Title of Invention

PRODUCTION OF ZEAXANTHIN BY PHAFFIA

Abstract

Disclosed is a process for producing zeaxanthin and ß-cryptoxanthin which comprises cultivating a recombinant microorganism which is expressing a ß-carotene hydroxylase gene and belonging to the genus Xanthophyllomyces (Phaffia) in an aqueous nutrient medium under aerobic conditions, and isolating the resulted carotenoids from the cells of said recombinant microorganism or from the cultured broth.

Full Text

Production of zeaxanthin by Phoffia The present invention relates to production of xanthophyll carotenoids, especially zeaxanthin and β-cryptotoxanthin by microorganism belonging to genus Phaffia. More particularly, the present invention provides a process for producing xanthophyll carotenoids, especially zeaxanthin and 3-cryptoxanthin by genetically modified recombinant microorganism belonging to genus Phaffia. By using methods of the present invention, it becomes possible to produce the useful xanthophyll carotenoids other than astaxanthin such as zeaxanthin and β-cryptoxanthin as a major xanthophyll carotenoid in the recombinant microorganism belonging to the genus Phaffia. Any strain which is able to produce β-carotene can be a suitable host strain. When the selected host strain is a normal Phaffia strain which is capable of producing astaxanthin from (3-carotene, production of mixture of astaxanthin and other xanthophyll carotenoids can be achieved after introduction and expression of a gene coding for β-carotene hydroxylase. On the other hand, when a strain which can not produce astaxanthin and is accumulating β-carotene is selected as the host strain, it is expected to produce maximum level of xanthophyll carotenoids such as zeaxanthin and β-cryptoxanthin, without accu- mulation of astaxanthin. Such a host strain which accumulates β-carotene can be obtained by mutagenesis of a strain of P. rhodozyma accumulating astaxanthin. Alternatively, such a host strain accumulating β-carotene can also be obtained by deactivating astaxanthin synthase which is an enzyme involved in the biosynthesis of astaxanthin from β-carotene and is disclosed in US 6,365,386. Disruption of the gene of astaxanthin synthase will be one of the most convenient ways to deactivate the enzyme. Furthermore, such a host strain accumulating β-carotene can also be obtained from public type culture collections. E.g., P. rhodozyma ATCC96815 accumulating β-carotene can be purchased from American Type Culture Collection (P.O.Box 1549, Manassas, VA 20108, USA). Isolating a new β-carotene accumulating strain of P. rhodozyma, which may be a derivative or a spontaneous mutant of astaxanthin producing strain, from nature will be another approach to prepare the host strain of the present invention. An aspect of the present invention is a process for producing zeaxanthin and β-crypto-xanthin which comprises cultivating a recombinant Phaffia strain which is expressing β-carotene hydroxylase. Said β-carotene hydroxylase catalyzes hydroxylation of β-carotene at positions 3 and 3' on the β-ionone ring to produce the xanthophyll, zeaxanthin via β-cryptoxanthin. Genes encoding this enzyme have been isolated from several species, e.g., crtZ from Flavobacterium species (US 6,124,113), crtZ from Erwinia uredovora, crtZ from Erwinia herbicola, crtZs from marine bacteria, Agrobacteriurri aiirantiacum and Alcaligenes sp., and Paracoccus marcusii (GenBank accession No. Y15112, 1997), and Paracoccus carotinifaciens sp.nov. β-carotene hydrox7lase genes can also be obtained from plant species. In the present invention, any gene encoding a protein having the β-carotene hydroxylase activity can be used. Preferably, said β-carotene hydroxylase gene can be obtained from a microorganism selected from microorganisms of the genera Flavobacterium, Erwinia, Agrobacteriurriy Alcaligenes, and Paracoccus having the β-carotene hydroxylase gene. More preferably, said β-carotene hydroxylase gene can be obtained from Flavobacterium sp. R1534 WT (ATCC21588) (GenBank accession No. U62808), £ uredovora ATCC1932I (GenBank accession No. D90087), E. herbicola ATCC39368 (GenBank accession No. M87280), Agrobacterium aurantiacurn (GenBank accession No. D58420), Alcaligenes PC-1 (GenBank accession No. D58422), Paracoccus marcusii MHl (GenBank accession No. Y15112), or the gram-negative bacteria E-396 (PERM Bβ-4283) (Jβ-A Hei 10-155497) having the β-carotene hydroxylase gene. Still more preferably, said 3-carotene hydroxylase gene can be obtained from Flavobncteri-urn sp. R1534 WT (ATCC21588) (GenBank accession No. U62808), or it can also be obtained as a DNA sequence which is substantially homologous thereto. The expression "a DNA sequence which is substantially homologous" refers with respect to the DNA sequence encoding the β-carotene hydroxylase to a DNA sequence which encodes an amino acid sequence which shows more than 60%, preferably more than 70%, more preferably more than 80%, and most preferably more than 90% identical amino acids when compared to the amino acid sequence of crtZ of Flavobacterium sp. R1534 WT (ATCC21588) and is the amino acid sequence of a polypeptide which shows the same type of enzymatic activity as the enzyme encoded by crtZ of Flavobacterium sp, R1534 WT(ATCC21588). By using the |3-carotene hydroxylase gene, it is possible to render a microorganism belonging to the genus PbaJJia an ability to produce zeaxanthin and |3-cryptoxanthin. The recombinant microorganism ofPhaffia expressing a β-carotene hydroxylase gene can be prepared by the well-known recombinant technology. The techniques used to isolate or clone a DNA encoding β-carotene hydroxylase of the present invention are known in the art and include isolation from genomic DNA. The cloning of the DNA sequence of the present invention from such genomic DNA can be effected by using the polymerase chain reaction (hereinafter referred to as PCR). The isolated or cloned DNA encoding [3-carotene hydroxylase can be preferably utilized after cloning on a suitable expression vector for expression of the enzyme in the host microorganism Phajfia. "Expression vector" includes vectors which are capable of expressing DNA sequences contained therein where such sequences are operably linked to other sequences such as control sequences which are capable of effecting the expression of said DNA sequences in a microorganism belonging to Phaffia. The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. The term "control sequence" is intended to include, ara minimum, components which are necessary for expression of the gene of interest, and may also include additional advantageous components. Generally the control sequences include promoters, terminators and, in some instances, enhancers, transactivators, or transcription factors. Constitutive promoters, such as the glyceraldehyde-3-dehydrogenase (GAP) gene promoter derived from P. rhodozyma (WO 97/23,633), may be used to obtain constitutive expression. Inducible promoters can be also used in order to achieve an exactly-controlled expression. One of the examples for the inducible promoters is the promoter of genes encoding heat shock proteins or amylase gene, and the like. Methods which are well known to those skilled in the art may be used to construct said expression vectors. It is implied, although not explicitly stated, that expression vectors must be replicable in the host organisms either as episomes or as an integral part of a chromosomal DNA. Generally, higher stability of the gene may be expected in the latter case. To integrate the expression vector into the chromosome of the host microorganism by a homologous recombination, a vector is prepared to contain at least a portion of the DNA fragment whose sequence is homologous to the host genomic DNA. For this purpose, rDNA gene fragment can be effectively used in a microorganism belonging to Phaffia, The rDNA is a kind of satellite DNAs which exist in multicopies on the genome. By using the rDNA fragment as a targeting DNA on the expression vector, the objective DNA to be expressed on said vector can be integrated into the host genome, and also can exist as multicopies. This may give the gene dosage effects which will contribute to the overexpression of the objective enzymes. In the Examples of the present invention, such rDNA fragment was conveniently used for this purpose. The invention is intended to include other forms of expression vectors which serve equivalent functions and which are, or subsequently become, known. An isolated DNA sequence encoding β-carotene hydroxylase may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleotide sequence encoding said (3-carotene hydroxylase prior to its insertion into an expression vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing cloning methods are well known in the art. Recombinant DNA consisting of said gene of (3-carotene hydroxylase which was cloned in the expression vector will be introduced into a host microorganism. Methods for the introduction of foreign DNA intojungal cells including a microorganism belonging to PHaffta are well known in the art. These include, e.g., transformation by the LiCl method, protoplast fusion, electroporation, particle-gun methods which comprises bombardment by particles coated with DNAs, and other methods known in the art. In the Examples of the present invention, the particle-gun method was applied as a transformation method for P. rhodozyma. The recombinant organism thus obtained is capable of overexpressing the DNA sequence encoding β-carocene hydroxylase. Thus, the recombinant organism of the present invention is useful in the production process of xanthophyll carotenoids, especially zeaxanthin and β-cryptoxnnthin. A further aspect of the present invention is a biological process for producing zeaxanthin and β-cryptoxanthin which comprises cultivating the recombinant microorganism of Phaffia in the presence of substrate for producing carotenoids in an aqueous nutrient medium under aerobic conditions, and isolating the resulting carotenoids from the cells of said recombinant microorganism or from the cultured broth. Carotenoids including the xanthophylls are normally produced by cultivating a strain of Phaffia in a medium which comprises suitable macro- and micronutrients for the cells, such as molasses, saccharose or glucose as a carbohydrate source for cell growth and also as a substrate for producing carotenoids, and nitrogen sources such as corn steep liquor, yeast extract, diammonium sulphate, ammonium phosphate, ammonium hydroxide or urea, phosphorus sources such as ammonium phosphate and phosphoric acid and added micronutrients or mineral salts such as magnesium sulphate, zinc sulphate and biotin or desthiobiotin. The preferable conditions for cultivation are in a pH range of from 4 to 8 and in a temperature range of from 15 to 26°C for 24 to 500 hours. More preferable conditions for cultivation are a pH in the range of from 5 to 7 and in a temperature range of from 18 to 22oC for 48 to 350 hours. In the cultivation, aeration and agitation usually give favorable results for the production of carotenoids. Once carotenoids were produced by cultivating the recombinant strain of Phaffia using the methods of the present invention, the carotenoids can be isolated either from the medium, in the case they are secreted into the medium, or from the cells of the microorganism and, if necessary, separated from other carotenoids that may be present in case one specific carotenoid is desired, by methods known in the art. Carotenoids produced in accordance with the present invention can be used in a process for the preparation of food or feed. A man skilled in the art is familiar with such processes. Such compound food or feed can further comprise additives or components generally used for such purpose and known in the state of the art. The following Examples further illustrate the present invention, but these are not thereby limiting the scope of the invention. The following materials and methods were employed in the Examples described below: Strains P, rhodozyrnn ATCC96594 (re-deposited under the accession No. ATCC 74438 on April 8» 1998 pursuant to the Budapest Treaty) Vectors pCR2.1-T0P0 (Invitrogen Corporation, Carlsbad, USA) pGEM-T (Promega Corporation, USA) Methods Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo (Ohtsu, Japan). Polymerase chain reaction (PCR) was performed with the thermal cycler from Perkin Elmer model 2400. E.Kh PCR condition is described in examples. PCR primers were purchased from a commercial supplier. Fluorescent DNA primers for DNA sequencing were purchased from Pharmacia. DNA sequencing was performed with the automated fluorescent DNA sequencer (ALFred, Pharmacia). An authentic sample for zeaxanthin and β-carotene was purchased from EXTRASYN- THESE S.A. (Genay Cedex, France) and WAKO (Osaka, Japan), respectively, p-crypto-xanthin was obtained from Roche Vitamins AG (Basle, Switzerland). Example 1: Cloning of the p-carotene hydroxylase gene {crtZ) from genomic DNA of I'lavobacterium sp. ATCC21588 At first genomic DNA was prepared. Cells of Flavobacterium sp. R1534 WT (ATCC21588) (US 6,124,113) were inoculated into a 50 ml medium containing 1% glucose, 1% tryptone (Difco Laboratories), 1% yeast extract (Difco), 0.5% MgSO4, and 3% NaCl, and cultivated at 27°C under aerobic condition. Overnight culture (50 ml) was centrifuged at 10,000 g for 10 min. The pellet was washed briefly with 10 ml of lysis buffer (50 mM EDTA, O.lM NaCl, pH 7.5), resuspended in 4 ml of the same buffer supplemented with 10 mg of lyso-zyme and incubated at 37°C for 15 min. After addition of 0.3 ml of N-Lauroyl sarcosine (20%), incubation at 37°C was continued for another 15 min before extraction of DNA with phenol, phenol/chloroform and chloroform. The DNA was ethanol precipitated at room temperature for 20 min in the presence of 0.3 M sodium acetate (pH 5.2), followed by centrifugation at 10,000 g for 15 min. The pellet was rinsed with 70% ethanol, dried and resuspended in 1 ml of TE (10 mM Tris, 1 mM EDTA, pH 8.0). Thus obtained genomic DNA was dialysed against H2O for 48 hours using collodium bags (Sartorius, Germany), ethanol precipitated in the presence of 0.3 M sodium acetate and resuspended in H2O. Using the prepared genomic DNA as template, a complete coding sequence for the β-carotene hydroxylase gene without excessive flanking region was obtained by PCR amplification using a thermal cycler (Perkin elmer 2400, USA). The synthetic primers used were: PZl (SEQ ID NO:l)(having a Sma 1 site CCCGGG) and PZ2 (SEQ ID NO:2)(having a BamH I site GGATCC). The PCR was performed using an Advantage-HF PCR Kit (CLONTECH Laboratories, Inc., USA). The PCR mixture was prepared according to the user's manual described by the manufacturer. The initial template denaturation step consisted of 1 min at 94°C. An amplification cycle of 30 seconds at 94°C and 4 min at 68°C was repeated for 25 times. After additional 3 min reaction at 68°C, the reaction mixture was kept at 15°C. By this reaction, DNA fragment containing a complete ORF of the β-carotene hydroxylase gene (522 bp) was amplified. This amplified β-carotene hydroxylase gene was ligated with a pCR2.1-TOPO vector and introduced into Ecoli TOP 10 cells by using a TOPO TA Cloning kit (Invitrogen Corporation, USA) according to an instruction manual prepared by the mnnufacturer. Several clones were selected for sequence analysis. The sequence of the cloned (3-carotene hydrox)'lase gene of each candidate clone was examined. One of the DNA clones that showed completely the same sequence as the β-carotene hydroxylase sequence of Flavobacterium sp. ATCC21588 (GenBank accession No. U62808) was named as pTOPO-P2;=23N. A 522 bp Sma I / BamH I fragment cut out from pTOPO-PZ#23N was used as a crlZ gene cassette of Flavobacterium sp. Example 2: Cloning of the β-carotene hydroxylase gene (crtZ) from genomic DNA of E-herbicola ATCC39368 Cells ofE herbicola (Escherichia vubieris) ATCC39368 were inoculated into a 10 ml medium containing 1% tryptone (Difco Laboratories), 0.5% yeast extract (Difco), 0.5% NaCl, and cultivated nt 26oC under aerobic conditions. Overnight culture (10 ml) was centrifuged at 10,000 g for 10 min. The pellet was used for the genomic DNA preparation using a Ql AGEN Blood & Cell Culture DNA Midi Kit (QI AGEN, Germany) according to a protocol prepared by the manufacturer. Using the prepared genomic DNA as template, a complete coding sequence for the β-carotene hydroxylase gene without excessive flanking region was obtained by PCR amplification using a thermal cycler (Perkin elmer 2400, USA). The two synthetic primers used were EZl (SEQ ID N0:3)(having an Sma I site CCCGGG) and EZ2 (SEQ ID N0:4)(having an Sal I site GTCGAC). The PCR was performed using an Advantage-HF PCR Kit (CLONTECH Laboratories, Inc., USA). The PCR mixture was prepared according to the user's manual described by the manufacturer. The initial template denaturation step consisted of 1 min at 94°C. An amplification cycle of 30 seconds at 94°C and 4 min at 68°C was repeated for 25 times. After additional 3 min reaction at 68°C, the reaction mixture was kept at IS'^C. By this reaction, DNA fragment containing a complete ORF of the β-carotene hydroxylase gene (543 bp) was amplified. This amplified β-carotene hydroxylase gene was ligated with a pCR2.1-T0P0 vector and introduced into E.coli TOP 10 cells by using a TOPO TA Cloning kit (Invitrogcn Corporation, USA) according to an instruction manual prepared by the mnnufacturer. Several clones were selected for sequence analysis. The sequence of the cloned β-carotene hydroxylase gene of each candidate clone was examined. One of the DNA clones thar showed completely the same sequence as the β-carotene hydroxylase sequence of E. bcrbicola ATCC39368 (GenBank accession No. M87280) was named as pTOPO-EZ,2. A 543 bp Sma ]/ Sail fragment cut out from pTOPO-EZ#2 was used as a crtZ gene cassette of E. herbicola. Example 3: Preparation of components of the expression vector The components of the expression vector, a G418 resistant gene, promoter and terminator region of glyceraldehyde-3-phosphate dehydrogenase gene (hereinafter referred to as GAP) of P. rhodozymay and rDNA fragment of P. rhodozyma were prepared. A G418 resistant gene cassette was prepared as follows. A Sac I- linker was ligated into the unique Hind 111 site of the vector, pUC-G418 (US Patent No. 6,365,386 Bl), which is harboring the G418 resistant gene cassette, and resulted vector was named as pG418Sa512. A 1.7 kb Kpn 1 / Sac I fragment cut out from pG418Sa512 was used as a G4I8 resistant gene cassette. Each of the promoter and the terminator of GAP gene and the rDNA fragment was obtained by PCR using the genomic DNA of P. rhodozyma ATCC 96594 as template. To obtain the genomic DNA, a QIAGEN Blood 8c Cell Culture DNA Midi Kit (QIAGEN, Germany) was used with the cells of P. rhodozyma ATCC 96594 obtained by overnight culture in YPD (Difco Laboratories) medium. Using the prepared genomic DNA as a template, PCR was performed using an Advantage-HP PCR Kit (CLONTECH Laboratories, Inc., USA) and a thermal cycler (Perldn elmer 2400, USA). The two synthetic primers used to amplify the promoter sequence of GAP were GAP#a (SEQ ID N0:5)(having a Not I site GCGGCCGC) and GAP#5 (SEQ ID NO:6)(having a Sma I site CCCGGCC). The initial template denaturation step consisted of 5 min at 94°C. An amplification cycle of 30 seconds at 94°C, 30 seconds at 55°C, and 1 min at 72°C was repeated for 25 times. After additional 10 min reaction at 72°C, the reaction mixture was kept at 4°C. By this reaction, DNA fragment containing the GAP promoter (398 bp) was amplified. This amplified GAP promoter was ligated with a pCR2.1-TOPO vector and introduced into E.Coli TOP 10 cells by using a TOPO TA Cloning kit (Invitrogen Corporation, USA). Several clones were selected for sequence analysis. The sequence of the cloned GAP promoter of each.candidate clone was examined. One of the DNA clones that showed completely the same sequence as the GAP promoter of P. rhodozyma (GenBank accession No. Y08366) was named as pTOPO-pGAP2#I. A 398 bp Not I / Sma I fragment cut out fi-om pT0P0-pGAP2#l was used as the GAP promoter cassette. The synthetic primers used to amplify the terminator sequence of GAP were GAP#33 (SEQ ID NO:7)(havmg a BamH I-Sal I site GGATCCGTCGAOand GAP#4 (SEQ ID NO:8) (having a Kpn I siteGGTACC). The PCR conditions were the same as those for the GAP promoter described above. By this reaction, DNA fragment containing the GAP terminator (302 bp) was amplified. This amplified GAP terminator was ligated with a pCR2.1-TOPO vector and introduced into Exoli TOP 10 cells by using the TOPO TA Cloning kit. Several clones were selected for sequence analysis. The sequence of the cloned GAP terminator of each candidate clone was examined. One of the DNA clones that showed completely the same sequence as the GAP terminator of P. rhodozyma (GenBank accession No. Y08366) was named as pTOPO-tGAP#l. A 302 bp BamH 1IKp1 fragment or a 296 bp Sal l/Kpnl fragment which was cut out from pTOPO-tGAP#l was used as the GAP terminator cassette. The two synthetic primers used to amplify the rDNA fragm'ent were R#l (SEQ ID N0:9) (having a Sac I site CAGCTC) and R#2 (SEQ ID NO:10)(having a Not l-Sac I site GCGGCCGCCAGCTC). The PCR conditions were the same as those for the GAP promoter described above. By this reaction, DNA fragment containing the rDNA (3126 bp) was amplified. This ampli- fied the rDNA was ligated with a pCR2.1-T0P0 vector and introduced into E.coli TOP 10 cells by using ihe TOPO TA Cloning kit. Several clones were selected for sequence analysis. The sequence of the cloned the rDNA of each candidate clone was examined. One of the DNA clones that showed completely the same sequence as the rDNA of P. rhodozyma (GenBank accession No. D31656, AF139632) was named as pTOPO-rDNA#l. A I960 bp SacW I Not I fragment cut out from pTOPO-rDNA#l was used as the rDNA cassette. Example 4: Construclion of the expression vector carrying the ^-carotene hydroxylase gent icrtZ) obtained from Flavobacterium sp. or E, herbicola To construct the expression vector carrying the crtZ of Flavobacterium sp., the rDNA cassette and the GAP promoter cassette (obtained in Example 3). the crtZ gene cassette of Flavobacterium sp. (obtained in Example 1), and the BamH I / Kpn I fragment of the GAP terminator cassette and the G418 resistant gene cassette (obtained in Example 3) were aligned by ligation in this order using a pGEM-T as a backbone. The resulting expression vector was named as pRPZG-ptGAP2. To construct the expression vector carrying the crtZ of £. herbicohy the rDNA cassette and the GAP promoter cassette (obtained in Example 3), the crtZ gene cassette of £. herbicola (obtained in Example 2), and Sal I / Kp7i I fragment of the GAP terminator cassette and the G418 resistant gene cassette (obtained in Example 3) were align'ed by ligation in this order using a pGEM-T as a backbone. Resulted expression vector was named as pREZG-ptGAP2. Example 5: Introduction of the expression vector carrying the β-carotene hydroxylase gene (crtZ) of Flavobacterium sp. or £. herbicola into P. rhodozyma Each of the expression vectors obtained in the Example 4, pRPZG-ptGAP2 and pREZG-ptGAP2, carrying the crtZ gene of Flavobacterium sp. and £ herbicola, respectively, was introduced into P. rhodozyma ATCC 96815, a mutant strain producing ^-carotene predominantly by using the particle-gun method as described in EP 1>158,051. YPD-agar medium containing 0.75 M D-sorbitol and D-mannitol, and 0,1 mg/ml of geneticin was used for direct s^leclion of the recombinant strains. As a result, hundretls of transformants which were resistant to 0.1 mg/ml geneticin were obtained by each of pRPZG-ptGAP2 and pREZG-ptGAP2, and 35 each transformants were selected randomly for further characterization. Claims 1. A process for producing zeaxanchin and β-cryptoxanthin which comprises cultivating a recombinant microorganism which is expressing a β-carotene hydroxylase gene and belonging to the genus Xanthophyllomyces (Phaffia) in an aqueous nutrient medium under aerobic conditions, and isolating the resulted carotenoids from the cells of said recombinant microorganism or from the cultured broth. 2. The process according to claim 1, wherein the recombinant microorganism is derived from Xanthophylbinyces dendrorhous {Phaffia rhodozyma) ATCC96815, or a mutant thereof. 3. The process according to claim 1 or 2, wherein the β-carotene hydroxylase gene is originated from a microorganism which is selected from the group consisting of microorganisms ot ihe genera Flavobacterium, Erwinia, Agrobacterium, Alcaligenes, and ParacoccuSy which aie having the 3-carotene hydroxylase gene. 4. The process according to claim 1 or 2, wherein the 3-carotene hydroxylase gene is originated from a microorganism which is selected from the group consisting of Flavobacterium sp. U1534 WT (ATCC21588), Envinia uredovora ATCC19321, Erwinia herbicola ATCC3936y, Agrobacterium aurantiacum, Alcaligenes PC-1, Paracoccus marcusii MHl, and a grnm-negative bacteria E-396 (FERM BP-4283) which are having the β-carotene hydroxylase gene. 5. The process according to claim 1 or 2, wherein the 3-carotene hydroxylase gene is originated from Flcivobncterium sp. R1534 WT (ATCC21588) or the DNA sequence of the 3-carotene hydroxylase gene is substantially homologous thereto. 6. The process according to any one of claims 1 to 5, wherein the 3-carotene hydroxylase gene is expressed in the recombinant microorganism using the control sequences. 7. The process according to any one of claims 1 to 6, wherein the cultivation is carried out at pH range from 4 to 8 and at a temperature range from 15 to 26°C for 24 to 500 hours. 8. The process according to claim 7, wherein the cultivation is carried out at pH range

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