Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Identification of the<Emphasis Type="Italic">bphC</Emphasis> gene for<Emphasis Type="Italic">meta</Emphasis>-cleavage of aromatic pollutants from a metagenomic library derived from lake waters

Useful genes can be screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs were extracted by the SDS-based freezing-thawing method, and then further purified using an UltraClean^TM kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested withEcoR I,BamH I, andSac II inEscherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13∼15 kb in size. ThebphC genes responsible for degradation of aromatic hydrocarbons viameta-cleavage were identified from the metagenomic libraries by colony hybridization using thebphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene (bphC), capable of degradation of 2,3-DHBP, was cloned and its nucleotide sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results indicated that thebphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.     

Purification and Characterization of Catechol 2,3-Dioxygenase from the Aniline Degradation Pathway of Acinetobacter sp. YAA and Its Mutant Enzyme,Which Resists Substrate Inhibition

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS–polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 μM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 μM, while the mutant enzyme loosened substrate inhibition.     

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.     

Degradation of phenanthrene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. C3: initial 1,2- and 3,4-dioxygenation and <Emphasis Type="Italic">meta</Emphasis>- and <Emphasis Type="Italic">ortho</Emphasis>-cleavage of naphthalene-1,2-diol

Burkholderia sp. C3 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Hilo, Hawaii, USA, and studied for its degradation of phenanthrene as a sole carbon source. The initial 3,4-C dioxygenation was faster than 1,2-C dioxygenation in the first 3-day culture. However, 1-hydroxy-2-naphthoic acid derived from 3,4-C dioxygenation degraded much slower than 2-hydroxy-1-naphthoic acid derived from 1,2-C dioxygenation. Slow degradation of 1-hydroxy-2-naphthoic acid relative to 2-hydroxy-1-naphthoic acid may trigger 1,2-C dioxygenation faster after 3 days of culture. High concentrations of 5,6-␣and 7,8-benzocoumarins indicated that meta-cleavage was the major degradation mechanism of phenanthrene-1,2- and -3,4-diols. Separate cultures with 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid showed that the degradation rate of the former to naphthalene-1,2-diol was much faster than that of the latter. The two upper metabolic pathways of phenanthrene are converged into naphthalene-1,2-diol that is further metabolized to 2-carboxycinnamic acid and 2-hydroxybenzalpyruvic acid by ortho- and meta-cleavages, respectively. Transformation of naphthalene-1,2-diol to 2-carboxycinnamic acid by this strain represents the first observation of ortho-cleavage of two rings-PAH-diols by a Gram-negative species.     

Simultaneous biodegradation of creosote-polycyclic aromatic hydrocarbons by a pyrene-degrading <Emphasis Type="Italic">Mycobacterium</Emphasis>

When incubated with a creosote-polycyclic aromatic hydrocarbons (PAHs) mixture, the pyrene-degrading strain Mycobacterium sp. AP1 acted on three- and four-ring components, causing the simultaneous depletion of 25% of the total PAHs in 30 days. The kinetics of disappearance of individual PAHs was consistent with differences in aqueous solubility. During the incubation, a number of acid metabolites indicative of distinctive reactions carried out by high-molecular-weight PAH-degrading mycobacteria accumulated in the medium. Most of these metabolites were dicarboxylic aromatic acids formed as a result of the utilization of growth substrates (phenanthrene, pyrene, or fluoranthene) by multibranched pathways including meta- and ortho-ring-cleavage reactions: phthalic acid, naphthalene-1,8-dicarboxylic acid, phenanthrene-4,5-dicarboxylic acid, diphenic acid, Z-9-carboxymethylenefluorene-1-carboxylic acid, and 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid. Others were dead-end products resulting from cometabolic oxidations on nongrowth substrates (fluorene meta-cleavage product). These results contribute to the general knowledge of the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted soils. The identification of the partially oxidized compounds will facilitate to develop analytical methods to determine their potential formation and accumulation in contaminated sites. An erratum to this article can be found at     

Tetrameric structure and cellular location of catechol 2,3-dioxygenase

Catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the TOL plasmid of Pseudomonas putida (pWWO) was investigated by electron microscopy. Negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. Monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in Escherichia coli (pAW31) and P. putida (pWWO), using the protein A-gold technique carried out as a post-embedding immunoelectron microscopy procedure. Our in situ labeling studies revealed a cytoplasmic location of the catechol 2,3-dioxygenase in both cell types.Abbreviations C23O Catechol 2,3-dioxygenase - 3MB 3 Methylbenzoate - AK1 Anti-C23O-IgG-antibody - G Gold particle     

Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil <Emphasis Type="Italic">Pseudomonad</Emphasis> by MALDI-TOF MS using a column separation

The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C23O) has been purified to homogeneity from a new bacterial strain, which belongs to genus Pseudomonas. The species was growing on phenol as carbon source. The C23O was detected and identified by absorption spectroscopy. The protein was isolated using sucrose density centrifugation and anion exchange chromatography. The purified protein showed a molecular mass of 32 kDa to sodium dodecyl sulfate polyacrylamid gel electrophoresis and an isoelectric point of 5 estimated by analytical isoelectrical focusing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mapping was attempted for the identification of the isolated protein and proteins involved in the metabolic pathway.     

Degradation of halogenated aromatic compounds

Due to their persistence, haloaromatics are compounds of environmental concern. Aerobically, bacteria degrade these compounds by mono- or dioxygenation of the aromatic ring. The common intermediate of these reactions is (halo)catechol. Halocatechol is cleaved either intradiol (ortho-cleavage) or extradiol (meta-cleavage). In contrast to ortho-cleavage, meta-cleavage of halocatechols yields toxic metabolites. Dehalogenation may occur fortuitously during oxygenation. Specific dehalogenation of aromatic compounds is performed by hydroxylases, in which the halo-substituent is replaced by a hydroxyl group. During reductive dehalogenation, haloaromatic compounds may act as electron-acceptors. Herewith, the halosubstituent is replaced by a hydrogen atom.Abbreviations CBz chlorobenzene - DCBz dichlorobenzene - TrCBz trichlorobenzene - TCBz tetrachlorobenzene - PCBz pentachlorobenzene - HCBz hexachlorobenzene - CBA chlorobenzoic acid - BBA bromobenzoic acid - FBA fluorobenzoic acid - IBA iodobenzoic acid - CP chlorophenol - CA chloroaniline - PCBs polychlorinated biphenyls - CB chlorobiphenyl - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Characterization of the meta-Cleavage Compound Hydrolase Gene Involved in Degradation of the Lignin-Related Biphenyl Structure by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 has the ability to transform a lignin-related biphenyl compound, 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), to 5-carboxyvanillic acid (5CVA) via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). In the 4.9-kb HindIII fragment containing the OH-DDVA meta-cleavage dioxygenase gene (ligZ), we found a novel hydrolase gene (ligY) responsible for the conversion of the meta-cleavage compound of OH-DDVA to 5CVA. Incorporation of ¹⁸O from H₂¹⁸O into 5CVA indicated there was a hydrolytic conversion of the OH-DDVA meta-cleavage compound to 5CVA. LigY exhibited hydrolase activity only toward the meta-cleavage compound of OH-DDVA, suggesting its restricted substrate specificity.     

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving the aromatic C-C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. Based on curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from P. aeruginosa ZD 4-3 able to degrade both single and bicyclic compounds via the meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it had one ORF, which showed a strong amino acid sequence similarity to the known C23Os of mesophilic gram-negative bacteria. The alignment analysis indicated that distinct difference existed between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases cleaving bicyclic aromatic compounds. The heterogenous expression of the pheB gene in Escherichia coli BL21(DE3) demonstrated that this C23O possessed a meta-cleavage activity.     

Degradation of biphenyl by a Micrococcus species

Summary A bacterium capable of utilizing biphenyl as the sole source of carbon was isolated from soil and identified as a Micrococcus species. The organism also utilized 4-chlorobiphenyl and several other aromatic compounds as growth substrates. 2,3-Dihydroxybiphenyl and benzoic acid were identified as intermediates by physico-chemical methods. The bacterium degraded biphenyl to 2,3-dihydroxybiphenyl followed by its meta-ring cleavage to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, which was then hydrolysed to give benzoic acid. Benzoate was further metabolised via a catechol meta-cleavage pathway by a Micrococcus sp.Correspondence to: H. Z. Ninnekar     

Catabolic degradation of 4-chlorobiphenyl byPseudomonas sp. DJ-12 via consecutive reaction ofmeta-cleavage and hydrolytic dechlorination

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of themeta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, andmeta-cleavage of protocatechuate. ThepcbC gene responsible for themeta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that ofPseudomonas sp. CBS3, yet only a 50% homology with that ofArthrobacter spp. However, thefcb genes for the hydrolytic dechlorination of 4CBA inPseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBP completely viameta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.     

Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters

Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.This publication is dedicated to the memory of Olga V. Maltseva, who contributed greatly to our current knowledge of biochemistry of degradative pathways for chloroaromatic compounds.This publication is dedicated to Prof. Dr. Hans G. Schlegel in honor of his 80th birthday.     

Degradation 1,2-dimethylbenzene by Corynebacterium strain C125

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Conjugative transfer of preferential utilization of aromatic compounds from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CSV86

Pseudomonas putida CSV86 utilizes naphthalene (Nap), salicylate (Sal), benzyl alcohol (Balc), and methylnaphthalene (MN) preferentially over glucose. Methylnaphthalene is metabolized by ring-hydroxylation as well as side-chain hydroxylation pathway. Although the degradation property was found to be stable, the frequency of obtaining Nap⁻Sal⁻MN⁻Balc⁻ phenotype increased to 11% in the presence of curing agents. This property was transferred by conjugation to Stenotrophomonas maltophilia CSV89 with a frequency of 7 × 10⁻⁸ per donor cells. Transconjugants were Nap⁺Sal⁺MN⁺Balc⁺ and metabolized MN by ring- as well as side-chain hydroxylation pathway. Transconjugants also showed the preferential utilization of aromatic compounds over glucose indicating transfer of the preferential degradation property. The transferred properties were lost completely when transconjugants were grown on glucose or 2YT. Attempts to detect and isolate plasmid DNA from CSV86 and transconjugants were unsuccessful. Transfer of degradation genes and its subsequent loss from the transconjugants was confirmed by PCR using primers specific for 1,2-dihydroxynaphthalene dioxygenase and catechol 2,3-dioxygenase (C23O) as well as by DNA–DNA hybridizations using total DNA as template and C23O PCR fragment as a probe. These results indicate the involvement of a probable conjugative element in the: (i) metabolism of aromatic compounds, (ii) ring- and side-chain hydroxylation pathways for MN, and (iii) preferential utilization of aromatics over glucose.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using -naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.