Patent Application: US-201314396317-A

Abstract:
it discloses a use of n - acetylneuraminic acid aldolase with an amino acid sequence as shown in seq id no : 2 in catalytic synthesis of n - acetylneuraminic acid . the preparation of n - acetylneuraminic acid is to use the n - acetylneuraminic acid aldolase with the amino acid sequence as shown in seq id no : 2 as a catalyst , and n - acetylmannosamine and pyruvic acid as substrates .

Description:
the present invention can be better understood based on the following examples . however , one skilled in the field will easily understand that the specific material ratio described in the examples , process conditions and their results are used to illustrate the present invention only and should not be used to limit the invention described in detail in the claims . the genome of corynebacterium glutamicum atcc 13032 was extracted , then pcr was carried out by using the extracted genome as the template . the primer adding enzyme digestion site used in expression vector was established , the sequence of the primer was as follow : cycle 30 times according to the following parameters : denaturation at 98 ° c . for 10 seconds , annealing and extension at 68 ° c . for 1 minute , finally extension at 72 ° c . for 10 minutes . the pet - 28a vector ( pet - 28a , purchased from novagen ( merck china )) was digested by bamh i and hind iii respectively , after conforming that the vectors were completely linearized , the target fragment of pcr and the linearized expression vector were extracted respectively , then with one - step clone kit ( clonexpress ), 10 μl of linking product pet - 28a - cgnal was added into 100 μl of rosetta ( de3 ) competent cells , and placed on ice for 30 minutes , heat shocked at 42 ° c . for 90 seconds , placed on ice for 5 minutes . a pre - heated 0 . 9 ml of lb medium was added . centrifuged at 200 rpm at 37 ° c . for 1 hour . a 200 μl of bacteria solution was added onto a lb plate containing 100 μg / ml kanamycin and chloramphenicol respectively , incubated at 37 ° c . overnight for 12 to 16 hours . the graph of establishment is seen in fig1 . the recombinant strain e . coli rosseta ( pet - 28a - cgnal ) was picked up into a lb liquid medium containing antibiotics , incubated under vibration at 37 ° c . overnight . then , inoculated to a fresh culture solution in a 1 ( v / v ) % inoculation amount , when incubated to od 600 of about 0 . 6 at 37 ° c ., iptg was added to a final concentration of 0 . 2 mmol · l − 1 , centrifuged at 200 rpm at 30 ° c ., induced expression for 10 hours , then centrifuged ( 4 ° c ., 10000 rpm , 10 minutes ). the collected bacterial sludge was re - suspended in a 100 mmol · l − 1 tris - hcl ( ph 7 . 5 ) buffer , and the cells were ultrasonically lysed ( power 300 w , sonicated for 3 seconds , interrupted for 5 seconds , totally 5 minutes ), centrifuged ( 4 ° c ., 12000 rpm , 15 minutes ), and supernatant was removed . the collected enzyme supernatant was added to a ni - nta column ( ni - nta his bind resin , novagen ), and incubated on ice for 30 minutes . after the supernatant flowed through the column , the mixed protein was washed away with a 100 mmol · l − 1 tris - hcl ( ph 7 . 5 ) containing 20 mmol · l − 1 imidazole . then , the target protein cgnal was eluted down with a 100 mmol · l − 1 tris - hcl ( ph 7 . 5 ) containing 500 mmol · l − 1 imidazole . use aldolase ecnal from escherichia coli as a control , and the purity and expression level of cgnal were detected by sds - page , which was shown in fig2 . the protein concentration of the purified cgnal was determined by bradford method . the enzyme activities of cgnal were divided into the enzyme activity of neu5ac synthesis reaction and the enzyme activity of neu5ac decomposition reaction . the enzyme activity on neu5ac synthesis reaction was defined as the enzyme amount required for synthesizing 1 μmol neu5ac per minute , and the enzyme activity on neu5ac decomposition reaction was defined as the enzyme amount required for decomposing 1 μmol neu5ac per minute , the enzyme activity detection solution for neu5ac decomposition reaction was 0 . 1 m pyruvic acid , 0 . 1 mol · l − 1 mannac and 0 . 1 mol · l − 1 tris - hcl ( ph 7 . 5 or 8 . 5 ); the enzyme activity detection solution for neu5ac decomposition reaction was 100 mmol · l − 1 neu5ac and 0 . 1 mol · l − 1 tris - hcl ( ph 7 . 5 or 8 . 5 ). the purified nal was added into 1 ml of enzyme activity detection solution to the final concentration of 30 μg / ml ( about 0 . 36 u / ml ). after reaction at 37 ° c . for 20 minutes , the tube was heated in a boiling water for 5 minutes to stop the reaction , centrifuged at 12000 g for 10 minutes , and the sample was filtered with a 0 . 22 μm filter . the substrate and the product were detected with bio - rad aminex 87 - h column by using agilent 1200 hplc ,( 5 mmol · l − 1 h2so4 as a mobile phase , flow rate 0 . 6 ml / min , differential refractive index detector ). the following buffer was used in effect of ph on cgnal : 0 . 1 m tris - hcl ( ph 7 to 8 . 8 ) and 0 . 1 m glycine - naoh buffer ( ph 9 . 0 to 9 . 5 ). the enzyme activity was detected in the enzyme activity detection solutions of different phs , in order to detect the effect of ph on the enzyme activity . the detection results showed that the activity of cgnal on neu5ac decomposition reaction between ph 7 . 5 and 8 . 4 was much higher than the activity on neu5ac synthesis reaction ; when the ph was between 8 . 6 and 8 . 8 , the decomposition activity of neu5ac was close to neu5ac synthesis activity ( fig3 ). the enzyme activities of both directions of the aldolase cgnal were detected by using a temperature gradient at ph 7 . 5 ( 25 ° c . to 60 ° c . ), in order to find the optimum reaction temperature . at ph 7 . 5 , the optimum temperature for cgnal was 40 ° c ., the optimum temperature for decomposition and synthesis reaction of neu5ac were identical . at ph 8 . 5 , the optimum temperature of neu5ac synthesis direction was 40 ° c ., and the optimum temperature of neu5ac decomposition direction was 45 ° c . ( fig4 ). the cgnal was suspended in a 0 . 1 m tris - hcl ( ph 8 . 5 ) buffer , then placed in a warm water bath at 37 ° c . for 48 hours , and the change of enzyme activities was detected during the warm water bath , in order to determine the stability of cgnal . within 10 hours prior to the warm water bath , neu5ac synthesis activity of cgnal showed a rising trend , after 36 hours of warm water bath it can still maintain about 80 % of starting activity ( fig5 ). 4 . effects of metal ions and surfactant on the cgnal activity to an enzyme activity detection solution of cgnal , 5 mm of cacl 2 , nacl , bacl 2 , fecl 3 , kcl , zncl 2 , cocl 2 , mgcl 2 , nh 4 cl , niso 4 , edta , ctab and sds were added , and a sample without adding any metal ion and surfactant was used as a control . the enzyme activities of cgnal at both ph 7 . 5 and ph 8 . 5 were detected . the enzyme activity of cgnal at ph 8 . 5 was much higher than the enzyme activity of cgnal at ph 7 . 5 , and the effects of metal ions on cgnal under different ph conditions were quite different . at ph 7 . 5 , zncl 2 , cocl 2 , niso 4 , ctab and sds all promoted reaction of cgnal in the synthesizing neu5ac direction , meanwhile they also inhibited the reaction in the decomposing neu5ac direction ( fig6 ). whereas the metal ions at ph 8 . 5 had no significant activation effect on cgnal . zncl 2 , ctab and sds promoted the superiority of neu5ac synthesis over neu5ac decomposition , but overall decreased the enzyme activity ( fig7 ). therefore , at suitable ph value and under effect of metal ions and surfactant , the rate of cgnal in neu5ac synthesis direction was greater than the rate in neu5ac decomposition direction . the enzymatic reaction kinetic constants of cgnal at ph 7 . 5 and ph 8 . 5 on the substrates mannac , neu5ac and pyruvic acid were determined at different concentration of substrates . when the kinetic constant on pyruvic acid was determined , the fixed mannac concentration was 100 mm , and the pyruvic acid concentration varied between 1 and 100 mm . when the kinetic constant on mannac was determined , the fixed pyruvic acid concentration was 100 mm , and the concentration of mannac varied between 1 and 400 mm . when the kinetic constant on neu5ac was determined , the concentration of neu5ac varied between 1 and 200 mm . the kinetic constants of cgnal on neu5ac , mannac and pyruvic acid at ph 7 . 5 and ph 8 . 5 were as shown in table 1 . when the ph value was increased from 7 . 5 to 8 . 5 , the km and vmax values of the substrate were greatly increased . in a 20 ml of reaction system , the reaction solution was 50 mm tris - 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