Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus.

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

Metabolism of mono- and dihalogenated C1 and C2 compounds by <Emphasis Type="Italic">Xanthobacter autotrophicus</Emphasis> growing on 1,2-dichloroethane

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.     

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.     

Kinetics of Bacterial Growth on Chlorinated Aliphatic Compounds

With the pure bacterial cultures Ancylobacter aquaticus AD20 and AD25, Xanthobacter autotrophicus GJ10, and Pseudomonas sp. strain AD1, Monod kinetics was observed during growth in chemostat cultures on 1,2-dichloroethane (AD20, AD25, and GJ10), 2-chloroethanol (AD20 and GJ10), and 1,3-dichloro-2-propanol (AD1). Both the Michaelis-Menten constants (K_m) of the first catabolic (dehalogenating) enzyme and the Monod half-saturation constants (K_s) followed the order 2-chloroethanol, 1,3-dichloro-2-propanol, epichlorohydrin, and 1,2-dichloroethane. The K_s values of strains GJ10, AD20, and AD25 for 1,2-dichloroethane were 260, 222, and 24 μM, respectively. The low K_s value of strain AD25 was correlated with a higher haloalkane dehalogenase content of this bacterium. The growth rates of strains AD20 and GJ10 in continuous cultures on 1,2-dichloroethane were higher than the rates predicted from the kinetics of the haloalkane dehalogenase and the concentration of the enzyme in the cells. The results indicate that the efficiency of chlorinated compound removal is indeed influenced by the kinetic properties and cellular content of the first catabolic enzyme. The cell envelope did not seem to act as a barrier for permeation of 1,2-dichloroethane.     

Identification of Chloroacetaldehyde Dehydrogenase Involved in 1,2-Dichloroethane Degradation

The degradation of 1,2-dichloroethane and 2-chloroethanol by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde, a reactive and potentially toxic intermediate. The organism produced at least three different aldehyde dehydrogenases, of which one is plasmid encoded. Two mutants of strain GJ10, designated GJ10M30 and GJ10M41, could no longer grow on 2-chloroethanol and were found to lack the NAD-dependent aldehyde dehydrogenase that is the predominant protein in wild-type cells growing on 2-chloroethanol. Mutant GJ10M30, selected on the basis of its resistance to 1,2-dibromoethane, also had lost haloalkane dehalogenase activity and Hg²⁺ resistance, indicating plasmid loss. From a gene bank of strain GJ10, different clones that complemented one of these mutants were isolated. In both transconjugants, the aldehyde dehydrogenase that was absent in the mutants was overexpressed. The enzyme was purified and was a tetrameric protein of 55-kDa subunits. The substrate range was rather broad, with the highest activity measured for acetaldehyde. The K_m value for chloroacetaldehyde was 160 μM, higher than those for other aldehydes tested. It is concluded that the ability of GJ10 to grow with 2-chloroethanol is due to the high expression level of an aldehyde dehydrogenase with a rather low activity for chloroacetaldehyde.     

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.     

Alteration of the substrate range of haloalkane dehalogenase by site-directed mutagenesis

We attempted to expand the range of chlorinated solvents degraded by Xanthobacter autotrophicus GJ10 to include trichloroethylene by the rational modification of the enzyme haloalkane dehalogenase. The amino acids Phe164, Asp170, Phe172 and Trp175 were individually replaced with alanine by site-directed mutagenesis. All substitutions produced enzymes with lower than wild type activity with 1,2-dichloroethane. The Phe164Ala and Asp170Ala mutants were 3 and 2 times more active than was the wild type enzyme in dechlorinating 1,6-dichlorohexane. The Asp170Ala mutant resembled the wild type enzyme in its relative activity against longer chain substrates. No mutant was active with trichloroethylene.     

A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo- alkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide (Janssen et al. 1994, Annu. Rev. Microbiol. 48, 163-191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26: evidence for product- and water-mediated inhibition

The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.     

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.     

Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the gamma-hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl-enzyme intermediate. LinB is believed to belong to the family of alpha/beta-hydrolases which employ a catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the beta-strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the beta-strand seven.     

Utilization of Trihalogenated Propanes by Agrobacterium radiobacter AD1 through Heterologous Expression of the Haloalkane Dehalogenase from Rhodococcus sp. Strain m15-3

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P_lac, P_dhlA, and P_trc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Development of a Fiber Optic Enzymatic Biosensor for 1,2-dichloroethane

There is a significant need for devices capable of measuring water contaminant concentrations in situ––continuously, rapidly, and without reagents, extraction, or other pretreatment. Toward this goal, we constructed and tested fiber optic biosensors for measurement of 1,2-dichloroethane (DCA) in aqueous solutions. The biocomponent was the haloalkane dehalogenase, DhlA, in whole cells of Xanthobacter autotrophicus GJ10. These cells were immobilized in calcium alginate on the tip of a fiber optic fluoresceinamine-based pH optode. The resulting biosensor could quantify DCA at 11 mg/l and had a linear response up to at least 65 mg/l. Total signal change was reached in 8–10 min, and measurements were reproducible (SE <9%). The sensor’s small size, potential for remote operation, and low cost make it of interest for further development.     

Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.     

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene.

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.     

细菌卤代烷烃脱卤酶基因在拟南芥菜中的高效表达

报道了细菌Xanthobacter autotrophicus编码卤代烷烃脱卤酶基因在拟南芥菜中的高效表达。以土壤农杆菌介导将该基因整合到拟南芥菜基因组中,经数代筛选得到了转基因纯合种子,Northern印迹和气相色谱检测表明,转基因的表达程度很高,酶量占细胞总可溶性蛋白的8%,酶活力达7.8mU·ml^-1提取物。转基因植株在含二氯乙烷的培养基上不能生长。