A highly efficient amidohydrolase for degrading ochratoxin A and its encoding gene, recombinant vector and application

Abstract

The invention discloses an amide hydrolase for efficiently degrading ochratoxin A and its encoding gene, recombinant vector and application, belonging to the technical field of enzyme engineering. The invention also provides the optimal pH of the amide hydrolase and its application in degrading ochratoxin A. The recombinant amide hydrolase treats milk and tea samples containing ochratoxin A in a buffer system of pH 7.5 at room temperature for 20-30 minutes, and the degradation rate of ochratoxin A reaches 100%.

Description

Technical Field

[0001] The invention relates to an amide hydrolase for efficiently degrading ochratoxin A and a coding gene, a recombinant vector, a recombinant bacterium and application thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] Mycotoxins are a class of secondary metabolites produced by various filamentous molds. They are commonly found in moldy grains and their products, and are extremely harmful to agriculture, food processing and animal husbandry. Well-known mycotoxins include aflatoxins (AF), fumonisins (FUM), zearalenone (ZEN), deoxynivalenol (DON) and ochratoxin A (OTA). Mycotoxins are nephrotoxic, hepatotoxic, neurotoxic, cytotoxic, immunotoxic, embryotoxic, carcinogenic and teratogenic. The intake of mycotoxins can delay the growth and development of animals at the mildest, and cause liver and kidney damage or even death at the worst.

[0003] Among them, ochratoxin A (OTA) is mainly produced by pure green Penicillium, Aspergillus ochraceus and Aspergillus carbonii. OTA is the culprit for poultry ochre poisoning and mycotoxic pig nephropathy; OTA can destroy the correct synthesis of proteins, hinder metabolic pathways, disrupt calcium homeostasis, and damage human and animal DNA. Long-term exposure to OTA in humans can lead to symptoms such as renal interstitial fibrosis and tubular atrophy. At present, OTA detoxification is divided into physical, chemical and biological methods. Among them, physical detoxification includes screening, washing, peeling, heating, repeated freezing and thawing, cold plasma treatment, ultraviolet irradiation, material adsorption, etc. The physical detoxification method has the disadvantages of product nutritional loss, high requirements for equipment and instruments, and high energy consumption. Chemical detoxification methods include ozone treatment, inorganic acid treatment, organic acid treatment, alkaline compound treatment, strong oxide treatment, salt treatment, etc., but there are problems such as chemical residues, destruction of food flavor and nutritional value, and production of harmful substances. The use of specific bio-enzymes to catalyze the degradation and detoxification of OTA has the advantages of low cost, safety, and high efficiency, and is considered to be the most promising method for removing OTA pollution. Therefore, obtaining more efficient OTA degradation and detoxification enzymes and their encoding genes through gene screening or molecular modification technology will provide an important biological solution for the efficient degradation and detoxification of OTA.

[0004] Amide hydrolases can hydrolyze OTA to generate non-toxic ochratoxin α (OTα) and phenylalanine. This hydrolysis pathway is considered to be the most effective detoxification method for OTA. The amide hydrolase ADH3 from Stenotrophomonas sp.CW117 is the most efficient OTA hydrolase reported so far, which can convert OTA into OTα and L-phenylalanine. In order to achieve more efficient degradation and detoxification of OTA, it is urgently needed to obtain more efficient OTA hydrolysis and detoxification enzymes through gene mining or molecular modification technology. Summary of the invention

[0005] To solve the above problems, the present invention uses bioinformatics technology to mine and obtain the amidohydrolase PwADH from Pseudoxanthomonas wuyuanensis from the National Center for Biotechnology Information (full name in English: National Center of Biotechnology Information, abbreviated as: NCBI). The present invention first discovered that PwADH can efficiently hydrolyze OTA to generate non-toxic OTα and phenylalanine. By comparing PwADH with the previously reported most efficient amidohydrolase ADH3 for degrading OTA, it was found that the hydrolysis activity of PwADH on OTA was 7.3 times higher than that of ADH3; further, through structural analysis, rational design and site-directed mutagenesis technology of PwADH, the amino acid sequence of PwADH was transformed, and it was found that the 88th glycine in the amino acid sequence of PwADH was mutated into aspartic acid, and the constructed G88D mutant had a further 1.2-fold increase in the hydrolysis activity of OTA, thereby having greater application value in industrial applications.

[0006] The first object of the present invention is to provide a recombinant amide hydrolase, the amino acid sequence of the amide hydrolase consisting of the amino acid sequence shown in SEQ ID NO.2.

[0007] The second object of the present invention is to provide a recombinant amide hydrolase G88D with improved catalytic activity, wherein the amino acid sequence of the amide hydrolase consists of the amino acid sequence shown in SEQ ID NO.4, wherein the amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the glycine at position 88 of the sequence shown in SEQ ID NO.2 to aspartic acid.

[0008] The third object of the present invention is to provide a gene encoding the recombinant amide hydrolase, a gene encoding a highly efficient recombinant amide hydrolase consisting of the amino acid sequence of SEQ ID NO.2, or a gene encoding a highly efficient recombinant amide hydrolase consisting of the amino acid sequence of SEQ ID NO.4.

[0009] Furthermore, the encoding gene is a recombinant amidohydrolase gene consisting of the nucleotide sequence shown in SEQ ID NO.1.

[0010] Furthermore, the encoding gene is a recombinant amidohydrolase gene consisting of the nucleotide sequence shown in SEQ ID NO.3.

[0011] The polynucleotide sequence encoding the amidohydrolase of the present invention can be obtained by a variety of methods. For example, the polynucleotide can be isolated using hybridization techniques well known in the art. These techniques include (but are not limited to): (1) using probes to hybridize with gene or cDNA libraries to detect homologous polynucleotide sequences and (2) activity screening of expression libraries to detect cloned polynucleotide fragments with common structural characteristics. The expression library may include an environmental metagenomic library or a clone library constructed from a pure culture strain. The DNA fragment sequence of the present invention can also be obtained by the following methods: (1) isolating double-stranded DNA sequences from genomic DNA;

[0012] (2) Chemically synthesizing a DNA sequence to obtain a double-stranded DNA of the amidohydrolase.

[0013] The third object of the present invention is to provide a recombinant vector carrying the gene.

[0014] Furthermore, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0015] In the present invention, the polynucleotide encoding the amidohydrolase or the recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. "Host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell.

[0016] Transformation of host cells with DNA sequence of the present invention or recombinant vector containing said DNA sequence can be carried out by conventional techniques well known in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, which is well known in the art, and MgCl2 can be used as an alternative. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection method can be selected: calcium phosphate coprecipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0017] The method for producing the recombinant amidohydrolase according to the present invention comprises at least the following steps:

[0018] (1) a step of expressing a polypeptide from at least one gene according to any one of claims 3 to 6; and

[0019] (2) A step of collecting the polypeptide expressed in step (1).

[0020] Utilizing the polynucleotide sequence of the present invention, expressing or producing the recombinant amidohydrolase by conventional recombinant DNA technology comprises the following steps:

[0021] (1) transforming or transfecting a suitable host cell with a polynucleotide encoding a recombinant amidohydrolase of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0022] (2) culturing host cells in a suitable culture medium;

[0023] (3) Isolate and purify proteins from culture medium or cells.

[0024] In step (2), the culture medium used in the culture can be selected from various conventional culture media according to the host cells used. The culture is carried out under conditions suitable for the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method, and the cells are cultured for a period of time.

[0025] In step (3), the recombinant enzyme can be encapsulated in the cell, expressed on the cell membrane, or secreted outside the cell. The recombinant protein is separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, affinity chromatography, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and various other liquid chromatography techniques and combinations of these methods.

[0026] The fourth object of the present invention is to provide the use of the recombinant amidohydrolase in degrading ochratoxin A. Furthermore, the use includes degrading ochratoxin A in milk or tea.

[0027] A fifth object of the present invention is to provide a method for removing ochratoxin A from food, comprising at least the following steps:

[0028] (A) mixing an aqueous solution containing the recombinant amidohydrolase of the present invention with a food containing ochratoxin A;

[0029] (B) reacting the mixture at a pH of 5 to 9.8 and a temperature not exceeding 80° C. until ochratoxin A in the mixture is detected to be below a threshold value.

[0030] The fifth object of the present invention is to provide an enzyme preparation comprising the recombinant amidohydrolase.

[0031] The beneficial effects of the present invention are as follows: the present invention provides a recombinant amide hydrolase and a gene encoding the same, and also provides the optimal pH of the amide hydrolase and the application of the amide hydrolase in hydrolyzing ochratoxin A and detoxifying ochratoxin A in milk or tea. The amide hydrolase treats milk or tea containing ochratoxin A in a buffer system of pH 7.5 at room temperature for 20-30 minutes, and the degradation rate of ochratoxin A reaches 100%, and this degradation efficiency is highly efficient in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the chemical reaction equation of PwADH hydrolyzing ochratoxin A to generate phenylalanine and ochratoxin α; Figure 2 The SDS-PAGE protein electrophoresis of the purified recombinant amide hydrolase ADH3, recombinant amide hydrolase PwADH and improved PwADH mutant G88D;

[0033] Figure 3 The HPLC chromatograms of the products of OTA degradation by purified amide hydrolase ADH3, recombinant amide hydrolase PwADH and improved PwADH mutant G88D;

[0034] Figure 4 The relative enzymatic activities of ADH3, PwADH and the improved PwADH mutant G88D in degrading ochratoxin A were compared;

[0035] Figure 5 To compare the degradation effects of ochratoxin A in milk by amide hydrolase ADH3, recombinant amide hydrolase PwADH and improved PwADH mutant G88D;

[0036] Figure 6 To compare the degradation effects of ochratoxin A in Fuzhuan tea powder by amide hydrolase ADH3, recombinant amide hydrolase PwADH and improved PwADH mutant G88D;

[0037] Figure 7 This is a graph showing the results of enzyme activity detection of recombinant amide hydrolase PwADH under different conditions. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] Example 1: Synthesis of amidohydrolase PwADH gene and construction of its mutants

[0040] The amide hydrolase PwADH derived from Pseudoxanthomonas wuyuanensis was mined from the National Center of Biotechnology Information (NCBI) through bioinformatics technology. The nucleotide sequence of PwADH is shown in SEQ ID NO.1, and the amino acid sequence of the PwADH amide hydrolase encoded by it is shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.1 was submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and cloned into the Escherichia coli expression vector pET46EK to obtain the recombinant plasmid pET46EK-PwADH.

[0041] By analyzing the amino acid sequence of the amidohydrolase PwADH, the glycine at position 88 of the sequence shown in SEQ ID NO.2 was mutated to aspartic acid to obtain a mutant G88D with improved OTA hydrolysis activity, the nucleotide sequence of the mutant G88D is shown in SEQ ID NO.3, and the amino acid sequence of the mutant G88D is shown in SEQ ID NO.4. The nucleotide sequence of the G88D mutant was submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and cloned into the recombinant vector pET46EK to obtain the recombinant plasmid pET46EK-G88D.

[0042] The amide hydrolase gene ADH3 was obtained by a total gene synthesis method according to the method described in ZL201910004511.3. The nucleotide sequence of ADH3 is shown in SEQ ID NO.5, and the amino acid sequence of the ADH3 amide hydrolase encoded by it is shown in SEQ ID NO.6. Then, the ADH3 gene was connected to the pET46EK vector using restriction endonucleases NcoI and XhoI to construct the pET46EK-ADH3 recombinant plasmid.

[0043] Example 2: Expression and purification of PwADH and mutant proteins in E. coli.

[0044] The recombinant plasmids pET46EK-ADH3, pET46EK-PwADH and pET46EK-G88D were transformed into competent E. coli BL21 (DE3) cells, respectively, and the strains were screened on LB plates containing 100 μg / mL ampicillin to construct E. coli engineered bacteria containing the recombinant plasmids pET46EK-ADH3, pET46EK-PwADH or pET46EK-G88D. 1.0 mL of seed bacterial solution was inoculated into 5 mL of fresh LB, and cultured overnight at 37°C and 180 r / min, and then expanded to 200 mL of LB medium. Finally, it was expanded to 10 L of LB medium. At the OD of the bacteria, 600 When the value reaches 0.6 to 0.8, the culture temperature is lowered to 16°C, and IPTG is added to a concentration of 0.3 mM to induce the expression of a large amount of protein. After another 16 hours of protein induction, the bacterial solution is centrifuged at 6000 rpm for 10 minutes to collect the E. coli cells. Then the cells are resuspended in a buffer (20 mM Tris-HCl, pH 8.0), and the cells are broken using a cell crusher, and then centrifuged at 16000 rpm for 30 minutes, and the supernatant is collected for the next step of purification.

[0045] The target protein was then eluted using a fast protein liquid chromatography instrument using a nickel ion chromatography column, and then dialyzed in 5L (20mM Tris-HCl), pH 8.0 at 4°C overnight, and then purified by an ion column. Finally, the purified target protein was concentrated in 20mM Tris-HCl, pH 7.5 buffer and stored at -80°C. Figure 2 This is the SDS-PAGE protein electrophoresis gel image of the purified recombinant amide hydrolase ADH3, PwADH and improved PwADH mutant G88D expressed in Escherichia coli.

[0046] Example 3: OTA hydrolysis activity determination:

[0047] In order to compare the differences in the activities of ADH3, PwADH and the improved PwADH mutant G88D, this example further determined the activities of ADH3, PwADH and the improved PwADH mutant G88D in hydrolyzing OTA.

[0048] The activity test method of OTA hydrolase is as follows: the mixture (100 μL) of each reaction is in 50 mM Tris-HCl, pH 8.0 buffer, and the mixture includes substrate 200 μg / mL OTA and 10 μL amide hydrolase (10 μg / mL). After the mixture is mixed, it is placed in a shaking metal bath at 40°C and 800 rpm for 20 minutes. Each reaction is performed in 3 parallels and repeated 3 times. After the reaction, an equal volume of acetonitrile is added to terminate the enzyme reaction, and then centrifuged at 12000 rpm for 10 minutes, and the supernatant reaction solution is filtered through a 0.22 μm filter membrane. Then, each group of reactions was subjected to product determination and analysis by high performance liquid chromatography (HPLC, Shimadzu SPD-M20A), with an analytical column of InertSustain C18 column (4.6×250 mm, 5 μm), mobile phase A: water, mobile phase B: 95% acetonitrile + 5% acetic acid, flow rate 1 mL / min, detection wavelength 330 nm, elution time 20 min, and elution concentration 50%. The high performance liquid chromatography detection results of the degradation products of OTA by ADH3, PwADH and G88D are shown in Figure 2. Figure 3 As shown, the peaks were eluted at retention time 4.6min and retention time 12.3min, respectively. Among them, the peak time of retention time 4.6min is consistent with the product standard OTα, so the substance with retention time 4.6min is OTα; and the peak time of retention time 12.3min is consistent with the substrate standard OTA, so the substance with retention time 12.3min is OTA. Then, the activity difference was calculated by comparing the peak areas of ADH3, PwADH and G88D hydrolysis product OTα.

[0049] from Figure 3 The data showed that the products of OTA hydrolysis by ADH3, PwADH and G88D were OTα and phenylalanine, and OTα could be detected. Figure 4 In this example, the peak area value of the hydrolysis product OTα of ADH3 was taken as 100%, and the peak areas of the hydrolysis products OTα of PwADH and G88D were compared as relative enzyme activities. Figure 4 As shown in the results, the activity of PwADH is 7 times that of ADH3, and the activity of G88D is 1.2 times that of PwADH. Therefore, the mutant protein G88D has a higher application value in the OTA degradation industry.

[0050] Example 4: Application of recombinant amidohydrolase in detoxification of milk contaminated with ochratoxin A

[0051] The standard stock solution of ochratoxin A was mixed with milk to prepare milk contaminated with ochratoxin A (final concentration was 400 μg ochratoxin A / mL milk), and the recombinant plasmids (pET46EK-ADH3, pET46EK-PwADH and pET46EK-G88D) were expressed and then broken, and the supernatant of the bacterial cell disruption liquid containing the recombinant amide hydrolase was collected; the supernatant was mixed evenly with the pre-prepared ochratoxin A contaminated milk, and the prepared samples were cultured at 37°C, and samples were taken at 0min, 5min, 10min, 15min, 20min, 25min and 30min, and 3 replicates were set for each degradation experiment. At the same time, phosphate buffer was mixed evenly with the pre-prepared ochratoxin A contaminated milk as a negative control. The sample size of the treatment group and the blank control group was the same each time, and the extraction, purification and analysis were carried out according to the standard detection method of ochratoxin A. The degradation rate of ochratoxin A by recombinant amide hydrolase was measured according to the toxin extraction and purification detection method described in ZL201910004498.1. The results are as follows: Figure 5 As shown, in the negative control group without enzyme addition, ochratoxin A was stable in phosphate buffer and was not degraded within 0-30 min; while the recombinant amide hydrolases ADH3, PwADH and G88D completely degraded ochratoxin A in the sample in more than 30 min, 25 min and 20 min, respectively, with a degradation efficiency of 100%; the degradation efficiency of G88D for OTA was significantly better than that of PwADH and ADH3, which was consistent with the relative enzyme activity determination of ADH3, PwADH and G88D in Implementation Case 3.

[0052] Example 5: Application of recombinant amidohydrolase in detoxification of tea leaves contaminated with ochratoxin A

[0053] The standard stock solution of ochratoxin A was mixed with the crushed Fuzhuan tea powder to prepare the Fuzhuan tea powder sample contaminated with ochratoxin A (the final concentration was 400 μg ochratoxin A / g Fuzhuan tea powder). The recombinant plasmids (pET46EK-ADH3, pET46EK-PwADH and pET46EK-G88D) were expressed and then broken, and the supernatant was collected; the supernatant was mixed evenly with the pre-prepared Fuzhuan tea powder contaminated with ochratoxin A, and the prepared samples were cultured at 37°C, and samples were taken at 0min, 5min, 10min, 15min, 20min, 25min and 30min, and 3 replicates were set for each degradation experiment. At the same time, phosphate buffer was mixed evenly with the pre-prepared Fuzhuan tea powder contaminated with ochratoxin A as a negative control. The sample size of the treatment group and the blank control group was the same each time, and the extraction, purification and analysis were carried out according to the standard detection method of ochratoxin A. The degradation rate of ochratoxin A by recombinant amide hydrolase was measured according to the extraction and purification detection method of ochratoxin A described in ZL201910004498.1. The results are as follows: Figure 6 As shown, in the negative control group without enzyme addition, ochratoxin A was stable in phosphate buffer and was not degraded within 0-30 min; while the recombinant amide hydrolases ADH3, PwADH and G88D completely degraded ochratoxin A in the sample in more than 30 min, 25 min and 20 min, respectively, with a degradation efficiency of 100%; the degradation efficiency of G88D for OTA was significantly better than that of PwADH and ADH3, which was consistent with the relative enzyme activity determination of ADH3, PwADH and G88D in Implementation Case 3.

[0054] Example 6: Optimization of enzymatic reaction conditions of amide hydrolase PwADH

[0055] Referring to the activity test method of OTA hydrolase shown in Example 3, the optimum temperature of PwADH was tested, and the metal bath temperatures used in the reaction were set to 10°C, 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, 70°C, and 80°C, respectively, and three replicates were set for each degradation experiment.

[0056] Referring to the activity test method of OTA hydrolase shown in Example 3, the optimal pH of PwADH was tested, and the buffer pH of the reaction mixture was set to 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0, respectively, and 3 replicates were set for each degradation experiment.

[0057] Referring to the activity test method of OTA hydrolase shown in Example 3, the effect of metal ions on PwADH was tested. 10 mM metal ions (LiCl, CaCl2, MgCl2, CoCl2, MnCl2, NiCl2, FeCl2, CuCl2, and ZnCl2) were added to the buffer of the reaction mixture, and 3 replicates were set for each degradation experiment.

[0058] Referring to the activity test method of OTA hydrolase shown in Example 3, the temperature tolerance of PwADH was tested. Before the reaction, PwADH was bathed in water at 0°C, 10°C, 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, and 70°C for 1 hour, and each degradation experiment was repeated 3 times.

[0059] from Figure 7 According to the data of A, PwADH has a high OTA degradation activity (>70%) in the range of 30-50°C, but the residual enzyme activity of PwADH drops to 20% and 30% at 10°C and 70°C, respectively. Figure 7 According to the data of B, PwADH has a high OTA degradation activity (>60%) in the pH range of 7.0-8.5. Figure 7 According to the data of C, Mn2+ 、Ni 2+ , Fe 2+ , Cu 2+ and Zn 2+ The OTA degradation activity of PwADH was significantly inhibited by other metal ions such as Ca 2+ Mg 2+ and Co 2+ It also showed a slight inhibitory effect. Figure 7 According to the data of D, when PwADH was cultured at below 20°C for 1 h, the enzyme activity remained above 90%. When the temperature increased to 30°C, 40°C, 50°C and 60°C, the remaining enzyme activity decreased to 68%, 43%, 32% and 3%, respectively.

[0060] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A recombinant amidohydrolase, characterized in that The amino acid sequence of the recombinant amidohydrolase consists of the amino acid sequence shown in SEQ ID NO. 4, wherein the amino acid sequence shown in SEQ ID NO. 4 is obtained by mutating the glycine at position 88 of the sequence shown in SEQ ID NO. 2 to aspartic acid.

2. A gene encoding a recombinant amidohydrolase consisting of the amino acid sequence of SEQ ID NO.

4.

3. The coding gene according to claim 2, characterized in that A recombinant amidohydrolase gene consisting of the nucleotide sequence shown in SEQ ID NO.

3.

4. A recombinant vector carrying the recombinant amidohydrolase gene construct according to claim 3.

5. A method for producing the recombinant amidohydrolase according to claim 1, comprising at least the following steps: (1) a step of expressing a polypeptide from at least one gene according to any one of claims 2 to 3; and (2) A step of collecting the polypeptide expressed in step (1).

6. Use of a recombinant amidohydrolase in the degradation of ochratoxin A, characterized in that: The amino acid sequence of the recombinant amide hydrolase consists of the amino acid sequence shown in SEQ ID NO. 4 or SEQ ID NO. 2, and the application is for purposes other than disease diagnosis and treatment.

7. A method for removing ochratoxin A from food, comprising at least the following steps: (A) mixing an aqueous solution containing the recombinant amidohydrolase according to claim 6 with a food containing ochratoxin A; (B) reacting the mixture at a pH of 5 to 9.8 and at a temperature not exceeding 80° C. until ochratoxin A in the mixture is detected to be below a threshold value.

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