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  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group

Definitions

• the invention relates to a method for improving the NADH specificity of preferably NADPH-dependent dehydrogenases, which in particular for the production of dehydrogenase - especially short-chain dehydrogenases and preferably alcohol dehydrogenases - with NADH dependency suitable for preparative purposes in accordance with a kcat / KM -Value for NAD + > 20 is useful and then includes available dehydrogenases and their use.
• Dehydrogenases and especially alcohol dehydrogenases are valuable catalysts for the production of chiral products by stereoselective reduction of prochiral ketones to the corresponding chiral alcohols.
• Appropriate enzymes from yeast (depending on NAD), horse liver (depending on NAD) and Thermoanaerobium brockii (depending on NADP) are commercially available, and for special substrates also steroid dehydrogenases (depending on NAD and NADP).
• the aim of the invention is therefore a method for improving the NAD + dependency of such ADHn, which can also be used quite generally for the corresponding improvement of dehydrogenases.
• the method of the type mentioned at the outset, which was developed for this purpose is essentially characterized in that the basicity of the enzyme in the coenzyme docking area is reduced by appropriate modification of the amino acid sequence using genetic engineering means. This reduction in the basicity of the amino acid residues of the dehydrogenase in the coenzyme docking area can be achieved in particular by exchanging positively charged amino acid (s) for uncharged amino acid (s).
• the basicity of the amino acids of the dehydrogenase in the coenzyme docking area can also be reduced by exchanging neutral or positively charged amino acid (s) for negatively charged amino acid (s), it being possible, of course, to provide combinations of these requirements to reduce the basicity.
• the ADH from Lactobacillus brevis (DSM 20 054), in which a coenzyme binding site in the N-terminus is suspected and whose sequence of the first 50 amino acids in the N-terminal area is assumed to serve as an implementation example: SNRLDGKVA-I ⁇ o-ITGGTLGIGL 20 -AIATK- FVEEG 3 oAKVMITGRHS 40 -DVGEKAAKSV 5 o
• the complete sequence of a subunit of this ADH which consists of four identical subunits, is published in the dissertation B. Riebel (1997, University of Düsseldorf), as is the associated DNA sequence, via which the mutation ultimately takes place.
• the method according to the invention for modifying dehydrogenases naturally requires knowledge or preliminary determination of the amino acid sequence to be modified of the enzyme to be improved, in order for a targeted exchange of basic amino acids by uncharged or also negatively charged amino acids or an exchange of uncharged amino acids by negative to be able to make charged amino acids.
• the exchange or exchange area useful for improving the NAD specificity then results - even without prior knowledge of the coenzyme binding site - according to the trial-and-error principle, the ready-made kits currently commercially available for the essential sub-steps of genetic engineering work to make this feasible without too much effort.
• lysine and arginine can be exchanged as basic amino acids, preferably against uncharged ones such as, for example, glycine, alanine, valine, leucine, isoleucine, methionine, serine, tyrosine or phenylalanine.
• uncharged ones such as, for example, glycine, alanine, valine, leucine, isoleucine, methionine, serine, tyrosine or phenylalanine.
• glutamic acid and aspartic acid are particularly suitable.
• the positive change in the mutated enzymes was demonstrated by determining the kinetic parameters for the coenzymes NAD + , NADP + , NADH and NADPH via the corresponding kinetic parameters for the ketone substrate (acetophenone) or in the oxidation reaction for the alcohol (phenylethanol ). Furthermore, using the example of the reduction of acetophenone, it was checked whether the enantioselectivity was retained. Otherwise, temperature optima and stability, pH optima and stability and the isoelectric point were determined and compared with the corresponding data for the non-mutated wild-type enzyme.
• the wild-type enzyme shows the following properties:
• Table 1 Kinetic data for the oxidized and reduced coenzymes when reacting with the ADH from Lactobacillus brevis.
• the temperature optimum of the wild-type ADH is 55 ° C, after 24 hours of incubation at different temperatures it shows 100% residual activity at 30 ° C and 50% at 37 ° C.
• the pH optimum for the reduction of acetophenone with NADPH is 6.5.
• the optimal range is very narrow, even at pH 6.0 or 7.0 there is only about 60% residual activity.
• the optimum for the oxidation of phenylethanol with NADP + is 8.0 with a wider optimum range of 7-9.
• the wild-type enzyme shows the highest stability when stored at pH 7.0 to 8.5. D) Determination of the isoelectric point
• the isoelectric point of the wild-type enzyme is 4.95.
• mutant 1 (A9G, R38L, K45I):
• arginine (R) in position 38 should be replaced by leucine (L) (R38L), lysine (K) in position 45 by isoleucine (I) (K45I) and alanine (A) at position 9 against glycine (G) (A9G).
• Mutant 1 can therefore be described in comparison to the wild type as follows: A9G, R38L, K45I.
• PCR polymerase chain reaction
• Each individual cycle of a PCR consists of 3 steps, a denaturation step at 94-96 ° C, where the double-stranded DNA is put into the single-stranded state, an annealing step at a selectable temperature, where so-called primers can bind to the now single-stranded DNA, and a polymerase step at 72 ° C (the common temperature optimum of thermophilic polymerases), where these polymerases bind to the primers and complete the single strand of the DNA again into a double strand. Since this is double-stranded DNA, primers for both strands, the sense and the antisense strand, must be added. These primers are called sense and antisense primers.
• the annealing temperature used in all fusion PCR steps is 52 ° C, in the other PCR steps in the production of mutants 1 and 2 a temperature of 52 ° C and the mutants 1/1 and 2/2 a of 56 ° C .
• the primers mentioned in step 2 are necessary for the correct preparation of the polymerase and also determine the specificity of the PCR.
• the desired DNA sections are amplified by the predetermined primer sequence.
• primers can only bind to the target DNA if they find a more or less complementary sequence in the target DNA.
• the complementary binding capacity is determined on the one hand by the sequence homology and on the other hand by the annealing temperature of the second step, the annealing temperature resulting from the melting temperature of the primer.
• the PCR was used to generate the individual cofactor mutants.
• the point mutations on the target DNA were introduced by using such primers, ie the primers did not show the correct homology to the DNA sequence in individual bases. However, since the homology of the primers was still 94% despite point mutations, it was possible to carry out the PCR (with a base length of the primers of an average of 33 bases, 2 bases were exchanged).
• the point mutations must be inserted on both strands of the double-stranded DNA for a correct change of the target sequence. This means that two single PCR reactions are required to introduce a point mutation in the gene. In one reaction a PCR is carried out from the 5 'end of the gene (sense primer) to the mutation (anti-sense primer), in the second reaction the mutation (sense primer) to the 3 end of the gene ( anti sense primer) performed a PCR.
• This PCR corresponds to that described above, only in this case 2 different templates are added, but which have 100% homology in the mutation area, namely for the length of the mutation primer previously used (generally approx. 30-40 bases) .
• This mutation region functions as a primer of PCR due to its homology, ie after denaturing the DNA into the single strands, the two starting strands can be found again in the subsequent annealing step, but the two different strands can also pair in the homology region.
• the polymerase can then complete the remaining DNA from the mutation region as the primer to a double strand. In order to allow only this variant as a possibility, no gene-specific primers are added to the fusion PCR in the first 5-10 cycles.
• the first mutant generated by this method contains three amino acid changes compared to the wild-type enzyme: arginine (R) in position 38 was replaced by leucine (L) (R38L), lysine (K) in position 45 by isoleucine (I) ( K45I) and (not specific to the invention) additionally alanine (A) in position 9 against glycine (G) (A9G).
• R arginine
• L leucine
• K45I isoleucine
• A9G alanine in position 9 against glycine
• Recombinant plasmid [recADHpkk-177-3H] was used as the template for the production of the point mutation fragments, from which the ADH gene (recADH) was excised by restriction analysis using restriction endonucleases Eco Rl and Hind III. The cut-out fragment was used in the PCR after purification on an agarose gel. The following were used as primers for mutant 1:
• R38LK46I acc ggc ctg cac agc gat gtt ggt gaa ata gca gct (36 bp) (5 ' primer sense) and
• BRAS gcgc aag ctt cta cta ttg agc agt gta gcc acc gtc aac tac aaa ttc aga (3 ' primer antisense), which give the large fragment B; as well as BRS: gcgc gaa ttc atg tct aac cgt ttg gat ggt (5 'primer sense) and R38LK46Irev: agc tgc tat ttc acc aac atc gct gtg cag gcc ggt (3 ' primer antisense), which result in the small fragment A.
• reaction mixture is supplemented with water to 100 ⁇ l, which applies to all PCR mixtures below.
• PCR fragments were purified and combined by the overlapping part in the fusion PCR (Table 3) to the complete gene with the point mutations contained.
• the amount of fragments depends on the size of the fragments; the same pmol amounts must be present at the free ends of the fragments for the fusion PCR, ie with the same concentration in ng, smaller fragments have a higher number of pmol ends than larger fragments.
• the ratio is 1: 4.4 (small to large fragment), ie 4.4 times less of the small than the large must be used.
• A9G ggt aag gta gga atc att aca (5 trimer) and BRAS: (3 'primer), which give the large fragment.
• BRS (5 'primer) and A9Grev: tgt aat gat tcc tac ctt acc (3' primer), which give the small fragment (components and concentrations see table 4).
• the amplified fragments resulting from the PCR are reassembled via fusion PCR (Table 5).
• the fragments were used in a ratio of 1: 14.5 (small to large).
• the product from this PCR represents mutant 1, after purification it was cloned into the expression vector pkk-177-3H and overexpressed in E.coli HB101 + (pUBS520).
• the mutated gene piece can of course also be inserted into vectors other than pkk-177-3H, for example pBTac (from Boehringer Mannheim), pKK-233 (from Stratagene) or pET (from Novagen).
• E. coli strains other than HB101 + ( ⁇ UBS520), such as E. coli NM 522, E. coli RRl, E. coli DH5 ⁇ or E. coli TOP 10 " from public strain collections or vector-distributing companies can also be used as the host organism are available.
• Cell mass for the processing of the individual mutants was always produced by 5 L shake flask fermentation, and the HB101 + strain was always used as the host for the mutated recADH plasmids, ie double selection pressure by ampicillin and neomycin is required for the cultivation.
• the enzyme was then induced by adding 1 mM LPTG.
• the cells were harvested and the gene expression after cell disruption was checked by activity detection and SDS-PAGE of the proteins.
• the cells obtained by fermentation can also be stored frozen at -20 ° C.
• Standard digestion methods can be used to release the enzyme.
• the recombinant organisms were disrupted by pulsed ultrasound.
• the cells resuspended in Tris buffer (0.1 M Tris-HCl, pH 7.5; 1 mM MgCl 2 ) were subjected to digestion with 2 x 25 sec cycles with a 30 sec interval for cooling using the pulsed sonifier (from Branson) Subjected to 25% intensity.
• the disrupted cells were centrifuged and the supernatant pipetted off, which is referred to below as the crude extract.
• this mutant enzyme (mutant 1) is relatively stable, especially in comparison to enzyme mutant 2.
• this mutant 1 Compared to the wild type, this mutant 1 has already shown a clear improvement in the reactivity to the coenzyme NAD + : The K M value has decreased significantly from 2.9 to 1.8 mM, while the activity (at) increased from 21 to almost 80. Taken together, both improvements show that this mutant already accepts NAD + much better. While the selectivity (kcat M) of the wild type for NAD + compared to NADP + is only approx. 2.5%, this value has increased to approx. 10% for mutant 1.
• the optimum temperature was recorded for reduction reactions at pH 7.0 with the two coenzymes NADH and NADPH. In both cases it is 55 ° C.
• the temperature stability was determined as residual activity after 25 h of incubation at various Temperatures determined. At 37 ° C 95% residual activity was obtained, at 42 ° C 34%.
• the pH optimum of the mutant for the reduction reactions is in the relatively acidic range between 5.0 and 6.0 both when using NADH and with NADPH. In comparison, only 4% activity was measured at pH 7.5. For the oxidation reactions with NAD and NADP, maximum activity is obtained at a pH of 7.0.
• the pH stability is expressed as residual activity after 25 hours of incubation at different pH values, using the same buffers as used to determine the optimum pH. 45 ⁇ l of the respective buffer were mixed with 5 ⁇ l of the enzyme solution and incubated at room temperature. Samples were taken after 30 min, 60 min, 6 h and 25 h.
• the isoelectric point after determination with isoelectric focusing (LEF) is at pH 4.28. This is remarkable in comparison to mutant 2 (see example 4).
• the LP is 4.65, although one lysine was exchanged in both mutants: in mutant 1 the lysine-45 (in isoleucine) and in mutant 2 the lysine-48 (in methionine).
• mutant 1/1 (A9G, R38L, K45M):
• mutant 1 The aim of this mutation was to replace the lysine at position 45 in methionine in place of isoleucine (mutant 1) compared to mutant 1.
• the other exchanges in positions 9 and 38 made in mutant 1 have been retained, so that mutant 1/1 can be described in comparison to the wild type as follows: A9G, R38L, K45M.
• mutant 1/1 is an extension of mutant 1, mutant 1 is therefore used as a template for the subsequent PCR.
• R38LK45M acc ggc ctg cac agc gat gtt gaa atg gca (5 primers) and BRAS (3 primers), which give the large fragment.
• the fragments were purified and used in the fusion PCR.
• the fragments were used in the ratio 1: 4.4 in the PCR.
• the product represents mutant 1/1, since mutation A9 mutation A9G was adopted.
• mutant 1 As with mutant 1, the mutations introduced were confirmed by sequencing the gene. The gene was then overexpressed in E. coli HB101 + (pUBS520) to obtain the enzyme mutant.
• Table 11 Kinetic data for the oxidized and reduced coenzymes for the enzyme mutant 1/1.
• the temperature optimum for the enzyme mutant 1/1 measured with NADPH is at least 65 ° C, higher temperatures were not reached due to measurement technology. If the optimum temperature is determined with the coenzyme NADH, an optimal value is found at 40 ° C. Presumably, NADH is no longer bound to the enzyme so well at higher temperatures, so that only reduced activity is then detectable.
• the measurement of the temperature stability shows that after 25 hours only 100% residual activity is detectable only at 25 ° C, and only 59% at 30 ° C. The temperature stability of mutant 1/1 has therefore deteriorated significantly compared to the wild-type enzyme.
• the pH optimum for the direction of reduction is approximately 6.0 with both NADPH and NADH.
• the optimum for oxidation is 7.0 (NADP + ) or 7.5 (NAD + ).
• the isoelectric point of the enzyme mutant 1/1 is 4.85.
• mutant 2 (A9G, R38L, K48M):
• mutant 2 was replaced the lysine in position 48 with methionine compared to mutant 1 - instead of the K45I exchange.
• the other exchanges in positions 9 and 38 made in mutant 1 have been retained, so that mutant 2 can be described in comparison to the wild type as follows: A9G, R38L, K48M.
• mutant 2 was produced independently of mutant 1, the wild-type ADH (recADH) gene was used again as a template.
• R38LK48M acc ggc ctg cac agc gat gtt ggt gaa aaa gca gct atg agt gtc (5 'primer) and
• BRAS (3 'primers) that give the large fragment
• R38LK48Mrev gac act cat agc tgc ttt ttc acc aac atc gct gtg cag gcc ggt (3 'primers), which give the small fragment.
• the fragments were purified and used in the fusion PCR.
• A9G ggt aag gta gga atc att aca (5 'primer) and BRAS: (3' primer), which is the big one
• BRS (5 ' primer) and A9Grev: tgt aat gat tcc tac ctt acc (3' primer) which is the small one
• the fragments were used in the ratio 1: 14.5 in the fusion PCR (Table 19).
• mutant 2 The product of this fusion PCR is mutant 2, which, like all other mutants, was cloned into the expression vector pkk-177-3H. The gene was then overexpressed in E. coli HB101 + (pUBS520) to obtain the enzyme mutant.
• Table 17 Kinetic data for the oxidized coenzymes for enzyme mutant 2.
• the temperature optimum is significantly lower than that of the wild-type enzyme, measured with NADP at 50 ° C, with NAD + at 33-37 ° C.
• the temperature stability shows that after 25 h only 100% residual activity is still present at 25 ° C, and only 40% at 30 ° C.
• the optimum pH for the reduction of acetophenone was 8.0 with NADPH, but 6.5 with NADH. This optimum at 6.5 corresponds to the wild-type enzyme, measured with NADPH. Comparable to the wild-type enzyme, the activity measured with NADH also shows only a narrow activity range; at pH 7.0 there is only 46% residual activity. The stability of the mutant enzyme at various pH values after 25 h shows a maximum at pH 7.0, at pH 6.0 and 8.0, 84% residual activity was still measured.
• the isoelectric point of the mutant enzyme is pH 4.65.
• Mutant 2/2 can therefore be described in relation to the wild type as follows: A9G, R38L, H39L, K48M.
• mutant 2/2 is a subsequent mutant of mutant 2, this was used as a template for the PCR.
• BRAS (3 primers) that make up the large fragment
• BRS (5 primers) and R38LH39Lrev: aac atc gct gag cag gcc ggt (3 primers), which give the small fragment.
• the fragments were purified and used in the fusion PCR in a ratio of 1: 4.4 (small to large).
• the K M value decreased from 2.9 to 0.96 mM compared to the wild-type enzyme.
• K M - as well as at-value deteriorated slightly The particularly good affinity of the mutant for NADH is striking, the K value is 0.07 mM.
• the optimum temperature of mutant 2/2, measured at pH 5.0, is 42 ° C, measured with NADPH and at 50oC when using NADH. These optima are relatively broad, measured with NADPH still gives 95% activity when measured at 50 ° C, measured with NADH at 60 ° C still 70%.
• the stability during storage (6h) at different temperatures is 67% for 30 ° C and only 7% for 37 ° C.
• the pH optimum for the reduction of actophenone with NADPH is 6.0, when using NADH it is 5.0. With NADH in particular, this mutant is actually only active in the acidic range; at pH 7.0, only 6% activity is obtained compared to the value at 5.0. The optimum for oxidation is also significantly slightly shifted to the acidic range; maximum activity can be found with both NADP + and NAD + at pH 7.5. At pH 8.0, the optimum of the wild type, only 77% (NADP + ) or 61% (NAD 4 ) activity can be found. If the enzyme is stored at different pH values and the residual activity measured after 6 h, 100% residual activity is still found at pH 7.0. At pH 8.5, only 58% is obtained.
• the isoelectric point of the enzyme mutant 2/2 is 4.47.
• Examples 1 to 4 show that the reduction in basicity in the coenzyme-relevant amino acid sequence region of the ADH protein of the NADP-preferred alcohol dehydrogenase from Lactobacillus brevis can be achieved by genetic engineering Replacement of basic by uncharged amino acid residues such as R38L, H39L, K45I, K45M or K48M in different combinations leads to a practice-relevant improvement in the NAD + specificity of the enzyme.
• amino acid sequences are known and can be researched in databases, it being easiest to start by searching in the similarity range of an already known coenzyme binding motif.
• a protein sequence database (“Swiss-Prot”) was used to search for enzymes which showed similarities to the sequence of the L. brevis ADH.
• the search motif was the sequence from amino acid 17 to amino acid 50 of the L. brevis - Entered ADH, the search program then finds all proteins that have sequence similarities.
• the program (reference: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402) can also insert gaps to clarify the similarity of sequence regions, assuming that deletions and insertions can naturally lead to such shifts.
• Table 22 shows amino acid sequences in the similarity range based on consensus sequences for NADP-dependent dehydrogenases.
• Sp-Q93761 means an oxidoreductase from Caenorhabditis elegans, "sp-P50161” a versicolorin reductase from Aspergillus parasiticus, "sp-O67610” an oxidoreductase from Aquifex aeolicus, “sp-P42317” an oxidoreductase from Bacillus subtilis and "sp-Q49721” an oxidoreductase from Mycobacterium leprae.
• Table 22 Comparison of sequence regions in NADP-dependent oxidoreductases which correspond to the presumed coenzyme binding site of the ADH from Lactobacillus brevis (LB ADH).
• Example 5 demonstrates the benefits of introducing a negatively charged amino acid residue in this way, in addition to replacing basic amino acids with neutral ones in the coenzyme-sensitive range.
• mutant 2 was started, which already has a high preference for NAD + over NADP + and in position 37 the glycine was exchanged for aspartic acid.
• the mutant 2G37D can thus be described in relation to the wild type as follows: A9G, G37D, R38L, K48M.
• mutant 2/3 is a subsequent mutant of mutant 2, this was used as a template for the PCR.
• G37DR38LK48M 5 acc gac ctg cac agc gat gtt gtt gaa aaa gca gct atg agt gtc3 '
• BRAS (3 trimers) that make up the large fragment
• BRS (5 ' primer) and G37DR38LK48M rev: 5' gac act cat agc tgc ttt ttc acc aac atc gct gtg cag gtc ggt 3 '(3' primer) which give the small fragment.
• a mutant2 100 ng 100 pmol 100 pmol 0.2 lO ⁇ l l ⁇ l
• B mutant 2 100 ng 100 pmol BRS 100 pmol 0.2 10 ⁇ l 1 ⁇ l
• the product of this fusion PCR is the complete mutant 2/3, the mutations R38L, K48M and A9G were taken from mutant 2.
• the gene was then overexpressed in E. coli HB101 + (pUBS520) to obtain the enzyme mutant.
• the correct mutagenesis was verified by sequencing the positive clone.
• the propagation of the E. coli strain, which contains the enzyme modified by mutagenesis, is carried out as described in Examples 1 to 4.
• the enzyme-containing supernatant (crude extract) is obtained.

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