of 50 mM TBS buffer at pH 7.0 were added to the 1 mL reaction system. A buffer without esterase protein was used instead of the esterase protein buffer as the control group to correct the effect of the natural degradation of butyl 4-nitrophenyl ester on the absorbance of the reaction system. After 5 minutes of reaction, the absorbance of the reaction system at 405 nm was measured using a UV spectrophotometer. Both the experimental group and the control group were repeated three times in parallel. According to the absorbance of the reaction system at 405 nm, the standard curve was regressed to calculate the amount of p-nitrophenol released in the system within 5 minutes.

[0076] The calculation formula for specific enzyme activity is:

[0077] The specific enzyme activity of GutestA was measured to be 2525.67 U / mg.

[0078] The in vitro experiments of this example show that GutestA has extremely high decomposition activity against ester compounds.

[0079] Example 4 Analysis of the Optimal Reaction Conditions of GutestA Butyl p-nitrophenyl was selected as the decomposition substrate for GutestA. Hydrolysis of butyl p-nitrophenyl produces p-nitrophenol and butyric acid, with p-nitrophenol exhibiting a characteristic UV peak at 405 nm. As the reaction proceeds, the system changes from colorless to yellow. Therefore, measuring the absorbance of the reaction system at 405 nm can reflect the amount of p-nitrophenol produced in the system and, in turn, determine the catalytic activity of the enzyme. First, the optimal reaction temperature of GutestA was determined at pH 8.0. Next, the optimal pH for its catalytic hydrolysis reaction was determined at 37°C. Experimental measurements revealed an optimal temperature of 85°C and an optimal pH of 9.5.

[0080] 4.1 Determination of the optimal temperature of GutestA Add the components shown in Table 6 to a 1 mL reaction system, and set up a set of reactions at 5°C intervals between 20°C and 100°C. Use a buffer solution without esterase as a control to correct the effect of the natural degradation of 4-nitrophenyl butyl ester on the absorbance of the reaction system. Preheat 4-nitrophenyl butyl ester and Tris-HCl buffer to the corresponding temperature in a water bath, quickly mix the reaction system, and measure the absorbance of the system at 405 nm after reacting in a water bath for 5 minutes. Perform three reactions in parallel for each temperature gradient, and calculate the average value. The enzyme activity at the optimal temperature is defined as 100%. The results are shown in Figure 2. Figure 4As shown in the figure, the optimum temperature of GutestA is 85℃. It has good enzyme activity in the range of 20℃-100℃. When the temperature is between 60℃-95℃, the enzyme activity remains above 60%.

[0081] The experiment of determining the optimum temperature of GutestA in this embodiment shows that GutestA is suitable for working in a high temperature environment.

[0082] Table 6

[0083] 4.2 Determination of the optimal pH of GutestA In order to facilitate the experimental operation and reflect the activity of GutestA, its optimal pH was determined at 45°C. Buffer solutions of pH 4-pH10.5 were prepared (trisodium citrate-disodium hydrogen phosphate buffer solution of pH 4.0-pH6.5, Tris-HCl buffer solution of pH 7-pH 9, sodium carbonate-sodium bicarbonate buffer solution of pH 9.5-pH 10.5). The concentrations of the three buffer solutions were all 50 mM. After preheating the reaction substrate and buffer solution to 45°C in a water bath, 50 μL of 20 mM butyl para-nitrophenyl ester acetonitrile solution, 30 μL of the purified GutestA solution prepared in Example 2, and 920 μL of each pH buffer solution were quickly added to 1 mL of the reaction system and mixed. After reacting for 5 minutes, the absorbance of the system at 405 nm was measured. Using a buffer solution without esterase as a control, three reactions were performed in parallel for each pH gradient, and the average value was calculated. The enzyme activity at the optimal pH was defined as 100%, and the results were shown as follows. Figure 5 As shown, GutestA has the highest activity at pH 9.5, and the enzyme activity remains above 50% of the maximum activity under pH 8.5-pH 10. This shows that GutestA is suitable for applications under alkaline conditions. The optimal pH experiment for GutestA in this example shows that GutestA has a wide pH adaptability.

[0084] This example demonstrates that GutestA has high activity over a wide range of temperature and pH.

[0085] Example 5 GutestA substrate specificity was analyzed using ester compounds of varying carbon chain lengths as substrates for enzymatic reactions. The degradation rates of GutestA for these substrates were determined by gas chromatography to investigate GutestA's preference for substrates of varying carbon chain lengths. These substrates included ethyl lactate (CH3CH(OH)COOCH2CH3), ethyl acetate (CH3COOCH2CH3), ethyl butyrate (CH3(CH2)2COOCH2CH3), ethyl valerate (CH3(CH2)3COOCH2CH3), amyl acetate (CH3COO(CH2)4CH3), ethyl hexanoate (CH3(CH2)4COOCH2CH3), and ethyl heptanoate (CH3(CH2)5COOCH2CH3). The following components were added to a 1 mL reaction system: ① 50 μL of a 20 mM solution of the corresponding ester, ② 30 μL of the purified GutestA solution (prepared in Example 2), and ③ 920 μL of 50 mM Tris-HCl buffer, pH 7. At the same time, enzyme-free buffer was used instead of protein solution as a control. After reacting at 37°C for 1 hour, gas chromatography was used to detect the remaining amount of corresponding esters and the degradation rate was calculated according to the control group. The gas chromatography column was SH-polarD, the injection port temperature was 250°C, the carrier gas was helium, the split ratio was 40 / 1, the injection volume was 1μL, the detector temperature was 250°C, the cross flow mode was 0.8 ml / min, and the column temperature program was 35°C starting, maintained at 35°C for one minute; then increased by 5°C per minute to 70°C; increased by 2°C per minute to 100°C; increased by 15°C per minute to 230°C, and maintained at 230°C for 6 minutes. The reaction of each substrate and the control group were carried out in parallel 3 times. The results are shown in the table below. Figure 6 It can be seen that GutestA tends to degrade carboxylates composed of long carbon chains, but still has good degradation activity for esters composed of short carbon chains.

[0086] Example 6 Enzymatic Stability Analysis of GutestA 6.1 Temperature stability analysis The GutestA prepared in Example 2 was dissolved in a Tris-HCl buffer solution at pH 8 to obtain an enzyme solution. After incubation at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C for 1 h, 5 μL of the enzyme solution was taken for enzyme activity reaction. The following components were added to the 100 μL reaction system: ① 5 μL of 20 mM p-nitrophenylbutyrate acetonitrile solution, ② 5 μL of enzyme solution after incubation at various temperatures, and ③ 90 μL of 50 mM Tris-HCl buffer solution at pH 7.0. At the same time, 50 mM TBS buffer without enzyme was used instead of the protein solution as a control. After reacting at 45°C for 5 minutes, the absorbance at 405 nm was measured using an enzyme reader. Three parallels were made for each temperature gradient and control group. The group with the highest enzyme activity was set to 100%, and the results were normalized. The results are shown in the figure. Figure 7 The results showed that after incubation at 80°C for 1 hour, GutestA's activity remained above 90%. After incubation at 100°C (where the protein solution was near boiling) for 1 hour, GutestA's activity in degrading p-nitrophenol butyrate decreased by only 13.87% compared to incubation at 4°C for 1 hour.

[0087] The temperature stability test in this example shows that GutestA can not only exert its maximum degradation activity in a high-temperature environment, but also has excellent thermal stability and has the potential to be applied in industrial catalysis and environmental governance.

[0088] 6.2 pH stability analysis The GutestA solution prepared in Example 2 was incubated at 45°C for 1 h in 50 mM sodium citrate-sodium bicarbonate buffer at pH 4, pH 4.5, pH 5, pH 5.5, pH 6, and pH 6.5; 50 mM Tris-HCl buffer at pH 7, pH 7.5, pH 8, and pH 8.5; and 50 mM Na2CO3-NaOH-NaCl buffer at pH 9, pH 9.5, pH 10, and pH 10.5. 5 μL of the enzyme solution was then sampled for enzyme activity. The following components were added to a 100 μL reaction system: ① 5 μL of a 20 mM solution of p-nitrophenylbutyrate in acetonitrile; ② 5 μL of the enzyme solution incubated in each pH buffer; and ③ 90 μL of 50 mM Tris-HCl buffer at pH 7. A buffer without enzyme was used instead of the protein solution as a control. After reacting at 45°C for 5 min, the absorbance at 405 nm was measured using a microplate reader. Three replicates were performed for each pH gradient and control group. The group with the highest enzyme activity was set as 100% and the results were normalized. Figure 8GutestA showed the highest stability at pH 9.5. At pH 9, pH 10, and pH 10.5, its stability remained above 90% relative to pH 9.5. GutestA was unstable in acidic environments. After incubation for 1 hour at pH < 7.0, its activity in degrading p-nitrophenol butyrate only retained approximately 50%.

[0089] From the above results, it can be seen that the GutestA provided by the present invention not only has high activity in a wide range of temperature and pH, but also has excellent thermal stability and pH stability, and is suitable for various complex industrial and natural environments, giving GutestA diverse industrial application prospects.

[0090] Example 7: Modification and Optimization of GutestA The amino acid sequence shown in SEQ ID NO. 1 was modified by site-directed mutagenesis, replacing some hydrophobic amino acids with hydrophilic ones to increase its water solubility. Three-dimensional structure prediction of the GutestA protein using Alphafold2 and docking simulation using AutoDock software revealed that the catalytic active center is composed of the HGG motif at positions 76, 77, and 78, and the serine at position 149. Serine at position 149 serves as the core catalytic amino acid, directly carrying out nucleophilic attack and cleaving the ester bond. When optimizing GutestA, changes in these amino acids and their repositioning should be avoided. Several hydrophobic amino acids located on the protein surface were selected for hydrophilic substitutions. AA at positions 32 and 33 were replaced with TT, and FAPLLA at positions 226-231 were replaced with KQPKKQ. This resulted in the optimized GutestA2 (amino acid sequence shown in SEQ ID NO. 10), which shares 97.4% homology with GutestA.

[0091] The GutestA sequence was site-directed mutagenesis by PCR on the entire vector pBAD-GutestA. This resulted in the plasmid pBAD-GutestA2. The pBAD-GutestA2 vector was transformed into Escherichia coli BL21, and GutestA2 was heterologously expressed according to the procedures in Example 2. GutestA2 was purified according to the procedures in Example 2. The concentration of GutestA2 was 0.51 ng / μL as measured by a BCA kit.

[0092] GutestA2 enzyme activity was assayed according to the procedures in Example 3. One unit of activity is defined as the amount of enzyme required to release 1 μM p-nitrophenol from a 1 mM solution of p-nitrophenyl butyl ester in 1 mL of reaction system within 1 minute under optimal reaction conditions. The final specific activity of GutestA2 was 1097 U / mg.

[0093] GutestA2 has a 97.4% homology to GutestA (amino acid sequence shown in SEQ ID NO. 1) and exhibits carboxylesterase activity, indicating that the modified enzyme can maintain activity while maintaining a certain degree of homology (not reaching the optimal 98% or higher, but close) with the original sequence (amino acid sequence shown in SEQ ID NO. 1).

[0094] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A carboxylesterase, characterized in that The carboxylesterase is the enzyme described in (a)-(c): (a) an enzyme having an amino acid sequence as shown in SEQ ID NO. 1; (b) an enzyme represented by an amino acid sequence having 70% or more homology to the amino acid sequence represented by SEQ ID NO. 1 and having carboxylesterase activity; (c) A derivative enzyme obtained by modifying the enzyme having the amino acid sequence shown in SEQ ID NO. 1 and having the same activity as the enzyme before modification.

2. The carboxylesterase according to claim 1, characterized in that The carboxylesterase is an enzyme represented by an amino acid sequence having 80% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more homology to the amino acid sequence represented by SEQ ID NO. 1, and having carboxylesterase activity; and / or The transformation includes: i. at least one of substitution, deletion and addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1; and / or ii. A tag for facilitating purification is connected to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 1; and / or iii. a signal peptide sequence that facilitates secretory expression of the enzyme is connected to the amino terminus of the amino acid sequence shown in SEQ ID NO. 1; Preferably, the tag that facilitates purification is at least one selected from Poly-Arg, Poly-His, FLAG, Strep-tag II and c-myc, more preferably Poly-His.

3. A gene encoding carboxylesterase, characterized in that The gene has a nucleotide sequence encoding the carboxylesterase according to claim 1 or 2.

4. The gene according to claim 3, characterized in that The gene has a homology of 70% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1; Preferably, the gene is the nucleotide sequence shown in SEQ ID NO.

2.

5. A recombinant vector, characterized in that The recombinant vector contains the gene according to claim 3 or 4; Preferably, the expression vector of the recombinant vector is a PBAD vector.

6. A recombinant strain, characterized in that The recombinant strain contains the gene according to claim 3 or 4 or the recombinant vector according to claim 5; Preferably, the expression strain of the recombinant strain is Escherichia coli BL21.

7. A method for preparing carboxylesterase, characterized in that: The preparation method comprises: inoculating the recombinant strain according to claim 6 into a fermentation medium containing arabinose for fermentation culture.

8. An enzyme preparation, characterized in that The enzyme preparation comprises the carboxylesterase produced by the method according to claim 7.

9. Use of at least one of the carboxylesterase according to claim 1 or 2, the gene according to claim 3 or 4, the recombinant vector according to claim 5, the recombinant strain according to claim 6, and the carboxylesterase prepared by the method according to claim 7 in at least one of pharmaceutical synthesis, industrial catalysis, chiral compound resolution, oil and fat processing, environmental remediation, and degradation of ester compounds.

10. The use according to claim 9, characterized in that The ester compound is selected from at least one of ethyl lactate, ethyl acetate, ethyl butyrate, ethyl valerate, amyl acetate, ethyl hexanoate and ethyl heptanoate; Preferably, the ester compound is at least one of ethyl butyrate, ethyl valerate, pentyl acetate, ethyl hexanoate and ethyl heptanoate; Preferably, the degradation conditions include: temperature of 60-95° C. and pH of 8.5-10.

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