Identification of a heroin esterase in Rhodococcus sp. strain H1.

A strain of a Rhodococcus sp. (termed H1) capable of utilizing heroin as its sole carbon and energy source was isolated by selective enrichment. An inducible heroin esterase was partially purified and shown to catalyze the hydrolysis of both of the acetylester groups of heroin. The enzyme displays optimum activity at pH 8.5 and appears to be a trimer of identical subunits with an M(r) or 39,000 and a native M(r) of 120,000.     

Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M_r of approximately 65,000. The apparent K_m of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

The Rhodococcus sp. cocaine esterase: a bacterial candidate for novel pharmacokinetic-based therapies for cocaine abuse

Cocaine is a powerful central nervous stimulant and among the most abused of drugs. Despite decades of efforts, however, no effective pharmacological treatments are available against cocaine addiction or toxic effects. Classical receptor-antagonist therapeutic approaches have not yielded significant effects, although cocaine targets are well known, thus fostering development of alternative therapeutic strategies. Recent evidence indicates that a sensible approach for treatment of cocaine abuse could be to interfere with cocaine pharmacokinetics, i.e. by preventing the drug from reaching the receptors responsible for its biological effects. Administration of cocaine binding antibodies as well as catalytic antibodies and enzymes that hydrolyze cocaine represent potential alternative therapeutic approach(es). The discovery of the cocaine esterase from the strain MBI of the bacterium Rhodococcus sp. (cocE) could be a major breakthrough in this field; cocE hydrolyzes cocaine faster than any known cocaine esterase and catalytic antibody.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6?kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53?269?Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205?kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80°?C; ammonia-dependent reaction, 90°?C).     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Gene cloning,sequencing, and expression of an esterase from Acinetobacter lwoffii I6C-1

The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI- SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X(1)-S-X(2)-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1.     

苯酚降解菌红球菌(Rhodococcus sp.) P1的鉴定及其在焦化废水中的应用

摘要:【目的】酚类物质的去除是焦化废水处理的关键问题,目的是从焦化废水中分离高效的苯酚降解细菌。【方法】以苯酚为唯一碳源筛选纯化降解苯酚细菌,菌株鉴定采用菌落形态和16S rRNA 序列分析方法,并研究其苯酚降解特性和在焦化废水中的除酚作用。【结果】菌落形态和16S rRNA序列比对分析表明分离的P1菌株为红球菌属(Rhodococcus sp.)细菌;其耐酚浓度高达1400 mg/L,苯酚降解的最适条件为32℃－42℃、pH 7.0和0－4%盐;苯酚降解动力学曲线符合Haldane动力学模型,qmax=0.517/h,Ks=77.487 mg/L,Ki=709.965 mg/L;不同重金属对红球菌P1菌株的苯酚降解抑制作用不同,Zn2+、Mn2+和低浓度的Pb2+对菌株降酚没有影响,Cu2+、Ni2+、Cd2+均抑制菌株对酚的降解;红球菌P1菌株2d内可完全降解1/3焦化原水中的279.9 mg/L酚类物质。【结论】P1菌株是1株高效的苯酚降解菌,具有生物处理焦化废水酚类物质的潜力。     

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.     

Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1

An NAD-dependent secondary alcohol dehydrogenase (ADH) produced by Rhodococcus sp. GK1 was purified about fivefold with a yield of 82% by hydrophobic interaction chromatography. This enzyme reduced monoketones, diketones and α-dicarbonyl compounds ; it oxidized secondary alcohols but not primary alcohols. Optimum pH was 7·0 or 8·5 for reduction or oxidation of substrates, respectively, and optimal temperature for activity was 55 °C. The apparent molecular mass of ADH was about 60 kDa by gel filtration chromatography.     

The aryldialkylphosphatase-encoding gene adpB from Nocardia sp. strain B-1: cloning, sequencing and expression in Escherichia coli.

Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-1, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene. Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-1 DNA for fragments containing adpB. A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined. Under control of the lac promoter of pUC19, adpB expression in E. coli cultures was approx. 15-fold higher than in strain B-1 under the native adpB promoter. Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

Identification of a cocaine esterase in a strain of Pseudomonas maltophilia.

A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer. The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively. Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1. The cocaine esterase of P. maltophilia MB11L showed no activity with atropine, despite the structural similarity of cocaine and atropine.     

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.     

芽胞杆菌(Bacillus sp.)YX-1耐有机溶剂葡萄糖脱氢酶的基因克隆与酶学性质

[目的] 从芽胞杆菌(Bacillus sp.)YX-1基因组中克隆出一种有机溶剂耐受型的葡萄糖脱氢酶基因,实现了该基因在大肠杆菌中的高效表达,研究了重组蛋白的酶学性质.[方法] 依据芽胞杆菌属中葡萄糖脱氢酶氨基酸序列的保守性,设计合理引物,钓取来源于Bacillus sp.YX-1的葡萄糖脱氢酶基因,构建诱导型表达载体pET28a-gdh,于大肠杆菌中进行表达.镍柱亲和层析法纯化重组蛋白,考察了重组蛋白的酶学性质.[结果] 葡萄糖脱氢酶基因全长为786 bp,编码261个氨基酸.酶学研究结果表明:该酶最适反应温度为45℃,最适pH值为8.0;具有良好的有机溶剂耐受性,于50％的辛烷、环己烷、癸烷中室温放置1h后,酶活仍能保持90％以上;具有较宽的底物谱,对多种糖均具有一定的催化活性,其中催化D-葡萄糖的活力最高,产生还原型辅酶因子;对辅酶NADH和NADPH具有相似的依赖性,对NAD+和NADP+的催化比活分别为8.37 U/mg和8.62 U/mg.[结论]利用生物信息学成功地挖掘出Bacillus sp.YX-1一种耐有机溶剂的葡萄糖脱氢酶,为氧化还原酶在有机相反应中的的辅酶再生循环提供了新型的生物催化剂.     

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.     

Identification and sequencing of beta-myrcene catabolism genes from Pseudomonas sp. strain M1.

The M1 strain, able to grow on beta-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One beta-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique beta-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on beta-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for beta-myrcene catabolism in Pseudomonas sp. strain M1.     

Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.