Cholesterol oxidase derived from kibdelosporangium, mutant thereof, expression method therefor, and use thereof in pest control

Abstract

Disclosed in the present invention are a cholesterol oxidase derived from Kibdelosporangium, a mutant thereof, an expression method therefor, and a use thereof in pest control. In the present invention, a cholesterol oxidase gene is isolated from Kibdelosporangium and cloned, and the cholesterol oxidase gene is mutated by means of directional mutation to obtain a mutant, wherein the amino acid sequence of the cholesterol oxidase is SEQ ID NO. 1, and the amino acid sequence of the mutant thereof is as shown in SEQ ID NO. 3. In the present invention, the cholesterol oxidase gene or a mutant gene thereof is further subjected to heterologous expression by means of a prokaryotic or eukaryotic expression method, and the expression level thereof is effectively improved by optimizing related induction conditions. Insecticidal activity test results show that the cholesterol oxidase or the mutant thereof provided by the present invention has extremely strong insecticidal activity on Monolepta hieroglyphica and Tenebrio molitor adults, with a mortality rate of 90% at 24 hours and 100% at 48 hours, and exhibits a certain insecticidal effect on Henosepilachna vigintioctopunctata.

Description

Cholesterol oxidase derived from *Pseudomonas*, its mutants, expression methods, and their application in insecticides. Technical Field

[0001] This invention relates to cholesterol oxidase and its applications, particularly to cholesterol oxidase derived from *Streptococcus*, mutants and their expression methods, and their applications in insecticidal and plasma total cholesterol content detection, belonging to the field of cholesterol oxidase and its applications. Background Technology

[0002] Cholesterol oxidase (COD) is a flavoprotein oxidoreductase that breaks down cholesterol into hydrogen peroxide and cholesterol-4-en-3-ol through a two-step reaction of dehydrogenation and isomerization. Clinically, it is used together with cholesterol lipase and catalase as a reagent to detect total plasma cholesterol levels, and is used to diagnose cardiovascular and cerebrovascular diseases such as coronary heart disease and atherosclerosis (Fazio S, F. Linton M. Proprotein convertase subtilisin / kexin type 9 as transducer of physiologic influences on cellular cholesterol[J]. Journal of the American College of Cardiology, 2012, 59(19)).

[0003] Besides its important role in the medical field, cholesterol oxidase also plays a certain role in insect control. Early studies have shown that cholesterol oxidase can kill cotton boll weevil larvae and affect the reproductive capacity of female adults, while Bt does not have sufficient activity to kill cotton boll weevils (David RC, Robert JG, Thomas EO, et al. Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants[J]. Plant Physiology, 2001, 126(3):1116-1128). At the same time, cholesterol oxidase also has a toxic effect on a variety of lepidopteran pests in cotton fields. Therefore, it is believed that cholesterol oxidase may have great potential in the control of coleopteran pests and in assisting and replacing Bt. Cholesterol oxidase has the potential to be a second-generation insecticidal protein after Bt. It can effectively compensate for the insecticidal spectrum of various insecticidal proteins in Bacillus thuringiensis and reduce the speed at which various pests develop resistance to insecticidal crystal proteins. However, there are few reports on the insecticidal activity of cholesterol oxidase. Currently, only a few reports indicate that cholesterol oxidase may have an insecticidal effect on Coleoptera pests, and its specific insecticidal activity against each pest needs to be studied.

[0004] For storage beetle pests such as mealworms, rice weevils, and red flour beetles, chemical control offers advantages such as rapid effectiveness, ease of operation, strong emergency response, long-lasting efficacy, and lack of geographical or seasonal limitations. However, long-term and excessive use of chemical pesticides can pollute soil, water sources, and the ecological environment, disrupting the ecological balance. Over-reliance on chemical pesticides can lead to pesticide resistance in pests, gradually reducing their effectiveness and necessitating the continuous development of new pesticides to combat resistant pests, thus increasing control costs. Some chemical pesticides may also have adverse effects on beneficial insects, humans, and other non-target organisms. Therefore, in the control of beetle pests, it is essential to actively promote low-toxicity, highly effective, and environmentally friendly biological pesticides to achieve integrated pest management and mitigate the negative impacts of chemical control. Summary of the Invention

[0005] One objective of this invention is to provide cholesterol oxidase derived from *Streptococcus* and its encoding gene;

[0006] The second objective of this invention is to mutate cholesterol oxidase from *Streptococcus* to obtain a cholesterol oxidase mutant.

[0007] A third objective of this invention is to provide a recombinant expression vector or recombinant host cell containing a cholesterol oxidase gene or a mutant gene derived from *Streptococcus*.

[0008] The fourth objective of this invention is to apply the cholesterol oxidase or its mutant derived from the aforementioned *Pseudomonas* species to kill pests, prepare reagents for detecting total cholesterol content in plasma, or reduce cholesterol content in food.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0010] One aspect of the present invention provides a cholesterol oxidase derived from *Pseudomonas*, wherein the amino acid sequence of the cholesterol oxidase is selected from any of the amino acid sequences shown in (a)-(d) below:

[0011] (a) The amino acid sequence shown in SEQ ID NO.1; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of cholesterol oxidase; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of cholesterol oxidase; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, wherein the protein still has the function or activity of cholesterol oxidase.

[0012] The cholesterol oxidase provided by this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0013] Another aspect of the invention provides a gene encoding cholesterol oxidase from *Pseudomonas*, wherein the nucleotide sequence of the CDS of the encoding gene is selected from any of the nucleotide sequences described in (a)-(e) below:

[0014] (a) The polynucleotide sequence shown in SEQ ID No. 2; (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; (c) The polynucleotide sequence capable of hybridizing with the polynucleotide sequence shown in (a) or (b) under stringent hybridization conditions, wherein the protein encoded by the polynucleotide sequence still has the function or activity of cholesterol oxidase; (d) The polynucleotide sequence having at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the protein encoded by the polynucleotide sequence still has the function or activity of cholesterol oxidase; (e) The polynucleotide sequence complementary to any of the polynucleotide sequences shown in (a)-(d), wherein the protein encoded by the polynucleotide sequence still has the function or activity of cholesterol oxidase.

[0015] The percentage of sequence identity described in this invention can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0016] In addition, those skilled in the art can optimize the nucleotides shown in SEQ ID NO.2 to enhance their expression efficiency in host cells.

[0017] Those skilled in the art can readily mutate the nucleotide sequence of the cholesterol oxidase gene derived from *Streptococcus* using known methods, such as directed evolution or point mutation. The mutations include substitution, deletion, and / or addition of one or more nucleotides in the nucleotide sequence of the cholesterol oxidase gene derived from *Streptococcus*. Specifically, the mutations can be obtained through physical mutagenesis, chemical mutagenesis, or gene editing.

[0018] In a preferred embodiment of the present invention, the present invention provides a mutant of cholesterol oxidase derived from *Streptococcus*, the amino acid sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of the mutant gene is shown in SEQ ID NO.4.

[0019] Another aspect of the present invention further provides an expression cassette, recombinant expression vector, or recombinant host cell containing the cholesterol oxidase gene or its mutant gene derived from the aforementioned *Streptococcus*; the recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector, preferably a recombinant yeast expression vector; the recombinant host cell may be a recombinant prokaryotic host cell or a recombinant eukaryotic host cell, preferably a recombinant yeast host cell.

[0020] Another aspect of the present invention provides a method for preparing cholesterol oxidase or a mutant thereof from *Streptococcus*, comprising: operably linking a cholesterol oxidase gene or a mutant gene from *Streptococcus* to an expression regulatory element to obtain a recombinant expression vector; transforming the recombinant expression vector into host cells to obtain recombinant host cells; inducing the expression of cholesterol oxidase or a mutant thereof in the recombinant host cells; and refolding and purifying the expressed recombinant protein to obtain the product.

[0021] When exogenous proteins are efficiently expressed in *E. coli*, they readily form insoluble, biologically inactive inclusion bodies. By optimizing induction culture conditions and reducing induction efficiency, it is possible to obtain active, soluble proteins. Under induction conditions of 37°C, cholesterol oxidase expressed in *E. coli* exists in the form of insoluble inclusion bodies. To obtain active cholesterol oxidase, this invention conducted single-factor optimization experiments on the heterologous expression conditions of cholesterol oxidase, including induction temperature, inducer concentration, and induction time. This invention optimized the induction conditions for heterologous expression in *E. coli* with the introduced cholesterol oxidase gene, ultimately determining that an induction temperature of 25°C, a working concentration of IPTG of 0.5 mM, and an expression time of 8 h yielded the highest yield of soluble protein and effectively inhibited inclusion body formation.

[0022] As a preferred embodiment of the present invention, a method for preparing cholesterol oxidase or its mutant derived from *Streptococcus* is provided, comprising: operably linking the cholesterol oxidase gene or its mutant gene derived from *Streptococcus* to a prokaryotic expression regulatory element to obtain a recombinant prokaryotic expression vector; transforming the recombinant expression vector into *Escherichia coli* host cells to obtain recombinant *E. coli* host cells; inducing the expression of cholesterol oxidase or its mutant in the recombinant *E. coli* host cells; and refolding and purifying the expressed recombinant protein to obtain the product; wherein the conditions for inducing the expression of cholesterol oxidase or its mutant in the recombinant *E. coli* host cells include: an induction temperature of 25°C, a working concentration of IPTG as the inducer of 0.5 mM, and an induction time of 8 h.

[0023] Cholesterol oxidase is often expressed in the large intestine as inclusion bodies. This invention found that expressing cholesterol oxidase or its mutant gene from *Streptococcus pyogenes* in yeast can greatly improve its soluble expression level. Enzyme activity analysis showed that the soluble protease activity can reach 25 U / mg.

[0024] As a preferred embodiment of the present invention, a method for preparing cholesterol oxidase or its mutant derived from *Streptococcus* is provided, comprising: operably linking a cholesterol oxidase gene or its mutant gene derived from *Streptococcus* to a yeast expression regulatory element to obtain a recombinant yeast expression vector; transforming the recombinant yeast expression vector into host yeast cells to obtain recombinant yeast host cells; inducing the expression of cholesterol oxidase or its mutant in the recombinant yeast host cells; and refolding and purifying the expressed recombinant protein to obtain the final product.

[0025] Insecticidal activity tests showed that the cholesterol oxidase provided by this invention exhibits extremely strong insecticidal activity against adult two-spotted leaf beetles and yellow mealworms, with a mortality rate of 90% after 24 hours and 100% after 48 hours. It also shows some insecticidal activity against 28-spotted ladybugs, with a mortality rate of 50% after 72 hours. However, it has almost no insecticidal activity against red flour beetles and rice weevils, showing no significant difference from the control group. The results indicate that the insecticidal activity of cholesterol oxidase against Coleoptera varies depending on the insect species and is not a universally consistent activity.

[0026] Another aspect of the present invention provides cholesterol oxidase derived from *Streptococcus* or its mutants or their encoding genes for use in the preparation of pesticides; wherein the pests are preferably Coleoptera pests, more preferably, the Coleoptera pests are two-spotted leaf beetles, yellow mealworms, or 28-spotted ladybugs, etc.

[0027] Those skilled in the art can use conventional biological pesticide formulation methods in the field to prepare cholesterol oxidase or its mutants from *Streptomyces* into various suitable biological pesticide formulations. These methods are well known to those skilled in the art.

[0028] In addition, the cholesterol oxidase or its mutant derived from *Streptomyces* provided by this invention has stable enzymatic properties and high enzyme activity, and therefore can be used together with catalase as a reagent for detecting total cholesterol content in plasma.

[0029] In a preferred embodiment of the present invention, the present invention provides a test kit for detecting total cholesterol content in plasma, comprising cholesterol oxidase and catalase, wherein the cholesterol oxidase is cholesterol oxidase derived from *Streptomyces* or a mutant thereof provided by the present invention.

[0030] The cholesterol oxidase or its mutant derived from *Streptomyces* provided by this invention can also be used to reduce the cholesterol content in food, for example, as a food additive to reduce the cholesterol content in high-cholesterol foods.

[0031] This invention isolated and cloned the cholesterol oxidase gene from *Streptococcus* and obtained a mutant by targeted mutagenesis. Furthermore, the invention heterologously expressed the cholesterol oxidase gene or its mutant gene through prokaryotic or eukaryotic expression methods. It was found that the cholesterol oxidase gene or its mutant gene, when expressed in *E. coli*, mainly exists in the form of inclusion bodies. Expression of the cholesterol oxidase gene or its mutant gene in yeast significantly increased its soluble expression level, with enzyme activity analysis showing a soluble protease activity of up to 25 U / mg. Insecticidal activity studies showed that it has extremely strong insecticidal activity against adult *Lepidoptera litura* and *Mealworm*, with a 90% mortality rate after 24 hours and a 100% mortality rate after 48 hours. It also showed some insecticidal activity against *Ladybug 28*, with a 50% mortality rate after 72 hours. However, it had almost no insecticidal activity against *Triplophysa rubrum* and *Rhizoctonia solani*, showing no significant difference from the control group. The results indicate that the insecticidal activity of cholesterol oxidase against Coleoptera varies depending on the insect species and is not a universally consistent insecticidal activity. Attached Figure Description

[0032] Figure 1 shows the agarose gel electrophoresis image of the recombinant plasmid of the target protein; M. DNA marker; 1. Verification of recombinant plasmid by NcoI / XhoI restriction enzyme digestion; 2. Recombinant plasmid.

[0033] Figure 2 shows the SDS-PAGE analysis of the expression of the target protein in Escherichia coli during induction at 37℃; M. marker; 1. CK precipitate; 2. CK supernatant; 3. Supernatant before induction; 4. Supernatant after induction; 5. Precipitate after induction; 6. All cells after induction.

[0034] Figure 3 shows the SDS-PAGE analysis results of cholesterol oxidase renaturation from *Streptococcus*; M. marker; renatured sample 1.1; renatured sample 2.2; renatured sample 3.3.

[0035] Figure 4 shows the optimal reaction pH for cholesterol oxidase derived from *Streptococcus*.

[0036] Figure 5 shows the optimal reaction temperature for cholesterol oxidase derived from *Streptococcus*.

[0037] Figure 6. Effect of induction temperature on cholesterol oxidase activity and yield in recombinant Escherichia coli.

[0038] Figure 7 Effect of final IPTG concentration on cholesterol oxidase activity in recombinant Escherichia coli. Figure 8 Effect of induction time on relative cholesterol oxidase production.

[0039] Figure 9. SDS-PAGE analysis after optimizing expression conditions.

[0040] Figure 10. Chromatographic spectra during elution after optimizing expression conditions.

[0041] Figure 11 shows the SDS-PAGE analysis of the induced expression of cholesterol oxidase from *Streptococcus* and its mutants; where 3-1-1 and 3-1-2 are mutants obtained by mutating the original amino acid sequence of cholesterol oxidase from *Streptococcus* and then optimizing the gene codons; 3-2 is the original amino acid sequence of cholesterol oxidase from *Streptococcus*.

[0042] Figure 12 shows the protein expression of *E. coli* after transformation with COD plasmids 3-2 and 3-1-2; where a represents the precipitate, b represents the supernatant, lane M represents the protein marker, lanes a 1-6 represent the precipitate after 3-2 induction (uninduced), 3-1-2 induction (30℃), 3-1-2 induction (30℃), 3-2 induction (25℃), and 3-1-2 induction (25℃), respectively; lanes b 1-6 represent the supernatant after 3-1-2 induction (25℃), 3-1-2 induction (25℃), 3-1-2 induction (30℃), 3-2 induction (30℃), 3-2 induction (30℃), 3-1-2 induction (uninduced), and 3-2 induction (uninduced), respectively; the target protein size is approximately 60.2 kDa.

[0043] Figure 13 compares the expression of cholesterol oxidase in Saccharomyces cerevisiae and Escherichia coli. Lane M represents the protein maker, lanes 1-2 represent the supernatant of Saccharomyces cerevisiae after lysis and the uninduced control after culture at 30℃ and 200 rpm, and lanes 2-5 represent the supernatant of Escherichia coli after lysis under IPTG induction at 20℃, 160 rpm, 0.5 mM, 0.25 mM, and 0.1 mM for 12 h. The target protein size is approximately 62.7 kDa in yeast and 60.2 kDa in Escherichia coli.

[0044] Figure 14 shows the SDS-PAGE assay of purified COD3-1-2 protein expressed in Saccharomyces cerevisiae; lane M is the protein marker, and lanes 1-3 are the purified COD3-1-2 protein, respectively. The target protein size is approximately 60.2 kDa.

[0045] Figure 15 shows the 24-hour mortality rate of Coleoptera insects under COD3-1-2 treatment; where ns indicates no significant difference between the experimental group and the control group, and the more *, the more significant the difference; (p<0.05).

[0046] Figure 16 shows the changes in the average mortality rate of Coleoptera insects under COD3-1-2 treatment over 24-72 hours.

[0047] Figure 17 shows the 72-hour mortality rate of Coleoptera under COD3-1-2 and control treatments; where ns indicates no significant difference between the experimental group and the control group, and the more *, the more significant the difference (p<0.05). Detailed Implementation

[0048] Example 1: Cloning of a cholesterol oxidase gene derived from *Streptococcus*, its expression in recombinant *Escherichia coli*, and analysis of its enzymatic properties.

[0049] 1. Materials and Methods

[0050] 1.1 Strains and Vectors

[0051] The tested strain was Escherichia coli.

[0052] The vector was the recombinant plasmid pET-28a(+)kana, which introduced the cholesterol oxidase gene. The gene source was Kibdelosporangium sp.

[0053] 1.2 Test Methods

[0054] 1.2.1 Amplification of cholesterol oxidase gene fragment

[0055] The target cholesterol oxidase gene was amplified using the existing reaction system in the laboratory.

[0056] Using plasmid pET28a(+)kana as a template, PCR was performed. Primers were designed as shown in Table 1, and the PCR reaction system was shown in Table 2. After thoroughly mixing all components, the mixture was placed in the PCR instrument, and the reaction program was shown in Table 3.

[0057] Table 1 Primer sequence list

[0058] Table 2 PCR reaction system

[0059] Table 3 PCR reaction procedure

[0060] After the polymerase chain reaction product was detected by agarose gel electrophoresis, the target DNA fragment was recovered according to the instructions on the DNA gel recovery kit, and the recovery effect was detected by agarose gel electrophoresis. The recombinant bacteria were inoculated into LB liquid medium (with 100 μg / mL Kana) and cultured at 37°C and 200 rpm for 10–12 h before plasmid extraction.

[0061] 1.2.2 Linking of target gene and vector

[0062] The amplified cholesterol oxidase gene and vector pET28a were digested with restriction endonucleases NcoI / XhoI, and the gene fragment and vector fragment were ligated at a concentration ratio of 3:1.

[0063] 1.2.3 Enzyme digestion and sequencing verification of the constructed vector

[0064] The cholesterol oxidase gene, amplified by PCR, was double-digested with NcoI / XhoI restriction endonucleases, verified by agarose gel electrophoresis, and sequenced. The result was then validated and compared using BLAST. The validated vector was ultimately used for subsequent research and experiments.

[0065] 1.2.4 Preparation of competent cells using the CaCl2 method

[0066] 1.2.5 Vector transformation and strain preservation

[0067] The constructed expression vector was transformed into competent BL21 cells.

[0068] 1.2.6 Small-scale shake-flask expression of recombinant E. coli with the target gene

[0069] 1.2.7 Expression and detection of cholesterol oxidase in recombinant Escherichia coli

[0070] 1.2.7.1 Induction expression assay of cholesterol oxidase

[0071] Single colonies were picked from freshly streaked plates and inoculated into LB broth containing kanamycin, and incubated overnight at 37°C with shaking. A 1% inoculum was then transferred to 20 mL of freshly prepared LB broth and incubated at 37°C with shaking until OD (occurrence depth) reached. 600 =0.4-0.6 (achieved in about 2-3 hours), add IPTG to the culture to a final concentration of 1 mM, and continue culturing for 8 hours. The control group did not receive IPTG induction, but other treatments were the same as the experimental group.

[0072] 1.2.7.2 Preparation of intracellular crude enzyme solution

[0073] 1.2.8 Refolding test of cholesterol oxidase in inclusion body form

[0074] During the expression and synthesis of cholesterol oxidase, the folding of the primary amino acid chain into a more complex spatial structure can lead to misfolding of the protein. This misfolding, occurring as inclusion bodies in the precipitate, can result from factors such as folding rate, IPTG inducer dosage, and protein kinetics. Consequently, the expressed protein loses its activity, leading to reduced protein expression in the supernatant during SDS-PAGE electrophoresis. Refolding typically involves first using a denaturing agent to break the protein down into its amino acid chains, followed by dilution and dialysis under mild conditions to allow the protein to refold. Due to protein kinetics, the protein will fold into its correct structure, thus refolding into an active protein. The cholesterol oxidase inclusion bodies were refolded using the centrifuged bacterial pellet (inclusion bodies obtained using conventional methods often contain cell debris, so the pellet needs thorough washing before use in this experiment).

[0075] 1.2.8.1 Washing inclusion bodies

[0076] 1.2.8.2 Dissolving Inclusion Bodies

[0077] 1.2.8.3 Refolding of Inclusion Body Proteins

[0078] 1.2.9 Purification and related properties study of cholesterol oxidase

[0079] 1.2.9.1 Purification of cholesterol oxidase

[0080] The crude enzyme solution was purified using a purification column with 7-chloroflavin mononucleotide (FMN) as the affinity ligand.

[0081] 1.2.9.2 Determination of protein concentration

[0082] The protein concentration of the relevant protein solutions obtained in the experiment was determined using a Nanodrop 2000C. Alternatively, the Bradford method can be used to determine the protein concentration.

[0083] 1.2.9.3 Detection of cholesterol oxidase expression by polyacrylamide SDS-PAGE method

[0084] The appropriate PAGE separating gel concentration was selected based on the molecular weight of the target protein. In this study, the PAGE separating gel concentration was 12%, the separating gel volume was 5 mL, and the stacking gel volume was 2 mL.

[0085] 1.2.9.4 Determination of cholesterol oxidase activity

[0086] The activity of cholesterol oxidase was determined using the method of Huang Yin et al. (Huang Yin, Ge Fei, Tao Yugui et al. Mutagenesis and selection of high cholesterol oxidase-producing strains and optimization of enzyme production conditions [J]. Journal of Anhui University of Engineering Science and Technology (Natural Science Edition), 2009, 24(04):8-11).

[0087] 1.2.9.5 pH stability analysis of cholesterol oxidase

[0088] Optimal reaction pH: Within the pH range of 5.0 to 9.0, enzyme activity is determined according to the enzyme activity detection method provided in 1.2.9.4. The highest enzyme activity measured in the experiment is recorded as 100%, and the relative enzyme activity under other pH conditions is calculated to determine the optimal pH value for the catalytic reaction.

[0089] 1.2.9.6 Temperature stability analysis of cholesterol oxidase

[0090] Using cholesterol as a substrate, cholesterol oxidase activity was measured at different temperatures ranging from 30 to 80°C under optimal pH conditions. The highest enzyme activity measured in the reaction was taken as 100%, and the relative enzyme activities at other temperatures were calculated and compared to determine the optimal reaction temperature.

[0091] 2. Experimental Results

[0092] 2.1 Validation results of the cholesterol oxidase recombinant plasmid

[0093] The recombinant plasmid was double-digested using NcoI and XhoI restriction enzymes. Agarose gel electrophoresis showed that lane 1 contained a gene fragment approximately 1600 bp in size, as shown in Figure 1. The deduced amino acid sequence of the original cholesterol oxidase from *Kibdelosporangium* is shown in SEQ No. 1. A BLAST analysis was performed to compare the sequencing results of the original cholesterol oxidase gene from *Kibdelosporangium* with the sequence of the cholesterol oxidase gene in the full-length *Kibdelosporangium* sp. strain. The comparison showed 100% homology between the sequencing results and the *Kibdelosporangium* sp. strain, indicating that the *E. coli* plasmid carrying the cholesterol oxidase gene was successfully constructed and can be used for subsequent protein expression experiments.

[0094] 2.2 Detection of cholesterol oxidase expression results

[0095] The activities of induced and uninduced cholesterol oxidase were detected. It was found that almost no cholesterol oxidase activity was detected in the intracellular supernatant of recombinant *E. coli* under uninduced or 37℃ 1mM IPTG induction conditions. SDS-PAGE analysis of intracellular protein expression under 37℃ 1mM IPTG induction conditions was performed, and the results are shown in Figure 2.

[0096] Under 37℃ and 1mM IPTG induction conditions, there was no significant difference in the type and expression level of intracellular soluble protein components between the cholesterol oxidase expressed by recombinant E. coli induced with an inducing agent and the cholesterol oxidase expressed by recombinant E. coli without an inducing agent. However, the intracellular insoluble portion of the induced recombinant E. coli showed a protein with significantly increased expression level at around 60kD, which is consistent with the size of cholesterol oxidase. This study suggests that this is cholesterol oxidase existing in the form of inclusion bodies.

[0097] 2.3 Refolding of Inclusion Bodies

[0098] The SDS-PAGE analysis of the refolding results is shown in Figure 3. The SDS-PAGE analysis results show that the amount of refolded protein in refolded sample 1 is similar to that in refolded sample 2, but sample 1 contains a small amount of extraneous protein bands. The amount of extraneous protein in refolded sample 3 is lower than that in refolded sample 2, but the amount of the target protein in sample 3 is also very low. Overall, refolded sample 2 ensures successful refolding of cholesterol oxidase while maintaining a low amount of extraneous protein. Therefore, for subsequent refolding operations involving inclusion bodies of cholesterol oxidase in precipitated form, the washing number and dissolving solution concentration used in refolding sample 2 should be selected.

[0099] 2.4 Results of cholesterol oxidase stability analysis

[0100] 2.4.1 Results of pH stability analysis of cholesterol oxidase

[0101] The adaptability of enzymes to the acidity or alkalinity of their surrounding environment has always been one of the biological characteristics of enzymes. Excessively high or low pH levels in the external environment will cause enzyme inactivation, which is irreversible and irreversible. As shown in Figure 4, the data in the figure shows that the relative enzyme activity of cholesterol oxidase is highest at pH 7.0. It is worth noting that at pH 7.5, the relative enzyme activity of cholesterol oxidase is still 82%. Therefore, relatively speaking, cholesterol oxidase is less likely to maintain its activity in acidic environments than in alkaline environments.

[0102] 2.4.2 Results of temperature stability analysis of cholesterol oxidase

[0103] Thermostability of enzymes has always been an important reference standard in industrial applications. Increased ambient temperature intensifies molecular collisions in solution, increasing the probability of substrate-enzyme collisions and accelerating the reaction rate. However, on the other hand, increased temperature can alter the spatial structure of proteins, leading to protein inactivation and consequently affecting catalytic efficiency. Temperature stability analysis of purified cholesterol oxidase showed that its relative enzyme activity was highest at 50°C, as shown in Figure 5.

[0104] Example 2: Optimization of Heterologous Expression Conditions for Cholesterol Oxidase

[0105] When exogenous proteins are efficiently expressed in *E. coli*, they readily form insoluble, biologically inactive inclusion bodies. By optimizing the induction culture conditions and reducing induction efficiency, it is possible to obtain active, soluble proteins. As shown in Figure 3, cholesterol oxidase expressed in *E. coli* exists as insoluble inclusion bodies under 37°C induction conditions. To obtain active cholesterol oxidase, single-factor optimization experiments were conducted on the heterologous expression conditions, including induction temperature, inducer concentration, and induction time.

[0106] 1. Experimental Methods

[0107] Single colonies were picked from fresh streaked plates and inoculated into 50 mL of LB broth containing kanamycin, and incubated overnight at 37°C with shaking. The following morning, 1% of the colonies were transferred to 50 mL of fresh LB broth supplemented with antibiotics and incubated at 37°C with shaking until OD (occurrence depth) was reached. 600 The induction conditions were optimized to achieve a reaction time of approximately 0.4-0.5 mM (reached in about 3 hours). Inducer optimization included adding IPTG at final concentrations of 1 mM, 0.75 mM, 0.5 mM, and 0.25 mM to induce enzyme activity and determine yield. Optimal temperatures were determined at 15℃, 20℃, 25℃, and 30℃. Optimal induction times were set at 0, 2, 4, 6, 8, 10, and 12 hours for measurement.

[0108] 1.1 Optimization of Induction Temperature

[0109] IPTG was added to the culture to a final concentration of 0.5 mM, and the culture was continued at 15℃, 20℃, 25℃, and 30℃ for 6 h to prepare crude enzyme solution. The supernatant was collected by centrifugation and analyzed by SDS-PAGE protein electrophoresis to compare expression levels.

[0110] 1.2 Optimization of IPTG final concentration

[0111] IPTG was added to the culture at final concentrations of 1 mM, 0.75 mM, 0.5 mM, and 0.25 mM. After culturing for 6 h at the optimal induction temperature obtained in 2.2.10.1, the intracellular supernatant was collected for SDS-PAGE protein electrophoresis for comparison.

[0112] 1.3 Optimization of Induction Time

[0113] IPTG inducer was added to the culture to a final concentration of 0.5 mM. After culturing at 25°C for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, the intracellular supernatant was collected for SDS-PAGE protein electrophoresis and comparison.

[0114] 2. Experimental Results

[0115] 2.1 Optimization of Induction Temperature

[0116] Of the four induction temperatures designed in this experiment, the highest cholesterol oxidase activity was observed at 25℃, reaching approximately 200 U / L. The cholesterol oxidase production decreased with increasing induction temperature. At 30℃, the recombinant *E. coli* enzyme activity reached its lowest value, with an expression level of only about 10 U / L. At 15℃ and 20℃, the low temperatures inhibited *E. coli* growth, resulting in reduced inclusion body production, but also relatively low total cholesterol oxidase production, as shown in Figure 6. Based on the experimental results, subsequent exogenous expression of recombinant *E. coli* should be performed at 25℃.

[0117] 2.2 Optimization of IPTG dosage as an inducer

[0118] In this experiment, among the four inducer concentrations (with an induction temperature of 25℃ as determined above), starting from an inducer substrate concentration of 1 mM, the cholesterol oxidase activity of recombinant *E. coli* gradually increased from 65 U / L as the inducer concentration decreased, reaching a peak at a final IPTG concentration of 0.5 mM, under which condition the highest cholesterol oxidase activity reached 257 U / L. Further reducing the IPTG inducer concentration to 0.25 mM subsequently caused a decrease in enzyme activity. Both excessive and insufficient inducer concentrations affected the production of active protein. At an inducer concentration of 0.5 mM, the amount of soluble cholesterol oxidase protein produced by exogenous expression in *E. coli* was the highest among these cases, as shown in Figure 7.

[0119] 2.3 Optimization of Induction Time

[0120] As shown in Figure 8, the activity of cholesterol oxidase in recombinant *E. coli* expressed at different induction times under 25℃ and 0.5mM IPTG induction conditions was measured. It was found that the activity of cholesterol oxidase continuously increased within 8 hours after induction, reaching a peak of approximately 350 U / L at 8 hours. By 10 hours, the activity of cholesterol oxidase in the bacteria began to decline, which may be related to the degradation by intracellular proteases.

[0121] 2.4 Results of optimized cholesterol oxidase expression

[0122] Based on the above optimization of exogenous cholesterol oxidase expression conditions, the optimal conditions for recombinant *E. coli* expression were found to be 25℃, 0.5mM IPTG, and 8h. Therefore, numerous expression and purification experiments were conducted under these conditions. The SDS-PAGE analysis of the expression results is shown in Figure 9, and the elution of the supernatant from the recombinant *E. coli* expression protein onto the column is shown in Figure 10. As shown in Figure 9, cholesterol oxidase is highly expressed at approximately 60kD in lane 5 (corresponding to 300mM imidazole elution). Compared to the results in Figure 2, the expression levels of other proteins were significantly reduced after optimization. Combined with Figure 10, the 300mM imidazole elution zone shows the highest absorption peak except for the 10mM imidazole zone (which eluted a large amount of other proteins, hence the higher absorption peak), confirming that the protein mainly eluted in this expression was the target protein—cholesterol oxidase. The protein concentration was measured to be 0.264 mg / mL using a Nanodrop 2000c spectrophotometer, and this protein was used in subsequent insecticidal activity tests.

[0123] Example 3: Cholesterol oxidase derived from *Streptococcus* was mutated, and the mutant gene was codon-optimized to obtain a cholesterol oxidase mutant. The expression effect of this mutant in *E. coli* was then investigated.

[0124] The coding gene of the original cholesterol oxidase (the amino acid sequence of which is shown in SEQ ID No. 1) from *Streptococcus* was codon optimized (according to the codon preference of the host *Escherichia coli*) to obtain the cholesterol oxidase optimized gene (3-2), the nucleotide sequence of which is shown in SEQ ID No. 2.

[0125] The original cholesterol oxidase from *Streptococcus* was mutated, and then the mutant gene was codon-optimized (according to the host *E. coli* codon preference) to obtain cholesterol oxidase mutants (3-1-1, 3-1-2; where the nucleotide sequence of 3-1-2 is shown in SEQ ID No. 4, and the amino acid sequence of its encoded protein is shown in SEQ ID No. 3). These mutants were induced to express in recombinant *E. coli* under the same conditions, and the results of SDS-PAGE analysis of the expressed recombinant protein are shown in Figure 11. As can be seen from Figure 11, compared to the original cholesterol oxidase sequence (SEQ ID No. 1), the optimized cholesterol oxidase mutant sequences (3-1-1, 3-1-2) obtained by mutating the amino acid sequence of cholesterol oxidase D from *Streptococcus* and then codon-optimizing the mutant gene significantly improved the expression of the soluble protein.

[0126] Experiment 1: Expression, enzyme activity analysis, and insecticidal activity assay of cholesterol oxidase gene and its mutants in Escherichia coli or yeast.

[0127] 1. Materials and Methods

[0128] 1.1 Test Materials

[0129] 1.1.1 Test Insects

[0130] Adult mealworms (Tenebrio molitor), rice weevils (Sitophilus oryzae), red flour beetles (Tribolium castaneum Herbs), and 28-spotted ladybugs (Henosepilachna vigintioctopunctata) were raised at the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. Adult leaf beetles (Monolepta hieroglyphica) were provided by Associate Professor Zhang Haiyan of Heilongjiang Bayi Agricultural Reclamation University in Daqing City.

[0131] 1.1.2 Strains and Plasmids

[0132] The competent Escherichia coli cells BL21 were purchased from Solarbio Science & Technology Co., Ltd. Recombinant E. coli protein expression plasmids (numbered 3-1-2 and 3-2, containing Kana resistance) infused with the Kibdelosporangium sp. cholesterol oxidase gene were provided by Researcher Wenli Sun from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing. Plasmid 3-2 is an optimized gene obtained by codon optimization of the original cholesterol oxidase nucleotide sequence from Kibdelosporangium sp. according to the host E. coli's preference (its nucleotide sequence is shown in SEQ ID No. 2, and its encoded protein amino acid sequence is shown in SEQ ID No. 1). Plasmid 3-1-2 is an optimized gene obtained by mutating the amino acid sequence of the Kibdelosporangium sp. cholesterol oxidase and then codon optimization of the mutant gene according to the host E. coli's preference (its nucleotide sequence is shown in SEQ ID No. 4, and its encoded protein amino acid sequence is shown in SEQ ID No. 3). The Saccharomyces cerevisiae strain and Saccharomyces cerevisiae protein expression vector (including Kana, Ampr, and G418 resistance) were provided by Researcher Wenli Sun from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0133] 1.2 Test Methods

[0134] 1.2.1 Escherichia coli-induced expression

[0135] The recombinant protein expression plasmid was transformed into competent E. coli BL21 cells using the heat shock method. IPTG was used to induce protein expression in E. coli, and the rotation speed, temperature, and IPTG concentration were adjusted according to experimental requirements. After 12 hours of induction, the cells were centrifuged at 8000 rpm for 30 minutes at 4°C to retain the cells. The cells were resuspended in 10 ml of 1×PBS buffer and then sonicated. The cells were centrifuged at 12000 rpm for 30 minutes at 4°C to obtain soluble protein supernatant and inclusion body precipitate. Each 0.1 g precipitate was resuspended in 1 ml of 1×PBS buffer. 2 μl of the precipitate was then mixed with 38 μl of 1×PBS and 10 μl of 5×protein loading buffer. 40 μl of the supernatant was mixed with 10 μl of 5×protein loading buffer and treated at 100°C for 10 minutes. Cholesterol oxidase expression was detected by polyacrylamide SDS-PAGE.

[0136] 1.2.2 Induced expression of Saccharomyces cerevisiae

[0137] The plasmid 3-1-2 or 3-2 and the *Saccharomyces cerevisiae* protein expression vector were double-digested using NcoI / XhoI restriction endonucleases. The cholesterol oxidase gene fragment was then ligated to the linearized *Saccharomyces cerevisiae* vector using T4 ligase. The recombinant plasmid was then transformed into *E. coli* DH5α competent cells and cultured, and the plasmid was extracted. Competent cells of *Saccharomyces cerevisiae* were prepared using the lithium acetate method and transformed with plasmids. After culturing at 30°C for 3 days, single colonies were picked and placed in 5 ml of YPD medium. 5 μl of 10 mg / ml carbamycin, 5 μl of 50 mg / ml ampicillin, and 20 μl of 100 mg / ml genimycin G418 were added. The culture was incubated at 30°C and 250 rpm for 6-8 hours. The entire culture was then transferred to 20 ml of fresh YPD medium, and 20 μl, 20 μl, and 80 μl of the three antibiotics were added respectively. The culture was incubated overnight at 250 rpm until the OD value reached approximately 1. The entire culture was then transferred to 30 ml of fresh YPD medium, and 30 μl, 30 μl, and 120 μl of the three antibiotics were added respectively. Ethanol was added to induce an induction concentration of 1%. After culturing at 30°C and 200 rpm for 1 day, 5 ml of the bacterial culture was collected, centrifuged at 4°C and 12000 rpm for 20 min to retain the bacterial cells, and 300 μl was extracted. Resuspend the cells in SLB buffer, grind the cells with glass beads, centrifuge at 12,000 rpm for 20 min at 4 °C, retain the supernatant, mix 80 μl with 20 μl of 5× protein loading buffer, treat at 100 °C for 10 min, and detect cholesterol oxidase expression by polyacrylamide SDS-PAGE.

[0138] 1.2.3 Cholesterol oxidase activity assay

[0139] The activity of cholesterol oxidase was determined using the method of Huang Yin et al. (Huang Yin, Ge Fei, Tao Yugui et al. Mutagenesis and selection of high cholesterol oxidase-producing strains and optimization of enzyme production conditions [J]. Journal of Anhui University of Engineering Science and Technology (Natural Science Edition), 2009, 24(04):8-11).

[0140] 1.2.4 Verification of the insecticidal ability of cholesterol oxidase

[0141] The expressed soluble cholesterol oxidase was purified by nickel column elution. The enzyme solution was fed to the tested Coleoptera insects using the immersion feeding method, with water as a control. Three experiments were set up for both the experimental and control groups. The mortality rate was observed and recorded every 24 hours. The data were processed and plotted using GraphPad Prism 8.

[0142] 2. Experimental Results

[0143] 2.1 Expression and Enzyme Activity Analysis of COD in Escherichia coli and Yeast

[0144] Under induction conditions of 30℃, 0.5mM, 160rpm, and 25℃, 0.5mM, 160rpm for 12h (Figures 12a and 12b), *E. coli* transformed with the COD plasmid showed no significant difference in the expression of the target soluble protein, with the target protein mostly existing in inclusion body form. The constructed *Saccharomyces cerevisiae* vector was transformed into *Saccharomyces cerevisiae* and cultured at 200rpm and 30℃ for 2d to induce protein expression. The soluble COD expressed by *E. coli* under IPTG induction conditions of 20℃, 160rpm, 0.5mM, 0.25mM, and 0.1mM for 12h was compared (Figure 13). *Saccharomyces cerevisiae* showed a very clear band at 60kDa, while *E. coli* showed no clear band under the three induction conditions. Figure 14 shows the SDS-PAGE analysis of purified COD protein expressed in *Saccharomyces cerevisiae*. Lane M is the protein marker, and lanes 1-3 represent the purified COD protein, with a target protein size of approximately 60.2kDa.

[0145] The purified COD protein was subjected to enzyme activity assay, and the activity of the original cholesterol oxidase (3-2) (SEQ ID No. 1) was found to be 25 U / mg. ， The cholesterol oxidase mutant (3-1-2) (SEQ ID No. 3) has an enzyme activity of 52 U / mg.

[0146] 2.2 Insecticidal activity of COD3-1-2 against different Coleoptera insects

[0147] The purified soluble COD3-1-2 enzyme solution was fed to adult mealworms, two-spotted leaf beetles, rice weevils, red flour beetles, and 28-spotted ladybugs using the immersion feeding method. The results showed that adult mealworms and two-spotted leaf beetles were highly sensitive to COD3-1-2, with an average mortality rate exceeding 70% after 24 hours, significantly higher than the control treatment, and all insects died after 48 hours (Figures 15 and 16). Rice weevils and red flour beetles, however, were not sensitive to this protein, with an average mortality rate of less than 10% even after 72 hours, showing no significant difference from the control group (Figures 15, 16, and 17). COD3-1-2 also exhibited some insecticidal activity against 28-spotted ladybugs, with an average mortality rate of approximately 50% after 72 hours (Figures 16 and 17).

[0148] Table 4. Insecticidal activity of COD3-1-2 against the two-spotted leaf beetle.

[0149] 2.3 Comparison of insecticidal activities of different cholesterol oxidases against different insects

[0150] Experimental Methods: E. coli expression vectors were constructed using the mutant cholesterol oxidase 3-1-2 (SEQ ID No. 4) and the codon-optimized original cholesterol oxidase gene 3-2 (SEQ ID No. 2), respectively. Prokaryotic expression was performed, and the soluble target protein in the supernatant was purified. This protein was then used to kill adult mealworms, two-spotted leaf beetles, twenty-eight-spotted ladybugs, rice weevils, and red flour beetles. The enzyme solution was fed to the tested Coleoptera insects using an immersion feeding method, with water as a control. The experimental results are shown in Table 5.

[0151] Table 5. Detection of insecticidal activity of different COD proteins against five species of Coleoptera insects.

Claims

1. A cholesterol oxidase derived from Kibdelosporangium sp., characterized in that, The amino acid sequence of the cholesterol oxidase is selected from any of the amino acid sequences shown in (a)-(d) below: (a) The amino acid sequence shown in SEQ ID NO.1; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of cholesterol oxidase; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of cholesterol oxidase; (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, which still has the function or activity of cholesterol oxidase.

2. The encoding gene for cholesterol oxidase from *Pseudomonas* as described in claim 1, characterized in that, The nucleotide sequence of the CDS encoding the gene is selected from any of the nucleotide sequences described in (a)-(e) below: (a) The polynucleotide sequence shown in SEQ ID No. 2; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, wherein the protein encoded by the polynucleotide sequence still has the function or activity of cholesterol oxidase; (d) A polynucleotide sequence that has at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), and the protein encoded by the polynucleotide sequence still has the function or activity of cholesterol oxidase; (e) A multinucleotide sequence that is complementary to any of the multinucleotide sequences described in (a)-(d), wherein the protein encoded by the multinucleotide sequence still has the function or activity of cholesterol oxidase.

3. The mutant of cholesterol oxidase derived from *Pseudomonas* as described in claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

3.

4. The coding gene of the mutant according to claim 3, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

5. A chimeric gene, expression cassette, recombinant expression vector, or recombinant host cell containing the encoding gene of claim 2 or 3; wherein, The recombinant expression vector is a recombinant yeast expression vector; the recombinant host cell is a recombinant yeast host cell.

6. A method for preparing cholesterol oxidase derived from *Pseudomonas* as described in claim 1 or the mutant as described in claim 3, characterized in that, include: A recombinant expression vector is obtained by operatively linking the cholesterol oxidase gene or its mutant gene from *Streptococcus pyogenes* to an expression regulatory element; the recombinant expression vector is transformed into host cells to obtain recombinant host cells; cholesterol oxidase or its mutant is induced to express in the recombinant host cells; the expressed recombinant protein is then renatured and purified to obtain the final product.

7. The method according to claim 6, characterized in that, include: A recombinant prokaryotic expression vector was obtained by operatively linking a cholesterol oxidase gene or its mutant gene derived from *Streptococcus pyogenes* to a prokaryotic expression regulatory element; the recombinant expression vector was transformed into *E. coli* host cells to obtain recombinant *E. coli* host cells; cholesterol oxidase or its mutant was induced to express in the recombinant *E. coli* host cells; the expressed recombinant protein was then renatured and purified to obtain the final product; wherein the conditions for inducing the expression of cholesterol oxidase or its mutant in the recombinant *E. coli* host cells included: an induction temperature of 25°C, a working concentration of IPTG of 0.5 mM, and an induction time of 8 h; Alternatively, a cholesterol oxidase gene or its mutant gene derived from *Streptococcus pyogenes* can be operatively linked to a yeast expression regulatory element to obtain a recombinant yeast expression vector; this recombinant yeast expression vector is then transformed into host yeast cells to obtain recombinant yeast host cells; the expression of cholesterol oxidase or its mutant in the recombinant yeast host cells is induced, and the expressed recombinant protein is renatured and purified to obtain the final product.

8. The use of the cholesterol oxidase derived from *Streptomyces* as described in claim 1, the encoding gene as described in claim 2 or 4, or the mutant as described in claim 3 in the killing of agricultural pests or in reducing the cholesterol content of food.

9. The application according to claim 8, characterized in that, The agricultural pests mentioned are Coleoptera pests; among them, the Coleoptera pests are two-spotted leaf beetles, yellow mealworms, or 28-spotted ladybugs.

10. A reagent kit for detecting total cholesterol content in plasma, comprising cholesterol oxidase and catalase, characterized in that, The cholesterol oxidase is the cholesterol oxidase derived from *Streptomyces* as described in claim 1 or a mutant of the cholesterol oxidase as described in claim 3.

Images