purification and characteriyation of a class i fructose 1,6+bisphosphate aldolase from...

8/6/2019 Purification and Characteriyation of a Class I Fructose 1,6+Bisphosphate Aldolase From Staphzlococcus Carnosus

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Appl M icrobiol Biotechnol (1990) 34:2875291Applied

MicrobiologyBiotechnology Springer-Verlag 1990

Purif icat ion and character izat ion of a class I f ructose1 ,6-b isphosphate a ldolase f rom Staphylococcus carnosus

H . P. B r o c k a m p a n d M . R . K u l a

Institu t ftir Enzymtechnologie der Heinrich-Heine Universit~it Dtisseldorf im Forschungszentrum Jtilich, Postfach 2050, D-5170 Jtilich,Federal Repu blic of Germany

Received 28 June 1990/Accepted 30 July 1990

S u m m a r y. A f r u c t o s e 1 , 6 - b i s p h o s p h a t e a l d o l a s e

(E.C.4 .1 .2 .13) f rom S t a p h y l o c o c c u s c a r n o s u s D S M2 0 50 1 w a s p u r i f i e d f o r t h e f i rs t t i m e . T h e e n z y m a t i c a c -t i v it y w a s i n s e n s i t iv e t o h i g h l e v e ls o f E D TA i n d i c a t i n gt h a t t h e e n z y m e i s a c l a s s I a l d o l a s e . T h i s e n z y m e e x h i -b i t s g o o d s t a b i l i t y a t h i g h t e m p e r a t u r e s a n d e x t r e m es t a b i l i t y o v e r a w i d e p H r a n g e . T h eKm f o r f r u c t o s e 1 ,6 -b i s p h o s p h a t e a s s u b s t r a t e w a s 0 .0 2 2 m M .T h e S . c a r n o -s u s a l d o l a s e i s a m o n o m e r i c e n z y m e w i t h a m o l e c u l a rm a s s o f a b o u t 3 3 k D a . I t e x h i b i t s a r e l a t i v e ly b r o a d p Ho p t i m u m b e t w e e n p H 6 .5 a n d 9 .0 . F u r t h e r m o r e , t h e a l-d o l a s e a c c e p t s o t h e r a l d e h y d e s i n p l a c e o f it s n a t u r a ls u b s tr a t e, g l y c e r a l d e h y d e 3 - p h o s p h a t e , a l l o w in g t h es y n t he s i s o f v a r i o u s s u g a r p h o s p h a t e s .

Introduct ion

A l d o l a s e s w e r e f i rs t o b s e r v e d n e a r t h e b e g i n n i n g o f t h i sc e n t u r y a s a n u b i q u i t o u s c l a s s o f e n z y m e s t h a t c a t a l y s et h e i n t e r c o n v e r s i o n o f h e x o s es a n d t h e i r t h r e e - c a r b o nc o m p o n e n t s ( M e y e r h o f e t a l. 1 93 6 ). A l l o rg a n i s m s p o s -s e s s a l d o l a s e e n z y m e s a n d t w o d i s t i n c t g r o u p s h a v eb e e n r e c o g n i z e d ( H o r e c k e r e t a l . 1 9 7 2 ) . Ty p e I a l d o l a s e ,f o u n d p r e d o m i n a n t l y i n h i g h e r p l a n t s a n d a n i m a l s,re -quires n o m e t a l c o f a c t o r, a n d c a t a l y s e s th e a l d o l c o n -

d e n s a t i o n t h r o u g h a S c h i f f b a s e i n t e r m e d i a t e . T h i s i n -t e r m e d i a t e c a n b e r e d u c e d w i t h b o r o h y d r i d e i n t h ep r e s en c e o f d i h y d r o x y a c et o n e p h o s p h a t e ( D H A P )w h e r e b y t h e a l d o l a s e i s i n a c t i v a t e d . Ty p e I a l d o l a s e sa r e u n a f f e c t e d b y E D TA . I n c o n t r a s t , c l a s s I I a l d o l a s e s ,w h i c h a r e f o u n d p r i m a r a l y i n b a c t e r i a a n d f u n g i , a r eZ n 2 + -, C a 2 + - o r F e 2 + - d e p e n d e n t e n z y m e s a n d c a n b ei n h i b i t e d b y c h e l a t i n g a g e n t s s u c h a s E D T A .

A l d o l a s e s a r e t h e m o s t u s e f u l e n z y m e s f o r t h e s y n -t h e si s o f m o n o s a c c h a r i d e s a n d r e l a t e d c o m p o u n d s( A k i y a m a e t a l . 1 9 8 8 ) , a l t h o u g h o n l y a s m a l l n u m b e r o ft h e k n o w n a l d o l a se s h a v e b e e n e x p l o i t ed f o r t h i s p u r -

Offprint requests to: M. R. Kula

p o s e . T h e m o s t t h o r o u g h l y s t u d i e d c l a ss I a l d o l a s e i s

t h e f r u c t o se 1 , 6 - b i sp h o s p h a t e a l d o l a s e f r o m r a b b i t m u s -c le ( R A M A ) . T h i s te t r a m e r i c e n z y m e w i th a m o l e c u l a rm a s s o f a b o u t 1 70 k D a a c c e p t s s ev e r a l a ld e h y d e s a ss u b s t r a t e s ( B e d n a r s k i e t a l. 1 9 89 ; E ff e n b e rg e r a n dS t r a u b 1 9 87 ) b u t i s n o t v e r y s t a b l e d u r i n g e n z y m a t i csynthes i s (Bossow-Berke 1989) . GOtz e t a l . (1980) de-s c r i b e d a v e r y h e a t - s t a b l e f r u c t o s e 1 , 6 - b i s p h o s p h a t e a l -d o l a s e f r o m S . a u r e u s , b u t n o t s u b s t r a t e s p e c i f i c i t y w a si n v e s t i g a t e d . We d e s c r i b e h e r e t h e f r u c t o s e 1 , 6 - b i s p h o s-p h a t e a l d o l a s e f r o m S . c a r n o s u s D S M 2 0 5 0 1 , w h i c h w a sc h o s e n a s a sa f e p r o d u c t i o n o rg a n i s m ( S c h l e i f er a n dFischer 1982) .

Mater ia l s and methods

O rg a n i s m a n d 9 r o w t h c o n d i t i o n s

The bacterium used in this study,S. carnosus DSM 20501, wasgiven to us by F. Grtz (Institut for Mikrobielle Genetik, Univer-sity of Ttibingen, F RG) who also supplied information on the cul-ture conditions. S. carnosus was cultivated under controlledaero-bic conditions (pOe , 5-10% ) in a 200-1 fermentation (Bioreaktor300 1; Chem ap, Volketswil, Swiss) at 37C. The growth m ediumcontained 1% glucose, 1% tryptone, 0.5% yeast extract a nd 0.5%NaC1 in a 0.1% sodium ph ospha te buffer, pH 7.2. The pro ductionfermen tor was inoculated with I01 of a preculture grown in thesame medium. Cells were cultivated to the end of the log phase,the culture reached an optical density at 660 nm of 16 after about5 h. Cells were harvested b y centrifugation (KA 6; Westfalia Sep-arator, ~}lde, FRG) and washed once with standard buffe r (60 mMTRIS-HC1 buffe r pH 7.5, containing 0 .1% mercaptoethanol). Ap-proxim ately 3 kg wet cells were obtained.

P u r i f i c a ti o n o f a l d o l a s e

Crude extract. The cell paste ob tained by ferm entation was mixedwith stan dard b uff er to give a 40% suspension. This suspensioncould be stored at -20C without any loss of activity. The cellsuspension was continuously disintegrated at 2500 rpm in a water-cooled cell mill (Netzsch, LME4, Selb, FRG). The grinding cham-ber was filled (85%) with glass bead s (0.3 mm diameter) pur chas edfrom H. Clauss (Nidderau, FRG). The supernatant obtaine d by

centrifugation at 8000 9 f or 10 rain served as the crud e cell-freeextract.


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Ammonium sulphatefractionation.The crude cell-free extract wasbrought to 40% ammon ium sulphate saturati on at 4 C. Precipi-tated protein was removed by centrifugation at 20000g for30 rain. The conc entrat ion of ammoni um sulphate in the supe rna-tant was adjusted to 80% saturation and precipitated protein wasagain removed by centrifugation. The supernatant was thenbrought to 100% ammon ium sulphate saturation and the pH wasadjusted to 5.0 with 7 M acetic acid. The precipita ted prote in wasseparated b y centrifugati on. Of the original aldolase activity 43%was recovered after dissolution of the sediment in standard buf-fer.

pHfractionation. The protein solution obtained in the ammoniumsulphate fractio n was cooled in ice a nd the pH lowered to 4.0 bydropwise addit ion of 7 M acetic acid unde r vigorous stirring. After1 h the precipitate was removed by centrifugat ion at 20 000 g for30 min. The pH of the supe rnata nt was lowered further to 3.5 andthen to 3.0 with 6 M HC1. The sup ernata nt at pH 3.0 obtain ed bycentrifuga tion at 15 000 g for 30 rain was foun d to conta in 31% ofthe original aldolase activity. This solution was diafiltered instandard bu ffer using an Amicon YM 10 ultrafilt ration membrane(Amicon, Witten, FRG).

DEAE-Sephadex A 25 chromatography.The diafiltrated proteinsolution was applied to a DEAE Sephadex A 25 column (2.6 by25 cm), previously equilibrated with standard buffer con taini ng inadditi on 0.1 M NaC1. The protei ns were eluted with a linear NaCIgradient (0.1-0.5 M). Aldolase was eluted at a sodium chlorideconcent ration of ab out 0.19 M. The fractions conta ining activitywere pooled, freeze-dried, a nd stored at - 2 0 C for further ex-periments.

Assa y o f aldolase activity

Aldolase assay was carried out by a spectrophotometric methodaccording to Blostein and Rutter (1963). The reaction mixturecontain ed 60 mM TRIS-HCI buffer (pH 7.5), 0.01 M EDTA,

0.28 mM NADH, 10 Ixg glycera ldehyde 3-phospha te dehydrogen-ase (G 3PDH)/triosephosphate isomerase (Boehringer, Mann-heim, FRG), 2.7 mM fructose 1,6-bisphosphate (F 1,6-BP) and en-zyme sol ution in a total volume of 1.3 ml. One un it (U) of activityis defined as the am oun t of enzyme required to oxidize 1 p~molNADH per minute. A molar adsorption coefficient of 6.3 1mmol -~ cm -~ was used for calcu lations (Boehringer 1989). Thephotometer cell and buffer solutions were thermostatted at37C.

Protein concentrations were assayed by the Bradford (1976)method using bovi ne serum albumi n (BSA) for calibration .

Sodium dodecyl sulphate (SDS) gel electrophoresis

This was car ried out by the method of L/immli (1970). The follow-ing proteins were used (protein calibra tion kit for gel electrophor-esis (Mr 20000-340000, Boehringer): macroglobulin [(Mr) red.170000l, phosphoryl ase B (Mr 97400), BSA (Mr 68000), gluta matedehydrogenase (Mr 36 500) and trypsin i nhibit or (Mr 20100). Pu-rified a ldolase (approxi mately 120 ~tg protein per ml) was sub-jecte d to electrophoresis. The gels were stained with Coomassiebri llant blue and silver reagent (Merril et al. 1981).

Inactivation of aldolase with D HA P and NaBH4

Inactivation was performed following the procedure of Lebherzand Rutter (1973). Aldolase (1 U /ml) was incub ated in 20 mM

DHAP (pH 6.0), the n 100 mM NaBH4 (in 0.02 M NaOH) was ad-ded slowly to the incub atio n mixture over a 30-60 min peri od

with constant stirring. The pH was adjusted , when necessary, byaddition of 2 M acetic acid. After the reaction, enzyme was sepa-rated from excess DHAP and NaBH4 by gel filtration on a Se-phadex G25 column. The remaining aldolase activity was assayedas described above.

Temperature stability

Samples of 12 Ixg puri fied aldolase in 1 ml o f 60 mM TRIS-HCIbuffer (pH 7.5) were kept in closed 1.5-ml Eppe ndorf tubes a ndheated at various temperatures in a water bath for 10 min. There-after the samples were cooled in an ice bath and the remainingactivity determined.

Substrate specificity

DHAP was synthesized by the method of Effenberger and Straub(1987). An aqueous solution of 20 mM DHAP, 200 mM of variousaldehydes and 1-4 U aldolase/ml were incubated for 5-7 h. Theresulting sugar phosphates were detected with acid molybdatereagent (Hanes and Isherwood 1949) after TLC [Polygram Cel 30020 x 20 cm from Macherey & Nagel, Dti ren, FRG ; developed with51.25 ml butanol :propa nol:ac etone: water (4:2:2:2.5)+3.75 mlformic acid + 12 g trichloroacetic acid].

R e s u l t s

Purification o f S. carnosus aldola se

Purif icat ion of the F 1,6-BP aldolase is summarized inTab le 1. The c onven t iona l , no t op t imized , p rocedur egave a ldo lase in high pur i ty (25 U/mg pro te in ) bu t lowyield (20%). The enzyme from this preparat ion ap-

pea red to be homog eneou s as judge d by SDS e lec t ro-phores i s and was used to de te rmine the molecu la rpropert i es of the aldolase.

Molecular m ass

The subun i t molecu la r mass o f pur i f i ed a ldo lase wasde te rmined by SDS ge l e l ec t rophores i s . The logar i thmof the re l a t ive e lec t rophore t i c migra t ions o f markerpro te ins were p lo t t ed aga ins t the logar i thm of the mo-lecu la r mass y ie ld ing a ca l ib ra t ion curve o flny =a l nx+ b ( ln, logar i thm na tu ra l i s ; y, molecu la r

w e i g h t / D a ; x, r e la t i ve m o b i l i t y / c m ; a = 1 , 3 ; b = 1 2 , 9 )

Table 1. Summary of Staphylococcus carnosusaldolase purifica-tion

Purif icat ion steps Specific Pur i- Yieldactivity fica tion (%)(U/rag) factor

Cru de cell-free extract 0.4 1.0 100(NH4)~SO4 fract ion 1.6 4.6 43pH frac tionati on 5.3 15.2 31DEAE-Sephadex A 25chroma tography 25.0 71.4 21

U, units


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with a correlation coefficient of -0.994. The molecularmass of the aldolase subunits was calculated from thedistance of migration (6.9 cm) as approxim ately 33kDa. The molecular mass o f the native enzyme was alsodetermined by gel chromatography using a SephacrylHR 200 column (eluent: 50 mM phosphate buffer con-taining 150 mM NaC1, pH 7.5). The result was identical

to that obt ained by SDS electrophoresis, indicating thatthe S. ca rnosus aldolase is a monomeric enzyme.

Pro of tha t S . carnosus a ldolase i s a c lass I enz yme

Effect of chelat in9 agents. T h e E D TA i n h i b i t i o n t e s tw a s c a r r i e d o u t w i t h p u r i f i e d a l d o l a s e . E D TA d i d n o ti n h i b i t t h e a l d o l a s e f r o m S. carnosus even at 0.1 M (Ta-ble 2) .

I n a ct iv a ti o n o f a M ol a se b y D H A P a n d N a B H 4 . Class Ifructose 1,6-bisphosphate aldolases form a Schiff baseintermediate with DHAP, which can be reduced with

NaBH4 to yield an inactive protein. This r educed com-plex has no catalytic activity. After reaction withDHAP and NaBH4, more than 95% of the aldolase ac-tivity was lost. In the absence of DHAP, NaBH4 didnot inhibit the aldolase activity (Table 2). Both results

Tab le 2. Act iv i ty of S. carnosus a ldolase in the presence of var iousreagents

Addi t ives Act iv i ty Inhi b i ton~ (U/ml) (%)

No ne 1 .08 - -ED TA (100 mM) 1 .10 - -

NaBH 4 (100 mM) 1 .06 2NaBH 4 (100 mM), DH AP (20 mM) 0 .05 95

D H A P, d i h y d r ox y a c e to n e p h o s p h a t e

rel. activity [fro]

1 0 0

8 0

6 0

4 0

2o ,0

2 4 6

- ' ~ citrate~ KPI

--X-- Trls-HCI

- B - glyetneI l

8 10

pH

12

Fig . 1 . pH o pt im um of f ruc tose- l ,6- b isphos phate (F-1 ,6-BP) a ldo-lase from Staphylococcus carnosus determined in var ious buffers

clearly show that the newly isolated aldolase from S.carnosus is a class I enzyme.

p H o p t i m u m

The pH-optimum for purified aldolase was measured in

citrate-, phosphate-, TRIS-HCI and glycine buffer. Likeother class I aldolases, S. ca rnosus aldolase exhibited abroad pH optimum, ranging from pH 6.5 to 9.0 (Fig.1).

tel. activity [%]1 0 0

9 0

8 0

70

60 ~ ~ ~3 0 5 0 7 0 9 0 11 0

temperature [~C]

Fig . 2 . Heat inac t iva t ion of F- I ,6-BP a ldolase f rom S. earnosus.

The pur i f ied enzyme was incubated a t var ious tempera tures for 5ra in . Af ter cool ing in an ice ba th the samples were assayed forremain ing ac t iv i ty

rel. activity i%]1 0 0

}

~ -a , - T-to 0"c ]~ T- 6 0 " C

8 0

6 0

4 0 .

2O

0 ~ i ~ ~

2 0 4 0 6 0 8 0 100

time |mini

Fig . 3 . Tem pera ture (T) s tab i l i ty of F- I ,6-BP a ldolase f romS. car-nosus a t 60 C and 100 C. The pur i f ied enzyme was hea ted a t the

indica ted tempera tures in a water ba th . Af ter cool ing in an iceb a t h t h e r e m a i n i n g a c t iv i ty w a s d e t e r m i n e d


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H e a t s t a b i l i t y

The aldolase from S . c a r n o s u s demonstrates remarkableresistance to heat inactivation. Figure 2 shows the rateof inactivation by incubating the purified enzyme in60 mM TRIS-HC1 buffer, p H 7.5, at various tempera-tures for 10 min. Figure 3 shows the rate of inactivationfor various incubat ion times at 60C and 100 C. Theremaining activity after 90 min at 60C was more than70%. Compl ete inactiv ation at 100C was obser vedonly after more than 90 min.

p H s t a b i l i t y

The S . c a r n o s u s aldolase exhibits extreme stabilityagainst acid and base (Fig. 4). The purified aldolasewas titrated with HC1 or NaOH to various pH values.After 5 min the aldolase activity was assayed spectro-photometrically. In the assay mixture the enzyme was

immediately brought to pH 7.5. There was no detecta-ble influence on aldolase activity by pretreatment be-tween pH 1.0 and 12.0.

K i n e t i c s t u d i e s a n d s u b s t r a t e s p e c i f i c i t y

The kinetic parameters, V r n a x and Km , with F 1,6-BPand fructose 1-phosphate (F l-P) as substrates were de-termined by measuring the reaction rate at various sub-strate concentrations. A Km of 0.022 mM and a Vmax of19 ~tmol min -1 mg -1 were calculat ed for F 1,6-BP assubstrate (Fig. 5) by linear regression of the Michaelis-

Menten equation. A Km of 18.8 mM and a Vmax of1.9 gm ol mi n -1 mg -1 were obtai ned with F 1-P. Be-

rel. activity 1%]120

11 0

100 ~ -"

9 0

8 0

7 0

60 i I i i i i

0 2 4 6 8 10 12 14p H

Fig. 4. p H stability of F-1,6-BP aldolase from S . c a r n o s u s . Purif-ied aldolase was titrated with HC1 or NaOH to the various pHvalues. After 5 min incubation at the indicated pH values the en-

zyme was diluted tenfold in standard buffer and assayed spectro-photometrically

[ U / m g ]25

20

15

10

~

O. I [ I I

0 0,2 0,4 0,6 0.8

c o n c e n t r a t i o n F - 1 , 6 - B P [ m m o l ]

Fig. 5. Michaelis-Men ten kinetics of F-1,6-BP from S . c a r n o s u sfor fructose 1,6-bisphosphate as substrates; U, units

sides glyceraldehyde 3-phosphate G 3-P several otheraldehydes were tested as substrates together withDHAP in the synthesis reaction. The additional alde-hydes that have been identified so far as substrates areD,L-glyceraldehyde, formaldehyde, acetaldehyde andmethylglyoxal.

D i s c u s s i o n

G0tz et al. (1980) described a very heat-stable F-1,6-BPaldolase from S. au reus , but did not investigate the sub-strate specificity. In our studies we used S . c a r n o s u s ,which is a non-pathogenic organism usually employedas starter culture in the preparation of sausages (DFG1987). Like the F-1,6-BP aldolase from S. au reus , thealdolase from S . c a r n o s u s was shown to be a class I en-zyme. In contrast to aldolases from animal sources(RAMA for example), which are tetramers with a sub-unit molecular mass of 30-40 kDa, the enzymes from S.c a r n o s u s and S . a u r e u s are monomers with a molecularmass of approximately 33 kDa.

The aldolases from S . a u r e u s and S . c a r n o s u s exhi-

bit extreme pH stability. This finding could perhaps berelated to the monomeric structure and fast renatura-tion rate un der assay conditions (Jaenicke 1984). TheK m for F 1,6-BP as substr ate (0.022 raM) is slightlylower for the S . c a r n o s u s than the S . a u r e u s enzyme(0.045 mM). The temper ature stability of the S . c a r n o s u saldolase is quite satisfactory but distinctly differentfrom that of the S . a u r e u s aldolase, which is fully activeafter 90 rain incuba tion at 100 C.

It has been shown that aldolase-catalysed conden-sations provide a practical route to a wide variety ofsimple sugars and sugar derivatives (Durrwachter et al.1986). Wong and Whitesides (1983) established the use

of rabbit muscle aldolase in the organic synthesis of un-natural sugar phosphates. The advantages of these


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r e a c t i o n s a r e w e ll k n o w n ( W h i t e s i d e s a n d Wo n g 1 98 5) .T h e e n z y m e f r o m r a b b i t m u s c l e , h o w e v e r , s u f f e r s f r o m( 1) i n s t a b i l i t y d u r i n g s y n t h e s i s o f s u g a r p h o s p h a t e s a n d( 2) a c t iv i t y l o s s in t h e p r e s e n c e o f m a n y r e a c t o r m a t e r i -a l s ( B o s s o w - B e r k e 1 98 9) .

B e c a u s e o f i t s o u t s t a n d i n g s t a b i l i t y a n d g o o d a c c e s -s i b i l i t y, t h e S . c a r n o s u s e n z y m e p o s s e s s e s c o n s i d e r a b l ep o t e n t i a l f o r c a t a l y s i n g a s y m m e t r i c a l d o l c o n d e n s a -t i o n s . T h e S . c a r n o s u s a l d o l a s e a l s o a p p e a r s t o e x h i b i t ar a t h e r w i d e s u b s t r a t e s p e c i f i c i t y f o r t h e a l d e h y d e c o m -p o n e n t s i m i l a r t o t h e e n z y m e f r o m r a b b i t m u s c l e . T h i sw i l l b e i n v e s t i g a t e d i n g r e a t e r d e t a i l i n f u t u r e w o r k .

Acknowledgements . We thank Prof. F. G6tz for his advice with re-gard to S. carnosus and the Deutsche Forschungsgemeinschaftand Fonds der Chemic for financial support.

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purification and characteriyation of a class i fructose 1,6+bisphosphate aldolase from...

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