Patent Application: US-201314434219-A

Abstract:
enzymes for transforming , in particular hydrolytically cleaving , ergopeptines , which ergopeptines are α / β - hydrolases hydrolytically cleaving ergopeptines in the cyclol ring , for the transformation of ergopeptines , and method for producing ergopeptine - metabolizing enzymes .

Description:
determination of the catalytic activity of the enzyme with the sequence id no . 1 the gene with the sequence id no . 2 , which codes for an α / β - hydrolase comprising a catalytic triad of s94 - d234 - h270 , was cloned into the expression vector pet28a (+) by applying standard methods , transformed and expressed in e . coli . following the expression in e . coli bl21 ( de3 ), the his - tagged enzyme was purified by affinity chromatography . the enzyme concentration was determined using a pierce bsa protein assay kit , and the enzyme was used in activity assays . the assays were carried out in 50 mm sodium phosphate buffer ( ph 7 . 0 ) at 25 ° c . in the context of the detoxification assays , enzyme concentrations of 0 . 079 μg / ml and ergotamine concentrations of 5 mg / kg were used . a further assay for reacting the six ergopeptines , namely ergotamine , ergovaline , ergocomine , ergocristine , ergocryptine or ergosine , and their respective isomeric forms , namely ergotaminine , ergovalinine , ergocominine , ergocristinine , ergocryptinine and ergosinine , used 1 . 58 μg / ml of the enzyme with sequence id no . 1 and 10 mg / kg ergotamine , or the equimolar ( summation ) concentrations of the remaining ergopeptines or their epimers . the results are indicated in fig2 . the samples were analyzed using hplc - fld or hplc - ms / ms , each by analytically determining the respective concentration of the sum of the respective epimers . simultaneously with the determination of the ergopeptine concentration during the enzymatic reaction , the formation of the ergo hydroxy acid ( metabolite 1 ) and of the ergoproline cyclodipeptide ( metabolite 2 ) was observed . during the continued reaction course , the conversion of metabolite 1 to ergine was detected . fig1 exemplarily shows the kinetics of the reaction of ergotamine with sequence id no . 1 . during said reaction , slight amounts of an instable intermediate product were detected , and the end production of the reaction was ergine . from fig1 , it is apparent that an almost complete degradation of ergotamine to ergine by sequence id no . 1 occurred within 4 hours . the reaction courses of all other ergopeptines , namely ergovaline , ergocomine , ergocristine , ergocryptine or ergosine , as well as their respective isomeric forms , namely ergovalinine , ergocominine , ergocristinine , ergocryptinine and ergosinine , are comparable . identification of the n - terminus of the enzyme with the sequence id no . 1 to identify the n - terminus of the enzyme with sequence id no . 1 , the genes having sequences id no . 2 and id no . 6 were cloned into pet28a (+) and transformed into e . coli using standard methods . following the expression , the bacteria cells were taken up in 50 mm sodium phosphate buffer and lyzed using a french press ( 20 , 000 psi ). the lysates were used in dilutions of 1 : 10 , 1 : 100 and 1 : 1000 in degradation batches of 5 mg / kg ergotamine . the batches were incubated at 25 ° c ., and the samples were analyzed using hplc - fld . the results of the degradation test indicated that both of the enzymes were able to transform ergotamine . however , the enzyme with the shorter nucleotide sequence displayed a significantly higher activity , this variant thus having been able to completely transform ergotamine even in the 1 : 1000 dilution , the longer variant displaying only little activity already in the 1 : 100 dilution . determination of the temperature range of the activity , and the temperature stability , of the enzyme with the sequence id no . 1 in order to determine the optimum temperature for the activity of the enzyme with the sequence id no . 1 , 0 . 1 μg / ml enzyme was incubated with 5 mg / kg ergotamine in teorell - stenhagen universal buffer ( ph 9 . 0 ) at varying temperatures ranging from 10 ° c . to 50 ° c . the enzyme displayed activity in a range of 10 ° c . to 35 ° c . with an optimum at 35 ° c ., based on the starting speed . in order to determine the temperature stability , the enzyme was incubated for 1 h at varying temperatures ranging from 10 ° c . to 60 ° c . after this , the enzyme solutions were incubated at concentrations of 0 . 1 μg / ml in teorell - stenhagen universal buffer ( ph 7 . 0 ) with 0 . 1 mg / ml bsa and 5 mg / kg ergotamine at 25 ° c . the results indicate that the enzyme is stable up to a temperature of 30 ° c ., still displaying some activity after incubation at 40 ° c ., yet showing a decrease of activity between 35 ° and 40 ° c . to sum up , it has turned out that the enzyme with the sequence id no . 1 substantially shows the temperature optimum at the temperature conditions found in the gastrointestinal tract . determination of the ph optimum of the activity , and the ph stability , of the enzyme with the sequence id no . 1 in order to determine the optimum ph range for the activity of erga , 0 . 1 μg / ml enzyme was incubated with 5 mg / kg ergotamine at varying ph values using teorell - stenhagen universal buffer at 25 ° c . said buffer was chosen , since the combination of citrate , phosphate and borate allows for the adjustment of the same buffer capacity in a range of ph 2 to ph 12 by hydrochloric acid . the enzyme displayed activity in a range of ph 6 to ph 11 with a small activity plateau at ph 8 to ph 9 . in order to determine the ph stability , the enzyme was incubated for 1 h at 25 ° c . at varying ph values ranging from ph 2 to ph 12 . after this , the enzyme solutions in concentrations of 0 . 1 μg / ml were incubated with 0 . 1 mg / ml bsa and 5 mg / kg ergotamine in teorell - stenhagen universal buffer ( ph 7 . 0 ) at 25 ° c . also in this case an activity plateau appeared , this time in the range of ph 6 to ph 9 , with a strongly decreasing activity outside this range . the activity in this range ensures the technological application of the enzyme with the sequence id no . 1 as a feed additive . expression of the enzyme with the sequence id no . 1 in picha pastoris the gene with the sequence id no . 2 was cloned into pgapz alpha c , transformed into p . pastoris , and expressed using standard methods . the expression vector pgapz alphac with the gene having the sequence id no . 2 is illustrated in fig3 . a degradation assay was carried out in 50 mm sodium phosphate buffer ( ph 7 . 0 ) with 5 mg / kg ergotamine at 25 ° c . from the culture supernatant , a 1 : 100 dilution was used . the samples were analyzed by hplc - fld . based on the results from sds - page and degradation assays , an expression of the enzyme with the sequence id no . 1 in the culture supernatant could be confirmed . expression of the enzyme with the sequence id no . 1 in bacillus subtilis the gene with the sequence id no . 2 was cloned into pht43 , transformed into b . subtilis , and expressed using standard methods . the expression vector pht43 with the gene having the sequence id no . 2 is illustrated in fig4 . a degradation assay was carried out in 50 mm sodium phosphate buffer ( ph 7 . 0 ) with 5 mg / kg ergotamine at 25 ° c . from the culture supernatant , a 1 : 10 dilution was used . the samples were analyzed by hplc - fld . based on the results from sds - page and degradation assays , an expression of erga in the culture supernatant could be confirmed . the activity of the ergot alkaloid - degrading enzyme of the enzyme with the sequence id no . 1 was tested in an in - vitro rumen model . to this end , fresh rumen juice was diluted 1 : 1 using a solution consisting of synthetic rumen juice , hay and a cereal mixture of wheat , maize and soy . to demonstrate the reaction of the ergopeptines , a batch was supplemented with the enzyme of sequence id no . 1 ( 1 μg / ml ) and 5 mg / kg ergotamine . fermentation tubes were used over septums , and the batches were incubated in water bath at 39 ° c . analytics by means of hplc / esi - ms / ms showed that ergotamine had been completely converted into ergine and lysergic acid in the rumen model . martinkova , l ., kren , v ., cvak , l ., ovesna , m . & amp ; prepechalova , i . 2000 . hydrolysis of lysergamide to lysergic acid by rhodococcus equi a4 . j . biotechnol ., 84 , 63 - 66 . kourist , r ., jochens , h ., bartsch , s ., kuipers , r ., padhi , s . k ., gall , m ., böttcher , d ., joosten , h .- j . & amp ; bornscheuer , u . t . 2010 , the α / β hydrolase fold 3dm database ( abhdb ) as a tool for protein engineering . chem bio chem , 11 , 1635 - 1643 . ollis , d . l ., cheah , e ., cygler , m ., dijkstra , b ., frolow , f ., franken , s . m ., harel , m ., remington , s . j ., silman , i . & amp ; schrag , j . 1992 . the alpha / beta hydrolase fold . protein eng ., 5 , 197 - 211 . schardl c . l ., panaccione d . g . & amp ; tudzynski p . 2006 . ergot alkaloids — biology and molecular biology . the alkaloids , 63 , 45 - 86 . yamada y ., matsuda m ., maeda k . & amp ; mikata k . 1995 . the phylogenetic relationship of methanol - 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