Patent Application: US-46781095-A

Abstract:
the invention relates to a multifunctional nucleoside didesoxyribosyl or nucleoside deoxyribosyl transferase which has one or more of the following additional activities nucleoside kinase , nucleoside reductase desaminase , or dna polymerase activity . utilizing the multifunctional enzyme results in a variety of nucleic acid products . these products can be prepared using sequential reactions in a single batch process wherein the sequential reaction can be caused to occur by varying process conditions in a manner which turns on or off the requisite activities causing the sequential reactions to occur . an example of a product prepared in this manner is dideoxyribofuranoside triphosphate . certain of the resultant products have pharmaceutical activities , e . g . antiviral agents . lactobacillus leichmannii is a source of the multifunctional nucleoside deoxyribosyl transferase .

Description:
150 l mrs - medium are inoculated with 1 l of a logarithmic preculture of lactobacillus leichmannii ( dsm 20076 ). dsm refers to deutsche sammlung von mikroorganismen and zellkulturen gmbh located at mascheroder weg 1b , d - 3300 braunschweig , federal republic of germany . the microorganisms were stirred at 37 ° c ., anaerobically until they attained the steady phase ( approximately 6 hours ). with cross - flow filtration equipment , the cell suspension was concentrated and then centrifuged off . the yield was 12 . 5 g / l wet cell weight . ______________________________________mrs medium______________________________________peptone for bacteriology 10 g meat extract 5 g yeast extract 5 g k . sub . 2 hpo . sub . 4 2 g sodium citrate 2 g sodium acetate × 3 h . sub . 2 o 6 g mgso . sub . 4 × 7 h . sub . 2 o 0 . 58 g mnso . sub . 4 × 4 h . sub . 2 o 0 . 28 g tween 80 1 g glucose 20 g______________________________________ water ad 1 l , adjust ph with hcl 1 n to 6 . 2 - 6 . 3 20 g dells ( wet weight ) were disintegrated in 30 ml buffer and 80 g of glass beads ( diameter 0 . 75 mm ) at 4000 rpm in a glass bead disintegrator . disintegration lasted no longer than 5 min . ______________________________________disintegration buffer______________________________________phosphate buffer ( na ) 20 mm , ph 6 . 5 nacl 50 mm edta 0 . 1 mm mercaptoethanol 1 mm______________________________________ the crude extract obtained , with an optimal protein concentration of 10 mg / ml ) is mixed , while stirring , at 4 ° c . with 1 % ( wet weight ) protamine sulfate solution , ph 6 . 5 , for one hour , until a final concentration of protamine sulfate of 0 . 4 to 0 . 45 % is attained . the proteins that have settled out are centrifuged at 11 , 000 rpm for 15 minutes . following this precipitation , relatively large aggregates of the proteins are formed , as a result of which a 30 % increase in activity of v 2 is effected . this is followed by a first ammonium sulfate precipitation between 0 and 50 % saturation ; that in turn is followed by a second ammonium sulfate precipitation at between 50 and 70 % saturation . in the last saturation range , the transferase precipitate out . the nucleoside deoxyribosyl transferase were separated from one another in a deae - sephacel column , which was equilibrated with 20 mm of phosphate buffer , 100 mm nacl , 0 . 1 mm edta , 1 mm mercaptoethanol . on the nacl gradient , v 1 is eluted between 160 and 190 mm , v 2 between 260 and 280 mm , and v 3 at the end of the breakdown with 100 mm of nacl ( see fig1 ). the fractions that show transferase activity were combined and dialyzed with 20 mm tris - hcl , ph 7 . 0 , 20 mm kcl , 4 . 0 mm dtt , 4 . 0 mm mgcl 2 , 0 . 1 mm edta . 3 ml 5 - amp agarose ( affinity matrix ) were packed in a column and equilibrated with the aforementioned buffer . the sample ( 5 ml ) containing v 3 was recirculated for one hour through the column by means of a peristaltic pump ( flow rate 10 ml / h ). the unbound enzyme was washed with 4 column volumes of 100 mm kcl in the same buffer . the v 3 is not eluted until a concentration of 300 mm of kcl in the tris - hcl buffer is reached . by this purification process , it was possible to increase the specific activity of v 3 to 20 units per milligram of protein . a homogeneous protein is therefore present . it was possible to isolate the following quantities of enzyme from the cells , per gram of wet cell weight : ______________________________________v 1 50 μg ( 1 unit ) v 2 160 μg ( 2 - 7 units ) v 3 550 - 600 μg ( 11 - 12 units ) ______________________________________ transferase activity was demonstrated for v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . here , the ddr is transferred to both position n 9 and position n 3 . in the same fashion a 2 &# 39 ;, 3 &# 39 ;- dideoxyribosyl residue is transferred to the following : transferase activity was demonstrated for v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________phosphate buffer ( sodium ) 20 mm , nacl 50 mm , ph 6 . 2 2 &# 39 ;- deoxyinosine 2 mm 8 - bromguanine 0 . 5 mm v 3 5 u / ml reaction mixturetemperature : 35 - 37 ° c . duration of reaction time : 24 to 30 hours end product : 8 - bromguanosine______________________________________ examples for the kinase activity of v 1 , v 2 and v 3 kinase activity was demonstrated for v 3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - chloride 50 mm , ph 7 . 2 mgcl . sub . 2 6 . 5 mm dithiotreitol ( dtt ) 2 . 5 mm adenosine 5 &# 39 ;- triphosphate 5 mm 2 &# 39 ;- deoxythymidine 3 mm v 3 5 u / ml reaction mixturetemperature : 35 - 37 ° c . end products : dtmp , dtdp , dttp ( traces ). ______________________________________ kinase activity was demonstrated for v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________2 &# 39 ;- deoxycytidine 5 &# 39 ;- triphosphate 5 mm 2 &# 39 ;, 3 &# 39 ;- dideoxyadenosine 3 mm buffer tris - hcl 50 mm , ph 7 . 2 mgcl . sub . 2 6 . 5 mm dtt 2 . 5 mmv 3 10 / u / ml reaction mixturetemperature : 35 - 37 ° c . equilibrium is reached in 5 - 7 days . end products : ddamp , ddadp , ddatp______________________________________ examples for the reductase activity of v 1 , v 2 and v 3 reductase activity was demonstrated for v 1 , v 2 , and v 3 enzymes obtained in a manner as described in example 1 by reacting a suitable quantity of each enzyme under the conditions described below . ______________________________________buffer : sodium acetate 60 nm k . sub . 2 hpo . sub . 4 3 mm atp ( adp or amp ) 1 mm coenzyme b 12 4 μm dtt 30 mm dgtp ( effector ) 1 mm enzyme ( v 1 , v 2 , v 3 ) 0 . 2 to 0 . 5 units / ml of reaction mixture duration of the reaction : 2 to 3 days end product : datp ( dadp or damp ) ______________________________________ example for the deaminase activity of v 1 , v 2 and v 3 deaminase activity was demonstrated for v2 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________phosphate buffer 20 mm , nacl 50 mm , ph 6 . 5 2 &# 39 ;- deoxycytidine 2 mm v 2 0 . 5 u / ml reaction mixture end product : 2 &# 39 ; deoxyuridine______________________________________ deaminase activity was demonstrated for v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________phosphate buffer 20 mm , nacl 50 mm , ph 6 . 5 2 &# 39 ;, 3 &# 39 ;- dideoxyadenosine 2 mm v 3 5 u / ml reaction mixture end product : 2 &# 39 ;, 3 &# 39 ;- dideoxyinosine______________________________________ the reaction mixtures must be incubated for a relatively long time at 37 ° c . before the deamination occurs . the duration of the deamination reactions is 2 to 4 days . de novo polymerase activity was demonstrated for either v2 or v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 60 mm , ph 7 . 2 dtt 2 . 5 mm datp 5 mm enzyme : v 3 ( v 2 ) 5 u / ml ( 1 u / ml ) reaction mixturetemperature : 35 ° c . duration of the reaction : 3 days product : poly ( da ) ______________________________________ polymerase activity was demonstrated for any one of v1 , v2 or v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 40 mm , ph 7 . 55 mgcl . sub . 2 5 mm dtt 1 mm bsa 50 μg / ml poly [ d ( a - t )] 1 . 5 a260 u datp 1 mm dttp 1 mm dgtp 1 mm dctp 1 mm enzyme : v 1 , v 2 , v 3 2 to 5 u / ml reaction mixturetemperature : 35 - 37 ° c . duration of the reaction : 3 to 5 days product : poly [ d ( a - t )] ______________________________________ polymerase activity was demonstrated for v3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 40 mm , ph 7 . 2 dtt 2 . 5 mm damp 1 mm mgcl . sub . 2 6 . 5 mm bsa 50 μg / ml poly [ d ( a - t )] 1 . 5 a260 u dttp 1 mm dgtp 1 mm dctp 1m enzyme : v 3 5 units / ml of reaction mixturetemperature : 35 ° c . duration of the reaction : 3 to 5 days product : poly [ d ( a - t )] ______________________________________ multifunctionality of adenosine deaminase obtained from calf spleen was demonstrated by reacting a suitable quantity of deaminase under the conditions described below . ______________________________________buffer : tris - hcl 100 mm , ph 7 . 5 damp 1 mm dctp 2 mm mgcl . sub . 2 10 mm dtt 1 mm bsa 100 μg / ml enzyme : adenosine deaminase from 2 units / ml of reaction mixture calf spleentemperature : 35 ° c . duration of the reaction : 2 to 30 hours product : dadp , datp______________________________________ multifunctionality of adenosine deaminase obtained from e . coli was demonstrated by reacting a suitable quantity of deaminase under the conditions described below . ______________________________________buffer : phosphate 50 mm , ph 6 . 2 da 2 mm enzyme : dna - polymerase i from 2 units / ml of reaction mixture e . colitemperature : 37 ° c . duration of the reaction : 5 days product : di , hypoxanthine______________________________________ multifunctionality of dna - polymerase obtained from micrococcus luteus was demonstrated by reacting a suitable quantity of polymerase under the conditions described below . ______________________________________buffer : phosphate 50 mm , ph 6 . 2 da 2 mm bsa 50 μg / ml enzyme : dna - polymerase from 1 unit / ml of reaction mixture micrococcus luteustemperature : 35 ° c . duration of reaction : 20 to 24 hours products : di , hypoxanthine______________________________________ multifunctionality of dna - polymerase obtained from micrococcus luteus was demonstrated by reacting a suitable quantity of polymerase under the conditions described below . ______________________________________buffer : tris - hcl 100 mm , ph 7 . 5 da 3 mm dctp 5 mm mgcl . sub . 2 5 mm dtt 2 . 5 mm bsa 50 μg / ml enzyme : dna - polymerase from 1 unit / ml of reaction mixture micrococcus leteustemperature : 35 ° c . duration of the reaction : 2 days products : damp , dadp , datp______________________________________ reaction sequences utilizing the multifunctionality of the v 3 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 40 mm , ph 7 . 55 mgcl . sub . 2 6 . 5 mm dtt 2 . 5 mm datp 5 mm dc 3 mm enzyme v 3 5 u / ml reaction mixturetemperature : 35 ° c . duration of the reaction : 2 days products : dcmp , dcdp a spontaneous de novo polymerization occurs in the next 3 days . product : poly ( da - dc ) ______________________________________ reaction sequences utilizing the multifunctionality of the v 2 enzyme obtained in a manner as described in example 1 by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 40 mm , ph 7 . 55 mgcl . sub . 2 6 . 5 mm dtt 2 . 5 mm datp 5 mm dg 3 mm enzyme : v 2 1 unit / ml of reaction mixturetemperature : 35 ° c . duration of the reaction : 2 days after de novo - polymerization , poly ( da - dg ) occurs . ______________________________________ reaction sequences utilizing the multifunctionality of the v 3 enzyme obtained in a manner as described in example 1 were demonstrated by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 40 mm , ph 7 . 2 da 3 mm dctp 5 mm mgcl . sub . 2 6 . 5 mm dtt 2 . 5 mm bsa 50 μm enzyme : v 3 5 units / ml of reaction mixtureafter three days , the following were added to this : poly [ d ( a - t )] 1 . 5 a260 u dgtp 1 mm dttp 1 mmtemperature : 35 ° c . duration of the reaction : 5 to 6 days products : damp , dadp , datp , poly d ( a - t ) ______________________________________ reaction sequences utilizing the multifunctionality of the v 3 enzyme obtained in a manner as described in example 1 were demonstrated by reacting a suitable quantity of the enzyme under the conditions described below . ______________________________________buffer : tris - hcl 50 mm , ph 7 . 5 amp 1 mm dgtp 2 mm coenzyme b12 4 μm dtt 30 mm bsa 50 μg / ml enzyme : v 3 5 units / ml of reaction mixtureonce the amp reduction to damp has attained equilibrium , 5 mm mgcl . sub . 2 are added to it . products : damp , dadp , datp______________________________________ 1 ) durham j . p ., ives d . h . biochim . biophys . acta 228 , 9 - 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