166results about How to "Clear genetic background" patented technology

The invention relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

The invention relates the construct method for transgenosis rhinestone algae bioreactor. The method comprises the following steps: using the rhinestone algae as transgenosis receptor biology, highly effective expressing the carrier construct by constructing the exogenesis gene containing screening mark expression frame, leading the destination genes in rhinestone algae with bead milling method, electric punching method and other methods, integrating them into nucleus genome or chloroplast genome, and adopting a series of gene expression regulate and control technology to construct the transgenosis rhinestone algae bioreactor. The transgenosis rhinestone algae bioreactor possesses the characters of fast growth rate, short breeding cycle, easy segregation, photoautotroph and large scale culture, fast and cheap producing medicinal protein, industrial raw materials, and feeding vaccination, and possessing the bioactivity secondary metabolite.

The invention discloses a Bacillus subtilis adenylosuccinate synthetase mutant gene purA and applications thereof. The sequence of the Bacillus subtilis adenylosuccinate synthetase mutant gene purA is shown in SEQ ID No.1. Engineering bacteria containing the Bacillus subtilis adenylosuccinate synthetase mutant gene purA (C725T) and built according to the invention are good in bio-safety and clear in genetic background, and the contained mutant gene purA (C725T) can significantly improve the riboflavin synthesis capacity of Bacillus subtilis, and under the condition of fermentation in a shaking flask, the level of riboflavin accumulation is increased by over 40%.

The invention discloses a genetically engineered bacterium for producing L-arginine and a construction method and application thereof. According to the invention, a gene for encoding carbamyl phosphate synthetase and a gene for encoding L-arginine biosynthetic pathway enzyme are integrated in escherichia coli; a synthetic route of arginine in escherichia coli and metabolic flux related to argininein a whole amino acid metabolic network are analyzed and reconstructed to obtain genetically engineered bacteria which are clear in genetic background, do not carry plasmids, are not mutated and canstably and efficiently produce L-arginine; and the genetically engineered bacteria have good L-arginine production capacity.

The invention provides a genetic engineering bacterium capable of producing uridine at high yield as well as a building method and application thereof. The genetic engineering bacterium is characterized in that pyrimidine nucleoside operons pyrBCAKDFE with the nucleotide sequence shown as SEQ ID NO:1 is integrated on a genome of colon bacillus; the starting is realized by a strong promoter P[trc];a uridine synthesis path is reconstituted; the self PRPP synthetase coding gene prsA on the genome is subjected to dual copying, and the starting is realized by the strong promoter P[trc]; meanwhile,the activity of udk, udp, and rihA, rihB and rihC is lacked; the thrA activity is lacked; the argF activity is lacked. The genetic engineering bacterium is applied to fermentation production of uridine; 40 to 67g / L of uridine can be produced after the fermentation is performed for 40 to 70h in a 5L fermentation tank; the maximum production intensity can reach 1.5g / (L*h); the glucoside conversionrate is 15 to 25 percent; the genetic engineering bacterium belongs to the highest level for producing the uridine by the fermentation method reported in the prior art.

The invention provides a biological method for synthesizing alpha-pinene or beta-pinene from acetyl coenzyme A and a reconstitution cell capable of synthesizing the alpha-pinene or beta-pinene, wherein the acetyl coenzyme A is obtained from a simple starting material like glucose. The method for synthesizing the alpha-pinene or beta-pinene by adopting a biological process comprises the following steps: transferring acetyl coenzyme A acyltransferase, 3-hydroxy-3-methyl glutaryl coenzyme A synthase, hydroxymethyl glutaryl coenzyme A reductase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyldiphosphate synthetase and pinene synthase into an appropriate host cell by adopting metabolic engineering technology, and culturing the obtained reconstitution cell, so that the target product, namely alpha-pinene or beta-pinene can be obtained.

The invention discloses a genetically engineered bacterium co-generating geraniol and nerol and a construction method and an application thereof, and belongs to the technical field of generic field. According to the genetically engineered bacterium disclosed by the invention, acetyl coenzyme A acyltransferase / hydroxymethyl glutaryl coenzyme A reductase, 3-hydroxyl-3-methyl glutaryl coenzyme A synthase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyl diphosphonic acid synthetase and geraniol synthetase or phosphatase are expressed. The metabolic pathways of geraniol and nerol synthesized in escherichia coli are successfully constructed by genetic engineering means, and glucose is biologically converted into geraniol and nerol.

The invention relates to an escherichia coli genetically engineered bacterium and a method for efficiently producing L-tryptophan by utilizing the escherichia coli genetically engineered bacterium. The genetically engineered bacterium is obtained by correspondingly transforming and combining L-tryptophan synthesis-related genes in escherichia coli through a metabolic engineering method (includinggene integration, point mutation and promoter substitution) and introducing a glutamine synthetase coding gene glnA from lactobacillus acidophilus on genome of the escherichia coli. The strain is usedfor carrying out shake-flask fermentation; 10 to 12g / L of L-tryptophan can be accumulated within 24 hours, reaches the maximum value reported in China and is improved by 15.1 percent compared with that of an existing L-tryptophan industrial strain carrying plasmid, which shows that the strain has good potential for industrially producing the L-tryptophan.

The present invention provides a high-yield L-histidine genetically engineered bacterium strain and a constructing method thereof. The bacterium integrates a nucleotide sequence of a corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant encoding gene hisG* represented by SEQ ID NO:1 based on genome of escherichia coli to enhance activity of histidine-synthesizing key enzyme HisG, also increases copy number of histidine operon genes in the genome to enhance a terminal synthesis pathway of histidine, and also integrates an encoding gene lysE of arginine / lysine transportprotein derived from the corynebacterium glutamicum to promote extracellular secretion of the intracellular histidine. The genetically engineered bacterium is used for producing the L-histidine by a fermentation method, can stably produce 40-55 g / L of the histidine by fermentation in a 5L fermentation tank for 40-50 h, has a production intensity reaching 1.0-1.5 g / (Lxh), and has a conversion rate of 0.18-0.22 g histidine / g glucose.

The invention relates to a heterodimer protein of a recombinant human bone morphogenetic protein and an efficient expression and renaturation method of the heterodimer protein. The method comprises the following steps: linking a BMP (Bone Morphogenetic Protein) 7 gene with a BMP2 gene through DNA fragments encoding (GlyGlyGlyGlySer)3 by virtue of a fusion PCR technology, inserting fusion gene into a pHis-NusA plasmid so as to construct a recombinant plasmid, introducing the recombinant plasmid into escherichia coli BL21 (DE3), culturing at 37 DEG C, and inducing the expression by virtue of 0.5mM IPTG. The results show that the BMP7 / BMP2heterodimer protein can be efficiently expressed by virtue of the method, the expressed protein is present in a form of an inclusion body and a large number of renaturated proteins can be obtained by virtue of simple purification and renaturation of the inclusion body. According to the method, the operation for obtaining the BMP active protein is simple, the preparation cost is greatly reduced and the method is suitable for large-scale fermentation production and provides the possibility of clinical wide application of BMP in the treatment of bone damage.

The invention provides a genetically engineered strain for high yield of ergothioneine and application. The stain takes escherichia coli as a host, and a coding gene hisG* of a corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant is integrated on a genome of the host; the copy number of a histidine operon gene hisDBCHAFI is further increased on the genome of the host; a mycobacterium smegmatis ergothioneine operon gene egtBCDE is further integrated on the genome of the host; an Escherichia coli glutamylcysteine ligase encoding gene gshA is integrated on the genome of the host,so that the synthesis of the ergothioneine is promoted; a neurospora crassa C-S lyase coding gene egtE<ncr> is further integrated on the genome of the host, so that the synthesis of ergothioneine isfurther promoted; and a gene egtB*<msm> of a sulfoxide synthase mutant is integrated on a genome of the host, so that synthesis of ergothioneine is further promoted.

The present invention provides a new strain of bacillus circulans WZ-12 which can degrade methylene chloride and an application of the bacillus circulans WZ-12 for microorganism to decompose and dispose the methylene chloride and preparing for methylene chloride dehalogenase. The bacillus circulans WZ-12 is conserved in the China Center for Typical Culture Collection; the conserving date is 19th, January, 2007; the conserving serial number CCTCC No. is M207006. The beneficial effect of the present invention mainly lies in providing the new strain of the bacillus circulans WZ-12 which can degrade the methylene chloride, the bacillus can degrade the methylene chloride fast under the aerobic condition, which provides important base for researching into the biological degradation mechanism of halohydrocarbon organic pollutant represented by the methylene chloride, and the bacillus can also be applied to a biological disposal technology of organic waste gas directly. Besides, the strain is wild type with very clear genetic background, which is fit for genetic improvement and is hopeful to improve the capability of disposing the organic waste gas greatly.

The invention provides a genetic engineering bacterium for L-theanine production and a fermentation method thereof. A construction method of the genetic engineering bacterium comprises the steps of carrying out single copying on an ribonucleic acid (RNA) polymerase gene T7RNAP derived from T7 phage on an Escherichia coli W3110 genome, wherein the gene is controlled by a xylose promoter; carrying out dual copying on a gamma-glutamyl methylamine synthetase gene gmas derived from methylovorusmays, wherein the gene is controlled by a T7 promoter; knocking out a xylose operon repressor protein genexylR; and knocking out a succinyl CoA synthetase gene sucCD. The genetic engineering bacterium has the beneficial effects that the yield of L-theanine fermented by using the genetic engineering bacterium can be up to 40g / L and the sugar acid conversion rate can be up to 25%; and the L-theanine production method provided by the invention has the advantages of being low in raw material price, shortin cycle, simple and convenient to operate, green and friendly to environment and high in yield and has good industrial application value.

A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene are disclosed. Acetyl coenzyme A used in the method is obtained from simple initial materials such as glucose. The method includes: A) a step of constructing the reconstituted cell capable of synthesizing the beta-caryophyllene from the acetyl coenzyme A, wherein the reconstituted cell comprises gene segments comprising acetyl-CoA acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A synthetase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, geranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, and beta-caryophyllene synthase; and B) a step of culturing the reconstituted cell in a culture medium containing glucose, inducing with a proper inducer, separating and purifying to obtain the beta-caryophyllene.

The invention provides an escherichia coli genetically engineered bacterium for the synchronous high yield of L-tryptophan and L-valine, and application thereof. The genetically engineered bacterium is obtained through the steps that on a genome of escherichia coli, trpE (S40F) mutation is introduced while a promoter of tryptophan operon is replaced with a Ptrc promoter; an aroG (S180F) gene controlled by the Ptrc promoter is integrated to a tyrR locus; a serA (H344A, N364A) gene controlled by a Plac promoter is integrated to a yjiV pseudo gene locus; a glnA gene controlled by the Plac promoter is integrated to a ycjV pseudo gene locus; and then a bacillus subtilis alsS gene controlled by the Ptrc promoter is integrated to a yghx pseudo gene locus. Shaking flask fermentation is conducted through a strain to be able to accumulate the L-tryptophan within 22-28 h to 10-14 g / L, meanwhile the accumulation amount of valine reaches 5-7 g / L, and the total acid-producing ability is improved by50% or so compared with that of a tryptophan producing strain; and meanwhile, strain OD600 is different slightly, growth problems are avoided, but the acid-producing ability per strain is obviously improved by 120%, and effective utilization of carbon sources and cells is improved greatly.

The invention discloses a bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and an application thereof. The bacillus subtilis-coded PRPP transamidase mutant gene pruF sequence is shown as SEQ ID No.1. An engineering bacterium which is established and contains the bacillus subtilis-coded PRPP transamidase mutant gene pruF is biologically safe and clear in genetic background, so that the capacity of the bacillus subtilis which synthesizes riboflavin can be greatly improved, and the accumulation level of riboflavin can be improved by over 20%.

The invention discloses escherichia coli capable of producing L-alanine through fermentation and application of the escherichia coli and belongs to the technical field of biological engineering. According to the escherichia coli, aspartase AspA and L-aspartic acid-beta-decarboxylase AsD are introduced into the escherichia coli capable of producing fumaric acid through the fermentation; furthermore, a YgaW gene is over-expressed to strengthen the transport ability of the L-alanine in recombinant bacteria, so that a novel strain capable of fermenting glucose to produce the L-alanine is constructed; when the recombinant strain is fermented in a fermentation tank for 45h, the yield of the L-alanine reaches 147g / L and the saccharic acid conversion rate is 79.0 percent.

The invention provides a preparation method of small molecular proteins or polypeptides. The method comprises the following steps: (a) establishing a gene engineering strain, wherein the gene engineering strain comprises a recombinant expression vector with a fusion protein coding nucleotide sequence, the fusion protein is an F-L-polypeptide, the F is fusion chaperonin and selected from aldosterone isomerase protein, aldosterone isomerase protein mutants, human glucagon-like peptide-1 and human glucagon-like peptide-1 mutants; L is a connecting peptide; the polypeptides are insulin or analogues thereof, GLP-1 or analogues thereof, GLP-2 or analogues thereof, antibacterial peptides or thymosin; (b) culturing the gene engineering strain so as to express the fusion protein; (c) purifying thefusion protein, and performing protease cleavage, thereby obtaining the small molecular proteins or polypeptides with biological activities. The method disclosed by the invention overcomes the defectthat the small molecular proteins or polypeptides are difficultly expressed, and provides a new thought for expressions of the proteins.

The invention provides a porcine reproductive and respiratory syndrome virus attenuated strain. The porcine reproductive and respiratory syndrome virus is a novel recombinant chimeric virus obtained by respectively substituting a Nsp9 gene and a Nsp10 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus strain with a Nsp9 gene and a Nsp10 gene of a classic porcine reproductive and respiratory syndrome virus attenuated strain. The porcine reproductive and respiratory syndrome virus attenuated strain can further contain a GP5 gene of the highly pathogenic porcine reproductive and respiratory syndrome virus attenuated strain. The invention also discloses a vaccine composition containing the attenuated strain and application thereof to preparation of drugs for preventing and / or treating porcine reproductive and respiratory syndrome. The attenuated strain overcomes the security problems of the artificial passage attenuated vaccine; the vaccine composition prepared from the attenuated strain not only can be used for the prevention of common porcine reproductive and respiratory syndrome but also can prevent highly pathogenic porcine reproductive and respiratory syndrome.

The invention relates to a method for breeding high-temperature-resistant Paralichthys olivaceus new varieties and belongs to the technical field of agricultural mariculture. In the method, Paralichthys olivaceus half sibs family and sibs family with a clear pedigree are established; multi-generation breeding is performed on the Paralichthys olivaceus family at half lethal temperature; inbreeding possibly occurring in the breeding process and tolerance of the Paralichthys olivaceus at different growth stages of juvenile fish and adult fish to the lethal temperature are taken into full consideration; and screening is repeated for two or more times, so that the high-temperature-resistant Paralichthys olivaceus new varieties can be bred effectively, and the problem that the Paralichthys olivaceus in some provinces in south China are difficult to aestivate.

The invention belongs to the biological field and particularly relates to preparation and application of recombinant porcine circovirus PCV2-Cap / Phage display particle. A preparation method of the capsid protein phage display particle of recombinant II porcine circovirus specifically comprises the following four steps: (1), optimizing and coding nucleotide of the capsid protein of the recombinant II porcine circovirus; (2), constructing a recombinant plasmid; (3), constructing a recombinant phage genome; and (4), preparing the capsid protein phage display particle of recombinant II porcine circovirus. An animal experiment proves that the recombinan display plasmid has good antigenicity, can stimulate a pig body to generate good humoral immunity and cellular immunity reaction, and can prevent and control II porcine circovirus infection; time needed for copying the process is short, production cost of the recombinant display particle is low, and product is convenient to use, simple and convenient to store and high in cloning yield.

The invention belongs to the field of material synthesis, and particularly relates to a biosynthesis method of a FeS-coated rGO composite material. According to the invention, dissimilatory metal reducing bacteria are utilized to decompose organic matters to generate electrons in the metabolic process, and GO is converted into rGO; and then water-soluble sulfate and ferric iron are used as final electron acceptors to obtain the biosynthetic FeS-coated rGO composite material, and the composite material can be directly used for removing Cr<6+>. The FeS nano-particles biosynthesized by the methodare regular in morphology and have a smaller size effect, and the technical breakthrough that the FeS nano-particles are uniformly anchored to rGO lamellas is realized. The biosynthesis method of theFeS-coated rGO composite material disclosed by the invention is wide in raw material source, simple and convenient to operate, high in reaction atom economy and environment-friendly, and the application prospect of the FeS-coated rGO composite material in the fields of environment, catalysis, optics, sensing, electrochemical energy storage performance and the like is further expanded.

The invention relates to an unmarked gene-deleted and attenuated mutant strain of a vibrio alginolyticus wild strain, which is the attenuated active vaccine of the vibrio alginolyticus wild strain and in which the sRNA molecular chaperone gene hfq of the vibrio alginolyticus wild strain is deleted. The vibrio alginolyticus wild strain is EPGS020401, and the collection number of the vibrio alginolyticus wild strain is CCTCC NO.AB 209306. The invention also provides the preparation of the unmarked gene-deleted and attenuated mutant strain of the vibrio alginolyticus wild strain and the use thereof in prevention and control of vibrio alginolyticus disease in cultured fish. The unmarked gene-deleted and attenuated mutant strain of the vibrio alginolyticus wild strain or the preparation thereof, which are disclosed by the invention, eliminates universal potential environment and product safety risks of the conventional attenuated vaccine and is a safe, effective and economic vaccine for preventing and controlling vibrio alginolyticus diseases in cultured fish.

The invention relates to the field of genetic engineering and the field of protein secretion expression and provides a method for constructing a pichia pastoris engineered strain used for preparing dextranase. The method comprises the following steps: (1) designing an oligonucleotides primer and taking lipomyces 1390 genome DNA as a template; cloning to obtain dextranase genes; (2) utilizing a secretion expression carrier pPIC9K to insert the dextranase genes into multi-cloning sites (EcoRI and NotI) of the pPIC9K so as to construct an integral secretion-type expression carrier pPIC9K-139; and(3) linearizing the reconstructed expression carrier pPIC9K-139 so as to realize the high-efficiency secretion expression of the dextranase, and obtaining the reconstructed pichia pastoris engineeredstrain GS115-139 by means of selection. The method successfully realizing the effective expression of the dextranase genes in pichia pastoris, constructs the engineered strain which can efficiently secrete and express the dextranase, and realizes the expansion of a fermentation process of the dextranase.

The invention belongs to the field of medical chemistry, and aims at providing a method for synthesizing trans-4-hydroxy-L-proline. According to the technical scheme, by virtue of genetiuc engineeringmodified escherichia coli, [alpha]-ketoglutaric acid, which is generated from autologous TCA circulation of a catalysis host, is converted into an L-proline gene, and the L-proline gene undergoes tandem and over-expression, so that the yield of L-proline is improved; and prolyl hydroxylase is introduced, so that a reaction that the L-proline is converted into the trans-4-hydroxy-L-proline and a reaction that the [alpha]-ketoglutaric acid is converted into succinic acid are coupled; therefore, the efficient synthesis of the trans-4-hydroxy-L-proline under a condition of not adding exogenous L-proline is achieved. The synthesis method of the trans-4-hydroxy-L-proline provided by the invention is optimized, so that the yield of the target product (the trans-4-hydroxy-L-proline) is greatly improved.

The invention discloses a chicken egg yolk antibody resisting human A rotavirus as well as a preparation method and application thereof. The method comprises steps of expressing and purifying structural protein VP7 or VP8 of human rotavirus in vitro, immunizing laying hens, collecting eggs, and separating and purifying egg yolk antibody of VP7 or VP8. The prepared chicken egg yolk antibody can be freeze dried and prepared into antibody dry powder for preventing and treating rotavirus. The preparation method has the beneficial effects that a large quantity of VP7 and VP8 antigens are expressed and recombined in vitro by a genetic engineering technology, the benefits are high, the cost is low, small field is required, and the method is environment-friendly; the potential risk of scattering virus caused by inactivation by rotavirus or attenuated vaccine in the traditional preparation process of egg yolk antibody is avoided, and the method meets the requirement of animal welfare. The egg yolk antibody prepared by the method can be prepared into oral antibody dry powder biological agent after sterility test, has obvious effect in treating rotavirus infectious diarrhea, takes effect rapidly and has low cost, and the prevention rate reaches over 95%.

The invention relates to a marker-free gene deletion attenuated mutant of a vibrio alginolyticus wild strain. The mutant is an attenuated live vaccine of the vibrio alginolyticus wild strain, wherein sRNA molecular chaperone protein gene hfq of the vibrio alginolyticus wild strain is deleted; and the vibrio alginolyticus wild strain is the vibrio alginolyticus wild strain EPGS020401 with a collection number of CCTCC No. AB209306. The invention also provides a preparation of the marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain and related application to prevention and treatment of vibrio alginolyticus diseases of cultured fishes. The marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain or the related preparation eliminates common potential security risk of environment and product existing in the conventional attenuated live vaccine, and is a safe, high-efficiency and economic vaccine aiming at the vibrio alginolyticus diseases of the cultured fishes.

The invention relates to an aquiculture animal disease prevention and control technology, in particular to an Edwardsiella tarda attenuated strain for preventing and controlling Edwardsiella tarda infection and application thereof. An Edwardsiella tarda mutant strain is MZLLSE40aroA which is preserved in CGMCC and has the preservation number: CGMCC No.2886 and the Edwardsiella tarda mutant strain is MZL LSE40 aroAesrB which is preserved in CGMCC and has a preservation number: CGMCCNo.2887. Bacterial cells of 106-109cfu / mL of Edwardsiella tarda strain MZL LSE40aroA or the Edwardsiella tarda strain MZL LSE40 aroAesrB and sterile phosphoric buffer salt solution are mixed as an attenuated live vaccine for preventing and controlling Edwardsiella tarda disease of culture fish. The strain has intraframe large-scale deletion of virulence related genes and nutrition metabolization related genes and non-recoverable virulence, does not contain a foreign gene segment and an antibiotics resistance marker and is safe for the environment and animals.

The invention discloses a method of target-integrating a foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rats and mice as well as application thereof. According to the invention, a stable cell line can be obtained for expressing various foreign genes through constructed TALEN plasmid, rat homologous recombination plasmid and mouse homologous recombination plasmid by means of respectively transferring corresponding plasmids to cells of rats and mice. The stable cell line obtained can be stably handed from generation to generation with clear genetic background, so that the biological function of the foreign gene is accurately represented.

The invention provides a delftia tsuruhatensis strain H1 with high efficiency and fast degradation of chloroaniline substances and an application thereof. The delftia tsuruhatensis strain H1 is collected in China Centre for Type Culture Collection, the address is Wuhan University, Wuhan, China, 430072, the collection number CCTCC No. is M 209249, and the collection data is November 4, 2009. The strain can realize fast degradation of chloroaniline pollutants under aerobic conditions, and play an important role in practices of chloroaniline industrial wastewater treatment. In addition, the strain is wild, has a clear genetic background, is applicable to genetic improvement and is expected to greatly improve the capacity of degrading the chloroaniline pollutants.