enzimatic synthesis of pyruvic acid and l-lactic

8/12/2019 Enzimatic Synthesis of Pyruvic Acid and L-lactic

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Greenhouse Gas C ontrol Technologies, Volume IIJ. Gale and Y. Kaya (Eds.) 2003 Elsevier Science Ltd . All rights reserved. 1451

E N Z Y M A T I C S Y N T H E S I S O F P Y R U V I C A C I D A N D L - L A C T ICA C I D F R O M C A R B O N D I O X ID E

Masaya Miyazaki , Hiroyuki Nakamura , and Hideaki MaedaMicro-space Chem ist ry Labora tory , AIST K yushu , Nat ional Inst itu te of Advance d Indust ria l

Science and Techno logy (AIST) , 807-1 Shuku, Tosu , Saga 841-0052, Japan

A B S T R A C TA new enzymat ic synthesis o f pyruvic ac id and L - lac tic ac id f rom aceta ldehyde and carbon d ioxide has beendeveloped . The react ion of pyruvic ac id synthesis u t i l izes reverse reac t ion of pyruvate decarboxylase insodium bicarbonate buf fer . The react ion proceeded a t h igh pH, and gave bet ter y ie ld in h igherconcentra t ion of buf fer . The ma xim um yie ld was ob ta ined in 500mM sodium bicarbonate buf fer a t pH I 1 .We a lso developed a one-pot , two-step enzymat ic synthesis o f L- lac t ic ac id f rom aceta ldehyde and carbondioxide . The react ion employ ing reversa l o f pyruvate decarboxylase and hydrogenat ion of pyruvate by L-lac tic dehydrogenase . The max imu m yie ld was ob ta ined a t pH 9 .5 in 500mM sodium bicarbonate buf fer .

I N T R O D U C T I O NBecause of recent g lobal warming , the emission of carbon d ioxide became ser ious problem. While carbondioxide gas i s no t in i t se l f a par ticu lar ly s t rong greenhouse gas , the contr ibu t ion to the a tmosphere s ince thebeginning of the indust ria l revolu t ion has been very considerab le . The increase in g lobal carbon d ioxidelevels over the las t hundred years or more have been repor ted . Fur thermore , there i s some ind ica t ion of ar is ing g lobal tempera ture . A number of meet ings, par t icu lar ly in Kyoto in December 1997 , haverecom mend ed reducing the to ta l emission of carbon d ioxide . Severa l e f for ts to im mob i l ize or u t i lize carbondioxide have been ach ieved . St i ll , no conclusive method is avai lab le for th is purpose . Recent in terest in theproblems of environmenta l po l lu t ion has forced the development of a g reen chemist ry process for thechemical indust ry [1] . Severa l environmenta l ly - f r iendly processes h ave b een developed . Biocata ly t icprocesses such as fermenta t ion and enzym e react ions have a t t rac ted a t ten t ion as environmen ta l ly safechemical process [2,3] .Biodegradable po lymers have a lso proved in teresting for environmenta l safe ty [4,5] . Am ong thebiodegradable p last ics , po ly(L- lac tic ac id) has been widely s tud ied . Severa l methods have been developedto produce lac t ic ac id f rom ma ny sources [6]. How ever , most o f them produce the racemic form and requirere la t ive ly longer time and mul t ip le s teps to ob ta in pure L- lac t ic ac id. These d isadvantages are problemat ic ,not only for large-scale produ ction but also for environ me ntal safety. Therefo re, a simp le metho d, that canprodu ce optica lly pure L -lactic acid, is desired.We are in terested in the development of novel enzymat ic reac t ions and reactor systems character ised byenvironmenta l ly safe chemical p rocesses . Pyruvate decarboxylase (EC 4 .1 .1 .1) is known as a ca ta lyst fo r


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the decarboxyla t ion react ion of pyruvic ac id , to produce aceta ldehyde, and has been u t i l ized for C-C bondformat ion such as ch i ra l G t-hydroxy ketones, wh ich are versa t i le bu i ld ing b locks for o rganic andpharm aceutica l chem istry [7, 8] . The reverse reaction of this enzym e is also of interest as a catalyticprocedure for carboxyla tion . Severa l stud ies have been per formed which imi ta te these enzyme react ionsusing CO2 as the reactan t . A prev ious s tudy using a- lac ty l th iamin show ed tha t p roduct ion of pyruvate wasachieved a t h igher pH (>10) [9] . How ever , these react ions require organic so lvents and severe condi tions.In the presen t s tudy , we dem onstra ted the usefu lness of the reverse reac t ion of pyruvate decarboxylase in theproduct ion of pyruvic ac id f rom aceta ldehyde and carbon d ioxide . W e a lso developed a one- reactor, two-step enzymat ic synthetic p rocedure for L- lac t ic ac id f rom aceta ldehyde, by com bin ing th is reverse reac t ion ofpyruvate decarboxy lase and reduct ion o f pyruvic ac id by L- lac t ic dehydrogenase to produce L- lac tic ac id .

M A T E R I A L S A N D M E T H O DGeneralPyruvate decarboxylase ( f rom brew er ' s yeast ) and L - lac tic dehydrogenase ( f rom rabbi t hear t ) were ob ta inedf ro m SI GM A (S t. Lo u is , MO , U. S .A . ) . Th iamin e p y r o p h o sp h a te an d NA DH wer e p u rch ased fr o m W ak oChem ical Ind. (Osaka, Japan). Sodium bicarbonate buf fers were prepared immed ia te ly before experiment .Al l exper im ents were per formed in thermo sta ted b ioshaker (Ti tech Co. , Tokyo Japan) wi th vor tex shaking .The HPLC analysis was per formed using Waters Al l iance 2596 system equipped wi th Wakosi l C22

analy t ica l co lumn (d~4.0 x h l 50 mm, W ako C hemical Ind .) . IH-NM R spectra was recorded by Bruker DSX-300 spect rophotometer .Synthesis o f Pyruvic AcidA typica l run was per form ed as fo l lows. To a so lu t ion of ace ta ldehyde (100 ~tM) in sodium bicarbonatebuffer (1 ml) in a 1.5 ml microcentr ifuge tube, pyruvate decarboxylase (1 unit) and thiamin pyrophosphate( the f ina l concentra t ion was 10 ~tM) were added a t 4 C. The react ion mix ture w as wa rmed to 25 C quick ly,and then shaken on a vor tex mixer a t room tempera ture . Af ter 1 h , the reac t ion mix ture was ch i l led on ice,and then subjec ted to RP-HPL C analysis immedia te ly . The am ount of pyruvic ac id was ca lcu la ted from thepeak area of RP-HP LC analysis ca l ib ra ted wi th commercia l ly avai lab le pyruvic ac id as s tandards. The y ie ldwas est im ated based on aceta ldehyde.Synthesis o f L-Lactic AcidThe reaction was init iated by adding pyruvate decarboxylase (1 unit) and L-lactic dehydrogenase (1 unit) to aso lu t ion of ace ta ldehyde (0 .1 laM) , th iamine pyrophosph ate (0 .1 k tM ) , NA DH (0 .2 ~tM) in var iousconcentra t ions and pHs of NaHCO3 Na2CO3buffer a t room tempera ture . The react ion was per formed 1 h ,and then the react ion was terminated by adding 1M HCI and analyzed by RP-HPL C. The am ount of eachcompound was est imated by peak area ca l ib ra ted by commercia l ly avai lab le s tandards. The abso lu teconf igura t ion of lac t ic ac id was conf i rmed by the op t ica l ro ta t ion , and the y ie ld was ca lcu la ted based onaceta ldehyde. For conf i rmat ion , the y ie lded lac tic ac id was sub jec ted to the analysis . The IH-NM R spectraand o pt ica l ro ta tion value were iden t ica l to the comm ercia l ly avai lab le L- lac t ic ac id .

R E S U L T SSynthesis o f Pyruvic AcidFirs t , we t r ied the reverse reac t ion of pyruvate decarboxylase (Scheme 1) . Prev ious organic synthesisshowed tha t hydro lysis o f lac ty lth iamin requires a h igher pH [9] . Thus, we c hose a sodium bicarbonatebuffer system, because n o t on ly is th is buf fer su i tab le a t h igher pHs, bu t i t a lso can be u sed as the source ofcarbon d ioxide .


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Pyruvatedecarboxylase OH3C-,,~ H + CO2 > H a C ~ o HO Thiamine O

Sch em e 1 : Synthet ic p rocess of pyruvic acid f rom carbon d ioxideW e f i r s t ly evaluated the ef fec t o f pH on the react ion . The react ion was per form ed using aceta ldehyde (100ktM), thiam in (0. 1 ~tM), and pyruv ate deca rboxy lase (1 unit) in 0.1 M NaHCO3/Na2CO3 buffer at variouspHs (pH 8 .5-11 .5) . The resu l t i s shown in Figure 1A. Higher pHs gave a be t ter y ie ld of pyruvic ac id . Thema xim um yie ld was ob ta ined a t pH 11 (61%). The presen t resu l t agreed wel l wi th a prev ious observat ionobtaine d from the hydrolysis of ct- lactylthiamin. In that case, the best yield from hydrolysis was obtained atpH 12. In our case , the best y ie ld was ob ta ined a t pH 11 , bu t the y ie lds decreased a t much h igher pH.Although the hydro lysis p roceeds a t a maxim um ra te a t pH 12 , the enzym e might no t be s tab le over pH 11.Therefore , the maxim um yie ld was ob ta ined a t pH 11. Thus, we decided to per form fur ther exper iments a tp H i l .

1 0 0

BO . . . . . . . . . . : . . . . . . . . . . ~ . . . . . . . . . . ~ . . . . . . . . . , ~

.o

40

o i i i8 B.5 9 9.5 10 10.5 11 11.5 12

pHFigure l " E f fec t o f pH o n the reverse reaction o f reverse react ion o f pyruva te decarboxy lase . Theexper ime nt was p er formed as descr ibed in M a t e r ia l a n d M e t h o d section.

. . . 6 0 ,

~ 4 0 , - - ' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

o . . . . . | . . . . . . . .1 o 1 o o 1 o o o

C o n c e n t r a t i o n r a M

F i g u r e 2: Ef fect o f ion ic s t rength of b icarbonate buffer on the reverse reac t ion of reverse reac t ion ofpyruvate decarboxylase . The expe r iment was per formed as descr ibed in Mat e r i a l s and Me t ho d section.


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Next , w e exam ined the e ffec ts of concent ra t ion o f b ica rbona te buffe r on the reac tion (F igure 1B). Higherionic s trength of the b ica rbona te buffe r s trongly inf luenced the yield , a s expec ted. The ma xim um yie ld ofthe reac t ion was 81% a t 500 mM NaHCO3/Na2CO3 buffe r . This y ie ld was suff ic ient to use as an organicprocess , and mu ch higher than tha t obta ined by the reac t ion in DM F u nder 20 a tm o f CO2 [9] . Not only doesthe la t ter reac tion requi re mul t ip le s teps , but the use of the organic solvent DM F is problemat ic forenvi ronmen ta l sa fe ty reasons . The enzym at ic reac t ion does not requi re any organic solvent and gave a be t te ryie ld . I t has been repo rted tha t the th iamin i t se l f could ca ta lyze a reac t ion ana logous to tha t of the enzyme,but preparat ion o f the intermed iate c~-lactylthiamin from acetaldehy de was unsuc cessful . Thu s, i t is difficultto reverse the reac tion wi thout an enzyme, and pyruva te decarboxy lase i s the bes t ca ta lys t for ca rboxyla t ionof ace ta ldehyde .Synthesis o f L- lac t ic ac idPyruvic ac id can be eas i ly hydrogena ted asym metr ica l ly by L-lac t ic dehydrogenase in the b iologica l sys tem.There fore , i t i s poss ible to produce L-lac t ic ac id f rom ace ta ldehyde and ca rbon dioxide by combining thetwo en zym e react ions , reverse reac t ion of pyruva te decarboxylase and L-lac t ic dehydrogenase (Sch eme 2) .In the present s tudy, we des igned and eva lua ted a one-pot , two-s tep enzymat ic synthe t ic procedure for L-lact ic acid from acetaldehyde as an ini t ia l experiment.

H3C ,,~H0

P y r u v a t e L - L a c t i cd e c a r b o x y l a s e O Oe h y d r o g e n a s e

C O 2 " - H 3 C O H ~ H 3 C O HO O H

Sch em e 2: Synthe t ic process of L- lac t ic acid f rom carbon dioxideFi rs t, the e ffec t of pH (7 to 10.5) on the reac t ion was examined, b ecause the reve rse reac t ion of pyruva tedecarboxy lase proceeds a t h igher pH, the yie lds should beco me b e t te r than tha t o f neut ra l pH. T he resul tswere su mm arized in F igure 3 . In the present s tudy, the y ie ld of lac tic ac id was max imized a t pH 9.5. M uchhigher pH gave lo wer y ie ld of L- lac t ic ac id and gave re la t ive ly be t te r recover y of pyruvic ac id . This resul tcan be expla ined as fo l lows. The f i rs t s tep proceeds a t h igher pH, whereas the second s tep, hydrogena t ion byL-lac t ic dehydrogen ase , gave low er y ie ld at much higher pH, y ie lded decrease of produc t ion of L- lac t ic ac idand the re fore the recovery ofpy ruv ic ac id was inc reased (F igure 3) .

100 . . . . . . . . . . . . . . . . . .

8 0 . . . . . . . . . : . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . ; . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . .

6 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : ....................................... ' .- i , ~ l k' - - , i [ : . A - -40 ............................................. -:.~ ....................................

. ~ A "

20 . . . . . . . . . i . . . . . . . ~ .. . . . . . ' . . . . . . . . . . . . . . . . . . .

O. , , - , ,7 8 9 10 1pHFig ure 3: Effec t of pH on the reac tion yie lds of the two-s tep reac tion. Each plot show the yie lds of pyruvicac id (O), L- lac t ic ac id (11) , and sum up o f both yie lds (A) . The exper im ents were perform ed as desc r ibed inMateria l and Method section.


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We a lso examined the ef fec t o f concentra t ion of b icarbonate buf fer a t pH 9 .5 , because the reverse reac t ion ofpyruvate decarboxylase prefers h igher concentra t ion of b icarbonate buf fer , s show n in Figure 4 , h igherconcentra t ion of b icarbonate gave bet ter yie ld, as expected . T he m axim um yie ld was ob ta ined a t 500 mMbicarbonate buf fer (51%). The reverse reac t ion o f pyruvate decarboxylase y ie lded about 43% of pyruvic ac idproduct ion in 500 m M bicarbonate buf fer a t pH 9 .5. How ever , the combined y ie ld of lac tic ac id and pyruvicacid exceeds 65%, mean ing the y ie ld of the reverse reac t ion of pyruvate decarboxylase was improved thanthat by a lone . This might resu l t f rom change o f equi l ib r ium condi t ion o f the reac tion , nam ely pyruvic ac idconsumption by the second step might promote better yield of the f irst step. Further studies are required toso lve the deta i ls o f this mecha nism.

B{ '] . . . . . . . . . .

70 - ; - , t . . . . . . . . . .

6 0 . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . " . . . . . . . . . , , ~ : . . . . . . . . . . . . . . . . . . . . . . . . .z/

~ o . . . . . i . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ."u' ~ 4 0: r - ~ . _ i

i . A ' - ' - -3 0 i ' i

11

I'1 , . t . . . . . . . . | . . . . . . . .1 0 1 O 0 1 0 0 0

C o n c e n t r a t i o n r a MFig ure 4 Ef fec t o f ion ic s trength on the react ion yie lds of the two-step react ion . Each p lo ts show the y ie ldsofpy ruvic ac id (0 ) , L- lac tic ac id (m) , and sum up of bo th y ie lds (A) . The exper iments were per formed asdescribed in aterial and ethod section.

D I S C U S S I O NAlthough the ef fec t is mu ch w eaker than methane, carbon d ioxide is considered to be a greenhouse gas , andtherefore i ts immobilization is desired. The methods currently reported are mainly catalytic ore lec t rochemical reac t ions, which require muc h energy [10-12] . One a l ternative method has been reported ,which u t i l ized carbonic anhydrase for the imm obi l iza t ion of carbon d ioxide [ 13]. This b iomim et ic approachneeds almo st no energy for the reaction. How ever, the carbonic anhydrase just im proves the solubili ty ofcarbon dioxide in aqueous media, and further treatment of dissolved gas is required.The b iodegradable po lymers have a lso been in terested for environmenta l safe ty . Am ong the b iodegradablep lastics , the po ly(L-lac t ic ac id) has been w idely s tudied . Severa l methods have b een developed to producethe lac tic ac id f rom man y sources . How ever , most o f them produce racemic form and require re la t ive lylonger t ime and multiple steps to obtain pure L-lactic acid. Thes e disadvantages are problem atic not only forlarge-scale production but also for the environmental safety. Therefore, a simple method, which can produceoptically pure L-lactic acid, is desired. Recent interest of poly(L-lactic acid) demands the effective method toproduce the material , L-lactic acid. The fermentation method has been focused as effective solution, becauseit can produce relative ly pure L -isom er [6].By our method , the carbon d ioxide in the aqueous phase can be condensed wi th aceta ldehyde to produce thepyruvic acid and can easily be converted into lactic acid, which is a mate rial ofbiod egra dativ e plastic. This


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method uti l izes an enzymatic reaction that proceeds in shorter t imes and gives the product in higher puri tythan that by fermentat ion. Although further studies are required to establish efficient pi lot-scale production,these features are advantageous than the classical fermentat ion method for lact ic acid production and thecarbon dioxide w il l be able to be imm obil ized into the biological cycles.In conclusion, we hav e dem onstrated the usefulness of the reverse reaction of pyruvate decarboxylase. Thisreaction might become a recommendable, environmentally safe carboxylat ion procedure for acetaldehyde.Also, we h ave develop ed a one-pot , two-step enzym atic synthesis method o f L-lact ic acid from acetaldehydeand carbon dioxide. This method might becom e a recom mend able, environ me ntally safe procedure for L-lact ic acid production. Further studies are in progress in our laboratory.

A C K N O W L E D G M E N TW e thank M itsukuni Shibue and Kazu ya Ogino for technical assistance.R E F E R E N C E S

10.11.12.13.

Anastas, P. T. and Warner, J. C. (1998) Gree n Chemistry: Theory and Practice, Oxford.Koller, K. M. and W ong, C.-H. (200 l) Nature 409, 232.Schm id, A., Dord ick, J. S., Haue r, B., Kiene r, M., W ubbo lts, M., and W itholt, B. (2001) Nature409, 258.Am ass, W., Amass, A., and Tighe, B. (1998) Polym. Int. 47, 89.Jacobsen, S., Degree, P. H., Fritz, H. G., Dubois, P. H., and Jerome, R. (1999) Polym. Eng. Sci. 39,1311.Lunt, J. (1998) Polym. Degred. Stab. 59, 145.Sprenger, G. A. and Poh l, M. (1999) J. Mo l. Catal. B: Enzym. 6, 145.Schorken, U. and S prenger, G. A. (1998) Biochim. Biophys. Acta 1385, 229.Foppen, M.-A. E., de Lange, Y. M., van Rantwijk, F., Maat, L., and Kieboom, P. G. (1990) Recl.Tray. Chim . Pay-Bas 109, 359.Li, Y., Xu, G.-H., Liu, C.-J., Eliasson, B., and Xue, B.-Z. (2001) Energy Fuels 15, 299.Aresta, M., D ibenedetto, A., and Tom assi , I . (2001) Energy Fuels 15, 269.Liu, C.-J., Xu, G.-H., and Wang, T. (1999) Fuel Processing Technol. 58, 119.Bond, G. M., Stringer, J. , Brandvold, D. K., Arzum Simsek, F. , Medina, M.-G., and Egeland, G.(2001) Energy Fuels 15, 309.

enzimatic synthesis of pyruvic acid and l-lactic

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