Application of amidase and phenol hydroxylase in the degradation of triclocarban and its intermediates

Abstract

This invention provides the application of amidase and phenol hydroxylase in the degradation of triclocarban and its intermediates, belonging to the field of pollutant degradation technology. Through genomic analysis and molecular biological manipulation and detection of strains BX2 and LY-1, this invention clarifies that amidase (TccG) and phenol hydroxylase (PhL) are key enzymes catalyzing the degradation of TCC and its intermediates 3,4-dichloroaniline (DCA) and 4-chloroaniline (4-CA). Compared with other methods, the application provided by this invention can effectively reduce the possibility of secondary pollution during TCC metabolism. This invention provides a new approach to the study of the microbial degradation mechanism of TCC, provides a reference for further utilizing microbial interactions to remediate TCC and its structural analogues, and provides a theoretical basis for the future application of enzyme resources.

Description

Technical Field

[0001] This invention belongs to the field of pollutant degradation technology, and in particular relates to the application of amidase and phenol hydroxylase in the degradation of triclocarban and its intermediate products. Background Technology

[0002] Triclocarban (TCC) is a typical diphenylurea compound widely added to personal care products and chemical products due to its broad-spectrum antibacterial properties. Because of its hydrophobic and stable chemical structure, TCC has extremely high detection rates in environments worldwide, particularly in wastewater, sludge, surface water, and sediments from wastewater treatment plants. Furthermore, TCC's main degradation products, 3,4-dichloroaniline (DCA) and 4-chloroaniline (4-CA), also have stable chemical structures and exhibit strong toxicity (higher than TCC) and resistance to degradation. Both TCC and DCA are identified as emerging endocrine disruptors and emerging organic pollutants (EOCs).

[0003] Currently, there are many methods for degrading triclocarban, but these methods are limited by technology, processing capacity, environment and other conditions, and cannot continuously process triclocarban parent compound and its intermediate degradation products. At the same time, there is the problem of immature degradation mechanism. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of amidase and phenol hydroxylase in the degradation of triclocarban and its intermediates, the application provided by the present invention being able to reduce the possibility of secondary pollution during the degradation of triclocarban.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of amidase in the degradation of triclocarban, wherein the amidase is TccG, and the gene nucleotide sequence of TccG is shown in SEQ ID No. 1.

[0007] The present invention also provides the application of phenol hydroxylase in the degradation of triclocarban intermediates, wherein the phenol hydroxylase is PhL, and the gene nucleotide sequence of PhL is shown in SEQ ID No. 3.

[0008] Preferably, the triclocarban intermediate includes 4-chloroaniline and 3,4-dichloroaniline.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] This invention constructs a microbial co-culture system for the synergistic degradation of TCC by strains BX2 and LY-1, and optimizes the degradation conditions. The degradation pathway is then inferred by detecting degradation products, and whole-genome sequencing is used to mine potential degradation genes in strains BX2 and LY-1, identifying enzymes involved in TCC degradation and key degradation enzyme genes. Finally, the key degradation enzyme genes are expressed to verify their effects, and the synergistic metabolic mechanism of TCC by strains BX2 and LY-1 in the co-culture system is analyzed. The results show that the microbial co-culture system constructed in this invention involves strain BX2 metabolizing TCC to obtain intermediate products 4-CA and DCA through its produced amidases (TccS, TccG), followed by the degradation of 4-CA by the phenol hydroxylase PhB produced by strain BX2, and the degradation of DCA by the phenol hydroxylase PhL produced by strain LY-1. This solves the problem of secondary pollution caused by the production of 4-CA and DCA during TCC degradation. This invention optimizes the biological pathway for TCC degradation by bacterial strains, reduces the possibility of secondary pollution during the degradation process, provides a reference for further utilizing microbial interactions to completely remediate TCC and its structural analogues, and lays the foundation for future applications of enzyme resources. Attached Figure Description

[0011] Figure 1 The pathway by which co-cultured strains BX2 and LY-1 synergistically degrade TCC;

[0012] Figure 2 This is a phylogenetic tree of amidase genes, with the same color indicating a high degree of similarity;

[0013] Figure 3 This is a phylogenetic tree of phenol hydroxylase genes; the same color indicates a high degree of similarity.

[0014] Figure 4 The co-degradation pathway of TCC by strains BX2 and LY-1 and the relative expression of TCC degradation gene mRNA; among which... Figure 4 In the diagram, A represents the degradation pathway. The numbers 1, 2, 3, 4, 5, 6, 7, 8, and 9 in A represent amidase, phenol hydroxylase, catechol 1,2-dioxygenase, adipodecanedioic acid cyclic isomerase, dienolactone hydrolase, mucinolone δ-isomerase, 4-carboxymucinolone decarboxylase, succinate semialdehyde dehydrogenase, and succinyl-CoA conjugation enzyme, respectively. Figure 4 In the equation B, the relative expression levels of enzymes related to the TCC degradation gene mRNA are represented. The numbers 1, 2, 3, 4, 5, 6, 7, 8, and 9 in B represent amidase, phenol hydroxylase, catechol 1,2-dioxygenase, adipodecic acid cyclic isomerase, dienolactone hydrolase, mucinolone δ-isomerase, 4-carboxymucinolone decarboxylase, succinate semialdehyde dehydrogenase, and succinyl-CoA conjugate, respectively.

[0015] Figure 5 Electrophoresis results for the tccS, tccG, phL, and phB genes; Figure 5 In the image, the first and last columns from left to right are both DNA Marker DL2000; 1 is the PCR product of the tccS gene; 2 is the PCR product of the phB gene; 3 is the PCR product of the tccG gene; and 4 is the PCR product of the phB gene.

[0016] Figure 6 For Kan R Cm R Amp R Electrophoresis results of genes; Figure 6 In the image, the first and last columns from left to right are both DNAMarker DL1000; 1 represents Kan. R PCR product of gene; 2 is Cm R PCR product of the gene; 3 is Amp R PCR products of genes;

[0017] Figure 7 For tccS-Kan R -tccS、phB-Amp R -phB、tccG-Cm R -tccG、phL-Kan R Electrophoresis results of -phL; Figure 7 In the image, the first and last columns from left to right are both DNA Marker DL2000; 1 represents tcc. S -Kan R -tcc S PCR product of the gene; 2 is phB-Amp R -phB gene PCR product; 3 is tccG-Cm R -tccG gene PCR product; 4 is phL-Kan R PCR product of the -phL gene.

[0018] Figure 8 Electrophoresis results for four gene deletion mutant strains; Figure 8 In the image, the first and last columns from left to right are both DNAMarker DL1000; 1 represents Kan. R PCR product of gene; 2 is Cm R PCR product of the gene; 3 is Amp R PCR product of the gene; 4 is Kan R PCR products of genes;

[0019] Figure 9The results of SDS-PAGE electrophoresis of purified amidase TccG and phenol hydroxylase PhL are shown. Figure 9 In the image, columns 1 and 4 from left to right are for maker; 1 and 2 are for purified TccG; 3 and 4 are for purified PhL.

[0020] Figure 10 The results show the effect of different temperatures on the specific enzyme activity of key enzymes;

[0021] Figure 11 The results show the effect of different pH values ​​on the specific enzyme activity of key enzymes;

[0022] Figure 12 Results showing the effects of metal ions and chemical substances on the specific enzyme activity of key enzymes;

[0023] Figure 13 The results show the degradation of triclocarban by amidase and phenol hydroxylase; yellow represents the degradation rate of TCC by TccG, and blue represents the degradation rate of 4-CA by PhL. Detailed Implementation

[0024] This invention provides the application of amidase in the degradation of triclocarban.

[0025] In this invention, the amidase preferably includes TccS and TccG, and more preferably TccG. The gene nucleotide sequence of the amidase TccG of this invention is shown in SEQ ID No. 1, specifically as follows:

[0026]

[0027] In this invention, the gene nucleotide sequence of the amidase TccS is shown in SEQ ID No. 2, specifically as follows:

[0028]

[0029] This invention also provides the application of phenol hydroxylase in the degradation of triclocarban intermediates.

[0030] In this invention, the phenol hydroxylase preferably includes PhL and PhB, and more preferably PhL. The gene nucleotide sequence of the phenol hydroxylase PhL of this invention is shown in SEQ ID No. 3, specifically as follows:

[0031]

[0032] In this invention, the gene nucleotide sequence of the phenol hydroxylase PhB is shown in SEQ ID No. 4, specifically as follows:

[0033]

[0034] In this invention, the triclocarban intermediate includes 4-chloroaniline and 3,4-dichloroaniline.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] By analyzing the TCC degradation products of *Rhodococcus rhodochrous* BX2 and *Pseudomonas sp.* LY-1 (both purchased from the China General Microbiological Culture Collection Center) under co-culture conditions using LC-MS, three TCC degradation pathways were deduced. Combined with whole-genome analysis, genes potentially involved in TCC degradation in both strains were identified and verified using RT-qPCR. The synergistic metabolic mechanism between strains BX2 and LY-1 was analyzed. The obtained metabolic mechanisms are as follows: Figure 1 As shown.

[0038] Depend on Figure 1 It is known that the amide bond (CN) of TCC is broken by amidases (chr-orf04940, chr-orf00841, chr-orf04781, chr-orf06854, LY-1_02220 and LY-1_04110), forming DCA and 4-CA, which reduces the concentration of TCC in the co-culture system, allowing strain LY-1 to adapt to environmental stress as quickly as possible. Furthermore, the generation of DCA and 4-CA provides the material basis for the growth and metabolism of strain LY-1.

[0039] Based on the differential expression results of degradation genes, it can be seen that in the first pathway, strain LY-1 utilizes phenol hydroxylases (LY-1_02562 and LY-1_01766) to hydroxylate and deaminate the product DCA, forming 4,5-DCC. The benzene ring of 4,5-DCC is ortho-cleaved by catechol 1,2-dioxygenases (chr-orf07022, LY-1_05271, and LY-1_01620) from the two strains to form 3,4-dichloromuconic acid. 3,4-dichloromuconic acid is then dechlorinated and esterified by muconic acid cyclic isomerases (chr_orf06981, LY-1_01623, and LY-1_05273) in LY-1 to produce 3-chlorodrinol. Subsequently, 3-chlorodrinol is hydrolyzed by drinolol hydrolase (LY-1_020). 32. LY-1_02964 and LY-1_03498 hydrolyze 3-chloro-4-hydroxymucoconic acid, which is then decarboxylated by 4-carboxymucoconyl lactone decarboxylases (chr_orf01301, chr_orf01896, chr_orf06982, LY-1_01137, LY-1_01625, LY-1_02637, and LY-1_02828) to form succinic acid and acetic acid, which subsequently enter the tricarboxylic acid cycle for further metabolism. Based on the number of genes involved and gene expression trends, strain LY-1 participates in all stages, but strain BX2, lacking a desylolone hydrolase gene, does not participate in the hydrolysis of 3-chlorodesyl lactone.

[0040] In the second pathway, DCA is first hydroxylated and deaminated by phenol hydroxylases (LY-102562 and LY-101766) of strain LY-1 to form 4,5-DCC. The benzene ring of 4,5-DCC is then cleaved at the ortho position and dechlorinated by catechol 1,2-dioxygenases (chr-orf07022, LY-105271 and LY-101620) of the two strains to form 3-chlorocis-muconic acid. 3-chlorocis-muconic acid is then esterified by muconic acid cyclic isomerases (chr_orf06981, LY-1_01623 and LY-1_05273) to form muconic acid lactone. Muconic acid lactone delta isomerases (chr_orf07019 and LY-1_05272) generate the transient intermediate metabolite β-adipic acid glycol ester-lactone. β-Glycol adipate-lactone is decarboxylated by 4-carboxymidine lactone decarboxylases (chr_orf01301, chr_orf01896, chr_orf06982, LY-1_01137, LY-1_01625, LY-1_02637, and LY-1_02828) to produce β-ketoadipic acid. β-ketoadipic acid is then converted into a coenzyme by succinyl-CoA synthases (chr_orf01657, chr_orf01659, LY-1_02024, and LY-1_02025), which then enters the tricarboxylic acid cycle. Based on the number of genes involved and gene expression trends, strains BX2 and LY-1 are involved in all stages of this pathway, and the intermediate metabolites are jointly utilized by the co-cultured strains.

[0041] In the third pathway, 4-CA is hydroxylated and deaminated by phenol hydroxylases (chr_orf07198, LY-102562, and LY-1_01766) to produce 4-CC. Subsequently, 4-CC is acted upon by catechol-1,2-dioxygenases (chr_orf07022, LY-1_05271, and LY-1_01620) to form 3-chlorocis-muconic acid. 3-chlorocis-muconic acid undergoes esterification to produce muconic acid lactone. Muconic acid lactone then undergoes cycloisomerization and delta isomerization to produce β-ketoadipic acid, which ultimately enters the tricarboxylic acid cycle. Based on the number of genes involved and their expression trends, strains BX2 and LY-1 are involved in all stages of this pathway.

[0042] In the three pathways described above, the products of TCC hydrolysis can exchange substances between co-cultured strains. Under the action of different enzymes produced by the two strains, they are eventually degraded into small molecules and enter the tricarboxylic acid cycle.

[0043] Example 2

[0044] By performing whole-genome analysis on the two strains, two key genes for the degradation of TCC and its intermediate product DCA were obtained. The nucleotide sequences of the genes encoding amidase and phenol hydroxylase were determined, as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0045] Example 3: RT-qPCR detection of TCC degradation gene expression

[0046] 3.1 RNA Extraction from Co-cultured Strains: Primers designed using Primer5 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. RT-qPCR experiments were performed in treatment and control groups. The co-cultured strains were inoculated into inorganic salt medium containing 15 mg / LTCC at 32.0℃, pH 6.9, a 3:2 inoculation ratio of strain BX2 to LY-1, and a total inoculation volume of 6.67% (v / v). These were the treatment groups (T1, T2, T3), while those inoculated into inorganic salt medium containing 1.2 mM glucose served as the control groups (CK1, CK2, CK3). Bacterial cells were collected at 0, 24, 48, 72, 96, 120, 144, and 168 h, and total RNA was extracted from the co-cultured strains BX2 and LY-1. RNA extraction was performed according to the instructions on the Total RNA Extractor (Trizol) kit (Thermo Fisher Scientific).

[0047] 3.2 Determination of expression of degradation genes inferred from the genome during TCC degradation: Using 16S rRNA as an internal control, RT-qPCR was used to detect the dynamic changes of each gene at the transcriptional level. The transcriptional level of the genes was measured using 2... -ΔΔCt The method was evaluated.

[0048] By reviewing literature and combining whole-genome data of strains BX2 and LY-1 with the TCC degradation pathway, it was inferred that the key degradative enzyme gene in the first step of TCC degradation by co-cultured strains BX2 and LY-1 is an amidase gene. Thirty-one amidase genes were annotated in the genomes of strains BX2 and LY-1. To determine which amidases play a role in co-culture, an amino acid sequence phylogenetic tree was constructed between these 31 amidase genes and the already characterized amidase gene (tccA:KU753911) with TCC degradation function. The constructed phylogenetic tree is shown below. Figure 2 As shown.

[0049] Phenol hydroxylase PhL (chr_orf07198) is an enzyme that degrades chloroaniline, acting as the second-step enzyme in TCC degradation. Eight phenol hydroxylase genes and related functional genes were annotated in the LY-1 genome. We constructed an amino acid sequence phylogenetic tree for these eight phenol hydroxylase genes and the phenol hydroxylase gene PhL (chr_orf07198), which has already been characterized for chloroaniline degradation. The results are shown below. Figure 3 As shown.

[0050] 3.3 Expression of 36 genes in co-culture of strains BX2 and LY-1: Based on whole-genome analysis and phylogenetic tree analysis, with glucose as a control, the expression of 36 genes involved in the 9-step enzyme metabolism process of strains BX2 and LY-1 in the co-culture system was verified by RT-qPCR. The results are as follows: Figure 4 As shown.

[0051] In summary, under co-culture conditions, when strains BX2 and LY-1 synergistically degrade TCC, except for the fifth step where only strain LY-1 participates, both strains are involved in all other reactions. Specifically, strain BX2 is the main participant in steps 1 and 8, while strain LY-1 plays a dominant role in steps 2, 3, 6, 7, and 9. During TCC degradation, the genes encoding amidases chr_orf04781 and chr_orf04940, and the genes encoding phenol hydroxylase chr_orf0719 and LY-1_01766 act in steps 1 and 2 of degradation, respectively. These two steps may be the rate-limiting steps of TCC degradation. Furthermore, these four genes exhibit high and stable expression levels. Based on the KEGG pathway annotated by gene annotation and relevant literature, it is speculated that these four genes may be key degradation genes in the synergistic TCC degradation process of strains BX2 and LY-1. Therefore, these four genes were selected as target genes for functional verification.

[0052] Example 4: Functional verification of key degradation genes tccS, tccG, phL, and phB

[0053] 4.1 Construction experiments of key degradation gene deletion mutants BX2△tccS, BX2△tccG, LY-1△phL, and BX2△phB, with the following specific treatment settings:

[0054] Strains BX2 and LY-1 were inoculated into LB medium and cultured at 30℃ with shaking at 150 rpm until the logarithmic growth phase. Genomic DNA was extracted from strains BX2 and LY-1 using an Ezup Column Bacteria Genomic DNA Purification Kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), strictly following the kit instructions. Using the extracted genomic DNA from strain BX2 as a template, the tccS, phB, and tccG genes were amplified by PCR; using the extracted genomic DNA from strain LY-1 as a template, phL was amplified by PCR. The primers are shown in Table 1. Results were detected by 1% agarose gel electrophoresis. The genomic DNA from strains BX2 and LY-1 was stored at -20℃. The results are shown below. Figure 5 As shown.

[0055] Table 1 Primers for gene amplification

[0056]

[0057] The results showed that the sizes of the tccS, phB, and tccG genes were 1503bp, 1044bp, and 1410bp, respectively; the size of the phL gene was 1194bp.

[0058] At 16℃, the amplified tccS, phB, tccG, and phL genes were ligated overnight into the pUC57 vector (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain ligation reaction products (the plasmids carrying the target genes tccS, tccG, phL, and phB were named pUC57-tccS, pUC57-tccG, pUC57-phL, and pUC57-phB, respectively). The ligation reaction products were transformed into competent E. coli DH5α cells (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain E. coli DH5α competent cells containing the tccS, phB, tccG, and phL genes, respectively. The next day, positive single clones were picked and cultured overnight. Plasmids were extracted from the bacterial culture, and PCR and single-enzyme digestion were performed using the extracted plasmids as templates for verification.

[0059] At the same time, select Kan respectively R Cm R Amp R As a disruptor gene, genes containing Kan were selected. R The gene plasmid pET-28a (purchased from Sangon Biotech (Shanghai) Co., Ltd.), containing Cm R The gene plasmid pKO3 (a kind gift from Professor Wang Jintong of Taiwan, China) and containing Amp RThe plasmid pUC19 (purchased from Baori Biotechnology (Beijing) Co., Ltd.) was used as a template to amplify the corresponding Kan gene using PCR. R Cm R Amp R After the gene reaction, electrophoresis was performed, and the target fragment was purified to obtain the purified product. The results are as follows: Figure 6 As shown.

[0060] The Kan R Cm R Amp R The primers for gene amplification are shown in Table 2.

[0061] Table 2 Kan R Cm R Amp R Gene amplification primers

[0062]

[0063] After verification, using pUC57-tccS, pUC57-tccG, pUC57-phL, and pUC57-phB as backbone vectors, enzyme digestion and ligation were performed sequentially between the target gene tccS, tccG, phL, and phB sequences to insert the cloned Kansas gene as the break-up gene. R Cm R Kan R Amp R Genes, thereby obtaining the recombinant fragment tccS-Kan R -tccS、tccG-Cm R -tccG、phL-Kan R -phL, phB-Amp R -phB. The obtained recombinant fragments were subjected to PCR and electrophoresis respectively. Results are as follows: Figure 7 As shown.

[0064] The recombinant fragment tccS-Kan obtained above R -tccS、tccG-Cm R -tccG, phB-Amp R -phB was added to the prepared BX2 competent cells to obtain mixture 1; the recombinant fragment phL-Kan obtained above was added to the mixture. R-phL was added to the prepared LY-1 competent cells to obtain mixture 2. Mixtures 1 and 2 were then transferred to pre-cooled electroporation cuvettes for electroporation. The parameters of the gene transfer instrument were set to 2250V and 4.5ms. Immediately after electroporation, the bacterial culture in the electroporation cuvette was resuspended in 1mL of SOC medium (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and cultured at 30℃ and 150rpm for 4h with constant temperature shaking. 100μL of the bacterial culture was then spread onto antibiotic LB agar plates and incubated upside down. The following mutant strains were obtained: BX2△tccS (tccS gene deletion mutant), BX2△tccG (tccG gene deletion mutant), LY-1△phL (phL gene deletion mutant), and BX2△phB (phB gene deletion mutant).

[0065] After the bacteria have grown on the plate, five single colonies are randomly picked from the plate using a sterilized toothpick and cultured at 30°C for 24 hours. The genomes of the selected tccS, tccG, phL, and phB gene deletion mutants are extracted, and the corresponding disrupted genes Kan are amplified. R Amp R Cm R Kan R The electrophoresis results are as follows Figure 8 As shown.

[0066] Table 3 Primers designed with recombinant fragments containing gene disruption.

[0067]

[0068] 4.2 Functional validation experiments of key degradation gene deletion mutants BX2△tccS, BX2△tccG, LY-1△phL, and BX2△phB: The specific settings are as follows:

[0069] Treatment 1: The BX2△tccS mutant containing the tccS gene deletion was inoculated at a rate of 3% (v / v) (OD600nm=2.0±0.1) into a basic culture medium containing 14.5 mg / L TCC inorganic salts.

[0070] Treatment 2: The BX2△tccG mutant containing the tccG gene deletion was inoculated at a rate of 3% (v / v) (OD600nm=2.0±0.1) into a basic culture medium containing 14.5 mg / L TCC inorganic salts.

[0071] Treatment 3: Strain BX2 was inoculated at a rate of 3% (v / v) (OD600nm = 2.0 ± 0.1) into a basic culture medium containing 14.5 mg / L TCC inorganic salts;

[0072] Treatment 4: The LY-1△phL mutant containing the phL gene deletion was inoculated at a rate of 2% (v / v) (OD600nm=2.0±0.1) into inorganic salt basal culture medium containing 7.5 mg / L DCA and inorganic salt basal culture medium containing 7.5 mg / L 4-CA, respectively.

[0073] Treatment 5: Strain LY-1 was inoculated at a rate of 2% (v / v) (OD600nm = 2.0 ± 0.1) into inorganic salt basal culture medium containing 7.5 mg / L DCA and inorganic salt basal culture medium containing 7.5 mg / L 4-CA, respectively.

[0074] Treatment 6: The BX2△phB mutant containing the phB gene deletion was inoculated at a rate of 3% (v / v) (OD600nm=2.0±0.1) into inorganic salt basal culture medium containing 7.5 mg / L DCA and inorganic salt basal culture medium containing 7.5 mg / L 4-CA, respectively.

[0075] Treatment 7: Strain BX2 was inoculated at a rate of 3% (v / v) (OD600nm = 2.0 ± 0.1) into inorganic salt basal culture medium containing 7.5 mg / L DCA and inorganic salt basal culture medium containing 7.5 mg / L 4-CA, respectively.

[0076] Treatment 8: The BX2△tccS mutant containing the tccS gene deletion was co-inoculated with strain LY-1 at an inoculation rate of 4% (v / v) (OD600nm = 2.0 ± 0.1) and 2.67% (v / v) (OD600nm = 2.0 ± 0.1) in a basic culture medium containing 14.5 mg / L TCC inorganic salts and cultured for 7 days.

[0077] Treatment 9: The BX2△tccG mutant containing the tccG gene deletion was co-inoculated with strain LY-1 at an inoculation rate of 4% (v / v) (OD600nm = 2.0 ± 0.1) and 2.67% (v / v) (OD600nm = 2.0 ± 0.1) in a basal culture medium containing 14.5 mg / L TCC inorganic salts and cultured for 7 days.

[0078] Treatment 10: The BX2△phB mutant containing the phB gene deletion was co-inoculated with strain LY-1 at an inoculation rate of 4% (v / v) (OD600nm = 2.0 ± 0.1) and 2.67% (v / v) (OD600nm = 2.0 ± 0.1) in a TCC inorganic salt basal medium containing 15 mg / L and cultured for 7 days.

[0079] Treatment 11: The BX2△phB mutant containing the phB gene deletion was co-inoculated with the LY-1△phL mutant containing the phL gene deletion at a 4% (v / v) (OD600nm=2.0±0.1) inoculation rate and cultured for 7 days in a TCC inorganic salt basal medium containing 14.5 mg / L.

[0080] Treatment 12: The BX2△tccS mutant containing the tccS gene deletion was co-inoculated with the LY-1△phL mutant containing the phL gene deletion at a 4% (v / v) (OD600nm=2.0±0.1) inoculation rate and cultured for 7 days in a TCC inorganic salt basal medium at a 2.67% (v / v) (OD600nm=2.0±0.1) inoculation rate.

[0081] Treatment 13: The BX2△tccG mutant containing the tccG gene deletion was co-inoculated with the LY-1△phL mutant containing the phL gene deletion at a 4% (v / v) (OD600nm=2.0±0.1) inoculation amount and cultured for 7 days in a TCC inorganic salt basal medium at a 2.67% (v / v) (OD600nm=2.0±0.1) inoculation amount.

[0082] For treatments 1 to 7, each treatment was set up in 3 parallel groups, and cultured at 30℃ and 150r / min for 5 days. The culture medium was taken out every 24 hours to determine the contents of TCC, DCA and 4-CA.

[0083] For treatments 8 and 10, the content of TCC was measured after 7 days of culture to verify the degradation ability of the co-culture system when the amidase genes tccS and tccG were missing.

[0084] For treatments 11 and 12, the content of TCC was measured after 7 days of culture to verify the degradation ability of the co-culture system when the phenol hydroxylase genes phB and phL were missing.

[0085] For treatments 12 and 13, the content of TCC was measured after 7 days of culture to verify the degradation capacity of the co-culture system when both amidase and phenol hydroxylase were absent.

[0086] The results showed that key degradation genes tccS, tccG, phB, and phL all played a role in TCC degradation, with TccS having a greater effect than TccG. PhL from LY-1 was mainly responsible for DCA degradation, while PhB from BX2 was mainly responsible for 4-CA degradation. The simultaneous absence of both amidase and phenol hydroxylase inhibited the hydrolysis of the amide bond in TCC and the deamination of DCA with 4-CA, having the greatest impact on TCC degradation in the co-culture system.

[0087] Example 5: Expression and Functional Verification Experiment of Four Key Genes

[0088] 5.1 SDS-PAGE Identification: The amidase (TccG) gene of strain BX2 and the phenol hydroxylase (PhL) gene of strain LY-1 were amplified by PCR using the primers in Table 1. These genes were then fused into NdeI / XhoI- and BamHI / saci-digested pET-28a(+) (purchased from Sangon Biotech (Shanghai) Co., Ltd.), respectively, to generate pET-tccG and pET-PhL. The constructed expression vectors were then transformed into competent cells of prokaryotic expression strain E. coli BL21(DE3) (purchased from Sangon Biotech (Shanghai) Co., Ltd.) using the heat shock transformation method. After overnight culture, positive clones were screened, and single colonies were selected for PCR identification. The E. coli BL21(DE3) containing the expression vector was preserved in glycerol, and the obtained products were named recombinant strains E. coli BL21(TccG) and E. coli BL21(PhL), respectively.

[0089] Recombinant strains E. coli BL21 (TccG) and E. coli BL21 (PhL) were inoculated into LB liquid medium containing 30 μg / mL kanamycin and cultured at 37℃ and 150 r / min for 4 h until the growth reached OD. 600nm =0.6, add IPTG solution to induce expression to a final concentration of 0.8 mM, incubate at 20℃ and 150 r / min for 12 h, centrifuge at 4℃ and 6000 r / min for 30 min, discard the supernatant, collect the cells, add 2 mL PBS buffer to wash twice, and resuspend the cells in 2 mL PBS buffer until OD600nm = 2.0; after resuspending the cells, place the centrifuge tube on ice and sonicate (power 300W, 5s interval, 2s working time, total time 30 min). Centrifuge the obtained recombinant enzyme TccG and PhL lysis buffers at 4℃ and 12,000 r / min for 30 min respectively. The obtained supernatant is the crude protein extract (named crude protein extract containing TccG and crude protein extract containing PhL respectively). Take 48 μL of the crude protein extract and add it to 12 μL of 5×SDS loading buffer for SDS-PAGE detection. The results are as follows. Figure 9As shown.

[0090] SDS-PAGE gel electrophoresis showed that the molecular weights of TccG and PhL were 50 and 43 kDa, respectively, which are consistent with the theoretical molecular weights of 50.37 kDa and 44.29 kDa.

[0091] 5.2 Thermostability assay of amidase TccG and phenol hydroxylase PhL: The crude protein extracts containing TccG and PhL obtained above were pre-incubated in 50 mM, pH 7.0 PBS at 15, 20, 25, 30, 35, 40, 50, 60, 70, and 80 °C for 60 min, respectively. Then, 10 mg / L TCC was added to the reaction system containing TccG, and 5 mg / L 4-chloroaniline (4-CA) was added to the reaction system containing PhL. The mixtures were incubated at 35 °C for 1 h. The results are as follows: Figure 10 As shown.

[0092] The results showed that amidase TccG exhibited relatively low degradation efficiency at pH 7.0 and 80℃, with a specific activity of only 1.50%. Its optimal degradation efficiency was achieved at 35℃, reaching 100%. Phenol hydroxylase PhL showed a similar pattern, with the lowest degradation efficiency at 80℃ and a specific activity of 0.12%. At 35℃, its optimal degradation efficiency was achieved, reaching 100%. In conclusion, the thermostability of both enzymes was optimal at 35℃.

[0093] 5.3 Acid-base stability assay of amidase TccG and phenol hydroxylase PhL: The crude protein extracts containing TccG and PhL obtained above were tested in six buffers at 35℃. The six buffers covered pH 2.0-11.0 to obtain the optimal pH range for amidase and phenol hydroxylase activities. The six buffers were: glycine-hydrochloric acid buffer (pH 2.0-3.0), citrate-sodium citrate buffer (pH 3.0-6.0), phosphate buffer (pH 6.0-8.0), Tris-hydrochloric acid buffer (pH 8.0-9.0), glycine-sodium hydroxide buffer (pH 8.0-10.0), and borate buffer (pH 9.0-11.0). The results are as follows: Figure 11 As shown.

[0094] The results showed that the specific enzyme activity of the crude protein extract containing TccG was 2.01% at pH 2.0, indicating poor degradation. While the specific enzyme activity was 35.25% at pH 10.0, showing some improvement compared to the acidic state, the optimal degradation effect was achieved at pH 7.0, reaching 100%. The crude protein extract containing PhL showed the worst degradation effect at pH 2.0 and the best degradation effect at pH 7.0. In conclusion, both exhibited the strongest stability at pH 7.0.

[0095] 5.4 Effects of Metal Ions and Other Chemical Substances on the Activities of Amidease TccG and Phenol Hydroxylase PhL: Different metal ions, such as Cu, were added to 10 mL reaction systems. 2+ Ca 2+ Fe 2+ Mg 2+ Zn 2+ and Ba 2+ Chemical detergents, such as SDS, Tween-80, and EDTA. Specific procedures are as follows:

[0096] 10 mL reaction system: Add 1 mL of crude protein extract containing TccG to 9 mL of PBS buffer containing 10 mg / L TCC, and incubate at 30°C for 60 min; add 1 mL of crude protein extract containing PhL to 9 mL of PBS buffer containing 10 mg / L DCA, and incubate at 30°C for 60 min. After the reaction, extract three times with 10 mL of dichloromethane. Concentrate the extracted organic phase by rotary evaporation, and finally dissolve in 1 mL of acetone, filter into a 2 mL HPLC vial, and analyze by HPLC. Results are as follows: Figure 12 As shown.

[0097] The results showed that Ba 2+ The inhibitory effect on amidase TccG was most significant, while SDS showed the most significant inhibitory effect on phenol hydroxylase PHL. It is worth noting that Cu... 2+ The enhancement effect on phenol hydroxylase PhL was significant, with a specific enzyme activity of 126.96%.

[0098] Example 6: Verification experiment on the degradation of triclocarban by TccG and PhL

[0099] Referring to Example 5, after expressing the amidase (TccG) of strain BX2 and the phenol hydroxylase (PhL) of strain LY-1, 1 mL of amidase was added to 9 mL of potassium phosphate buffer (50 mM, pH = 7.5) to start the reaction, resulting in a 10 mL reaction system. TCC was added to the reaction system until the final concentration of TCC in the reaction system was 15 mg / L. After the reaction was completed, 10 mL of dichloromethane was added to extract the TCC. The sample was analyzed by HPLC and MS / MS, and the results are as follows. Figure 13 As shown.

[0100] 1 mL of phenol hydroxylase was added to 9 mL of potassium phosphate buffer (50 mM, pH = 7.5) to initiate the reaction, yielding a 10 mL reaction system. 4-CA was added to the reaction system until the final concentration of the above substance was 7.5 mg / L. After the reaction was complete, 10 mL of dichloromethane was added to extract 4-CA. The sample was analyzed by HPLC and MS / MS, and the results are as follows. Figure 13 As shown.

[0101] The results showed that, under conditions of pH 7.0 and 35℃, the degradation rate of TCC by amidase reached 75.68%, and the degradation rate of 4-CA by phenol hydroxylase reached 71.53%. Figure 13 It can be seen that TccG degrades TCC, and the degradation product is 4-CA. PhL degrades 4-CA, which leads to the conclusion that amidase and phenol hydroxylase can take turns degrading TCC, thus enhancing the TCC degradation effect.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of phenol hydroxylase in the degradation of triclocarban intermediates, characterized in that, The phenol hydroxylase is PhL, and the gene nucleotide sequence of PhL is shown in SEQ ID No. 3; The triclocarban intermediates are 4-chloroaniline and 3,4-dichloroaniline.

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