Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus

ELSEVIER

Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus Duncan A. N. Edlin,* Arjan Narbad,+ Michael J. Gasson,+ J. Richard Dickinson,* and David Lloyd* *Microbiology Group, School of Pure and Applied Biology, University of Wales College of CardifJ; Card@ United Kingdom, ‘Institute of Food Research, Norwich Research Park, Norwich, United Kingdom

The yeast, Brettanomyces anomalus, produces a hydroxycinnamic acid decarboxylase which is active toward ferulic acid, p-coumaric acid, and caffeic acid. The enzyme transforms these hydroxycinnamic acids to hydroxystyrenes by the removal of the carboxyl group from the C3 side chain. We have pur$ied this enzyme 235fold from B. anomalus NCYC 615 using Mono Q ion exchange, Phenyl Superose. and Superose 12 column chromatography. Enzyme activity was found to be optimal at 40°C and pH 6.0 and was enhanced by EDTA, Mg*+, and C?‘. Fe3+, Ag+, and SDS completely inhibited the activity. Kinetic studies indicated a K, of 1.15 rnMand a V,, of 13,494 nmol min-’ mg-’ for ferulic acid and a K, of I.55 mM and a V,, of 22.256 nmol min-’ mg-’ for p-coumaric acid. Using gel$ltration, an apparent molecular mass of 39.8 kDa was estimated. The decarboxylase was inactive toward both o- and m-coumaric acid and toward cinnamic acid, indicating that the para-hydroq group is essential for activity. 0 I998 Elsevier Science Inc.

Keywords: Hydroxycinnamate

decarboxylase;

ferulic acid; caffeic acid; p-coumaric

acid; Bretkznomyces

unomalus;

flavor

compounds

Introduction Hydroxycinnamic acids such as ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid are common phenolic compounds found universally in plant tissue. Due to the abundance of these naturally produced aromatics, there is scientific interest in utilizing them as substrates for the production of novel phenolic flavor and fragrance chemicals. The use of microbial enzymes for the production of these compounds would mean that the products of the reactions could be labelled ‘natural’ as opposed to chemically synthesized, and therefore fetch a premium price on the market.’ A number of species of microorganisms have been reported to decarboxylate hydroxycinnamic acids. Previously, we have reported on the decarboxylative ability of the yeast, Brettanomyces anomalus, toward ferulic acid, p-

Address reprint requests to Dr. D. A. N. Edin, Univ. of Wales College of Cardiff, School of Pure and Applied Biology, Microbiology Group, P.O. Box 915, Cardiff CFI 3TL, UK Received 14 April 1997; revised 28 July 1997; accepted 5 August 1997

Enzyme and Microbial Technology 22:232-239, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

coumatic acid, and caffeic acid.’ This activity has also been noted in Saccharomyces cerevisiae3 as well as in numerous strains of bacteria and fungi.4-12 The products of the decarboxylation of hydroxycinnamic acids are hydroxystyrenes. These compounds give off strong smoky and aromatic odors and flavors and are regarded as the source of phenolic off-flavors in many beers and wines.‘3T’4 Ferulate and p-coumarate decarboxylases (no EC number) have been purified from Lactobacillus plantarum and from two strains of Bacillus pumilus.‘5-‘7 The strain of Pseudomonas jluorescens used for the purification of ferulate decarboxylase by Huang et al. ” has since been identified as a B. pumilus. In B. pumilus, the purified decarboxylase was found to exist as a homodimer with an apparent molecular mass of 45 or 40.4 kDa when analyzed by size exclusion chromatography. In L. plantarum. the purified p-coumarate decarboxylase also showed activity toward caffeic acid. Size exclusion chromatography indicated that in L. plantarum, the decarboxylase had an apparent molecular mass of 93 kDa and exists as a tetramer consisting of four 23.5 kDa subunits. We report for the first time the partial purification and characterization of a hydroxycinnamate decarboxylase from

0141-0229/98/$19.00 PII SO141-0229(97)00169-5

Brettanomyces CH=CH-COOH &

z

JH

Figure 1 Hydroxycinnamate decarboxylase from B. anomalus catalyzes the decarboxylation of hydroxycinnamic acids to hydroxystyrenes. Where R = H, the substrate and product are pcoumaric acid and 4-hydroxystyrene, respectively. Where R = OH, the substrate and product are caffeic acid and 3,4-dihydroxystyrene, respectively. Where R = OCH,, the substrate and product are ferulic acid and 3-methoxy-4-hydroxystyrene, respectively

B. anomalus. This wild yeast has been shown to decarboxylate p-coumaric acid. caffeic acid, and ferulic acid to 4-hydroxystyrene, 3,4-dihydroxystyrene, and 3-methoxy, 4_hydroxystyrene, respectively. These compounds are then subsequently reduced to ethyl derivatives by this yeast.*

Materials and methods Materials

D. A. N. Ecilin et al.

In order to assay large numbers of fractions during the purification procedure, a much simpler and faster spectrophotometric monitoring of the differences in absorbance of the substrate and product at 285 nm and 260 nm was performed. Samples which contained only p-coumaric acid produced a 285 nrn/260 nm ratio greater than 2 wheras samples with decarboxylase activity gave a ratio less than 2. In samples with a high decarboxylase activity, the ratio went as low as 0.3.

Protein determination The total protein concentration was determined using the Bradford method Bio-Rad protein assay with BSA as the standard.” Mono

Q separation

Crude extract (50 ml) was dialyzed overnight with 20 mM Bis-Tris-HCl buffer pH 6.0 and injected into a Mono Q column using a Superloop. The column was washed at a flow rate of 1.O ml min- ’ with 30 ml of 20 IIIM Bis-Tris-HCI buffer pH 6.0. A gradient of O-O.6 M NaCl was applied over 15 min and the column then flushed with 1.0 M NaCl for 15 min. The 35 X I ml fractions collected were assayed for decarboxylase activity. The active fractions were pooled and (NH&SO, added to give a final concentration of 1.7 M.

Phenyl Superose separation

Mono Q, Phenyl Superose. and Superose 12 columns were manufactured by Pharmacia LKB Biotechnology (Uppsala, Sweden). Microconcentrators were obtained from Amicon Ltd. (Stonehouse, Glocs, UK). Ferulic acid, p-coumaric acid, caffeic acid, dithiothreitol, and the SDS-PAGE silver staining kit were purchased from Sigma-Aldrich (Poole, Dorset, UK). HPLC grade methanol and 0.45 mm glass beads were obtained from BDH (Merck Ltd., Lutterworth, Leics, UK). Protein assay dye reagent and SDS-PAGE kit were supplied by Bio-Rad (Bio-Rad Laboratories Ltd.. Hemel Hemstead, Herts, UK).

Microorganism B. anomalus (NCYC 615) was obtained from the National Collection of Yeast Cultures (NCYC, Norwich, UK).

Preparation

anomalous:

of cell extracts

Cultures of B. anomalus were grown either in Y.E.P.D. (10 g yeast extract, 20 g bacteriological peptone, 20 g glucose, 0.1 g adenine. and 0.1 g uracil I-‘) or in a defined yeast medium (D.Y.M.; 16.7 g Difco vitamin-free yeast base, 20 g glucose, 2 pg biotin, 2 pg inositol, and 0.4 mg thiamin hydrochloride I-‘). The cells from the culture were harvested by centrifugation at 1,600 g for 15 min. They were washed with 20 ml 40 mM phosphate buffer pH 7.3 and resuspended in the same volume of buffer containing 0.1 mM dithiothreitol. Extracts were prepared using a Braun homogenizer (6 X 30 s bursts) using 0.45 mm glass beads. The supematant obtained was ultracentrifuged to remove any cellular debris at 100,000 K for 90 min at 4°C. The extract was then stored at -70°C prior to enzyme analysis.

Decarboxylase

assay

Assays were conducted in a 0.5 ml reaction volume containing 50 mM KH,PO,/K,HPO, buffer pH 6.0 and 2 mM substrate. The reaction was started by the addition of 5-50 p,l extract and was performed at 40°C for 10 min. The reaction was stopped by heating to 70°C for 10 min. After centrifuging at 12,000 g for 2 min, samples were injected directly into HPLC and the product measured at 280 nm as described previously.”

Pooled samples from Mono Q separation were used for Phenyl Superose column chromatography. The column was washed at a flow rate of 1.0 ml min-’ with 15 ml 1.7 M (NH&SO, 20 mM Bis-Tris-HCI pH 6.0. A gradient of 1.7-0.0~ (NH&SO4 in 20 mM Bis-Tris-HCl pH 6.0 was then applied over 35 min. The column was washed with 20 mM Bis-Tris-HCl pH 6.0 and the fractions obtained were assayed for decarboxylase activity.

Superose 12 separation The active pooled and Concentrate the proteins pH 6.0.

fractions from the Phenyl Superose separation were concentrated using Amicon 30 microconcentrators. (200 ~1) was injected into a Superose 12 column and eluted at 0.5 ml min-’ with 20 mM Bis-Tris-HCI

Second Mono Q separation Active fractions from the Superose 12 separation were pooled and injected into a Hi-trap Mono Q ion-exchange column. Proteins Table 1 Specific activities of hydroxycinnamate decarboxylase prepared from cultures of t?. anomalus Gflown on either YEPD, DYM, or DYM + 2 mM pcoumaric acid at different times during growth Specific activity (nmol min-’ mg-’ protein)

Growth phase

Cells grown on YEPD

Cells grown on DYM

Lag phase Mid-log phase Late-log phase Stationary phase

12.1 13.7 17.7 0.2

2.4 6.8 3.7 11.4

2 + t +

7.7 2.1 1.7 0.3

+ 1.4 -c 3.1 5 3.2 r 5.5

Experiments were conducted in triplicate show mean t standard deviation

Enzyme Microb. Technol.,

Cells grown on DYM +2 mM pcoumaric acid 25.4 33.3 21.1 23.9

t + 2 +

2.2 3.1 8.7 5.4

(n = 3) and results

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233

Papers --

(a)

*280nm Activity *285nm/*260nm M NaCl - 1.2

2.4

1

0.6

- 1.0 2.2 -0.8

2 z I

-0.6

- 0.4 1.8 - 0.2

1.6

- 0.0

Elution volume (ml) -

280nm

-

Activity A285nm/A260nm

-.--

(NH4)2S04

(M)

W 0.2 z Gj cu % 2

0.1

0.0 10

20

30

40

50

Elution volume (ml) Figure 2 Elution profiles of proteins from 5. anomalus on Mono Q (a), Phenyl Superose (b), and Superose 12 chromatography (c). Mono Q -the sample was applied with a Superloop, the column was washed with 30 ml of 20 mM Bis-Tris-HCI pH 6.0, and a gradient of 0.0-0.6 M NaCl was run to elute the proteins (a). Fractions were assayed using a spectrophotometric method (see MATERIALSAND METHODS).Active fractions gave an A285nm/k&,,nm ratio of less than 2.0. Phenyl Superose -the pooled active fractions from Mono Q were injected into a Phenyl Superose column (b). The column was washed with 15 ml of 1.7 M NH,(SO,), 20 mM Bis-Tris-HCI pH 6.0 and a gradient of 1.7-0.0 M NH,(SO& was used to elute the proteins. The 1.0 ml fractions collected from the column were assayed using a spectrophotometric method. Superose 12 gel filtration - the concentrated active sample from Phenyl Superose was injected into a Superose 12 column and proteins were eluted with 30 ml 20 mM Bis-Tris-HCI pH 6.0 (c)

234

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1998, vol. 22, March

Brettanomyces Cc)

anomalous:

b

A280nm

-

Activity @moles min“ )

D. A. N. Edlin et al.

0.08

E t 8

0.06

-?

4

0 20

10

Elution Volume (ml)

Figure 2

Continued

were eluted using the same conditions Mono Q separation.

as described

for the first

Specificity of hydroxycinnamate decarboxylase from B. anomalus

Characterization of the purified hydroxycinnamate decarboxylase from B. anomalus Inhibitor/activator studies. Crude extract was used to assay for the effect of a number of potential inhibitors or activators of the decarboxylase. The assay was performed as above but with the addition of a number of test compounds all at 1 mM final concentration.

The activity of the decarboxylase was tested toward p-coumaric acid, ferulic acid, caffeic acid, cinnamic acid, hydrocaffeic acid, sinapic acid, phenylalanine, p-hydroxybenzoic acid, o-coumaric acid, m-coumaric acid, S-hydroxyferulic acid, iso-ferulic acid, 3,4-methylenedioxycinnamic acid, and p-methoxycinnamic acid using the assay conditions described above. Activity toward pyruvate by the purified decarboxylase was also assayed by coupling the activity to alcohol dehydrogenase and measuring the decrease in A-+,,,“,,,.

Effect of temperature on activity. Crude extract was used to assay for activity at 4, 12, 20, 30, 37, 40, 50 and 60°C using the assay conditions described above.

SDS-PAGE analysis

Effect of pH on activity. Acetic acid/acetate buffer was used to assay activity over the pH range; 3.0-5.5, KH,PO,/K,HPO, buffer between pH 6.0-7.5, Tris-HCI for pH 7.5-9.0, and boric acid/sodium tetrdborate for pH 8.0-9.0. All buffers were present at 50 mivt final concentration.

SDS-PAGE (12% w/v resolving gel) was performed at 200 V for 45 min using Bio-Rad mini protein II cells and a low molecular weight standard set from Life Science Technologies (Paisley, Strathclyde, UK). The gels were stained with Coomassie blue, or where necessary, with a silver stain kit.

Table 2

Purification

Purification

Step

Crude extract Mono Cl Phenyl Superose Superose 12 2nd Mono 0

of hydroxycinnamate

decarboxylase

from 13. anomahs

Volume (ml)

Protein Concentration (mg ml-‘)

Total activity (U)=

Specific (U mg-‘)

Purification factor

% Yield

47.00 19.00 19.50 5.00 0.20

22.50 9.41 1.69 0.40 0.15

16,540 8,990 8,580 660 110

15.6 50.3 260.4 328.0 3,666.7

1.0 3.2 16.6 21.0 232.0

100.00 54.30 51.90 4.00 0.67

a One unit of enzyme activity (U) is defined as the amount

of enzyme that causes 1 nmol of product to be formed

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min -’

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235

Papers 1

68

-

43

-

29

-

different media and at various stages of growth (Table I). The data suggests that the hydroxycinnamate decarboxylase was expressed constitutively at low levels when the yeast was grown in the absence of the substrate; however, we found that induction above the basal constitutive level occurred in cells grown in the presence of p-coumaric acid. The specific activity of the enzyme was greatest in samples prepared from the mid-log phase of growth from cells grown on DYM + 2 mM p-coumaric acid.

18.4

-

Protein purification

14.3

-

Lane

97.4

2

3

-

Figure 3 SDS-PAGE analysis of samples taken in the purification of hydroxycinnamate decarboxylase from 6. anomalus. Lane 1. Standard protein molecular mass markers (200 kDa, 97.4 kDa, 68 kDa, 43 kDa, 29 kDa, 18.4 kDa, and 14.3 kDa), lane 1; crude extract, lane 2; purified sample after second Mono Q separation, lane 3

Results Hydroxycinnamate decarboxylase transforms hydroxycinnamic acids to styrene-like compounds. In our initial studies with cultures of B. anamalus, the enzyme was found to be active toward the hydroxycinnamic acids p-coumaric acid, caffeic acid, and ferulic acid (Fire 1). The cultures used in this work were grown on a defined yeast medium containing biotin, inositol, and thiamin. We investigated the specific activity of the enzyme in extracts from yeast grown in

120

A crude extract was prepared from B. anomalus NCYC 6 15 grown to mid-log phase in 20 1 of DYM supplemented with 2 mM p-coumaric acid. This extract was dialyzed with 20 mM Bis-Tris-HCl pH 6.0 and purified further by Mono Q, Phenyl Superose, and Superose 12 column chromatography (Figure 2). The purification of the enzyme is summarized in Table 2. The separation of the pooled sample by Phenyl Superose gave a five-fold purification. The matrix of this column is composed of aromatic rings which bind tightly to the hydroxycinnamate decarboxylase, thereby allowing the protein to be eluted at the end of the run. Superose 12 chromatography led to a considerable loss of enzyme yield but gave appropriate size separation of the sample and provided accurate information on the size of the native decarboxylase. After the second Mono Q separation, a 235fold purification and a final yield of 0.67% was obtained from the whole purification scheme.

Determination

of molecular mass

The molecular weight of the purified enzyme was estimated using Superose 12 gel filtration at 39.8 kDa. When the purified protein was analyzed on SDS-PAGE (Figure 3), the most likely candidate for the decarboxylase had an esti-

1

1

100

1

6

PH Figure 4 Optimum pH curve for hydroxycinnamate min-’ mg-’ protein

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1998, vol. 22, March

from B. anomalus where 100% activity is equivalent to 15.81 nmol

Brettanomyces

0

20

10

30

40

Temperature

Figure 5 Optimum temperature 18.56 nmol min-’ mg-’ protein

curve for hydroxycinnamate

decarboxylase

mated molecular mass of 21.8 kDa. If the most intense band is the hydroxycinnamate decarboxylase, then this result would suggest that the native enzyme in B. anomalus is a homodimer.

EfSect of pH and temperature The optimal pH and temperature for the decarboxylase were found to be 6.0 and 40°C respectively (Figures 4 and 5). The decarboxylase is active in the pH range 4-8. Activity declines rapidly above 40°C; however, 12.5% of the activity at 40°C could be detected at 60°C. The enzyme stability was maintained for longer in extracts frozen at -70°C or -20°C (Table 3). The activity was lost rapidly from extracts stored at 4°C or 20°C. Only 49.3% of the initial activity remained after 4 days at 20°C wheras 46.8% of the initial activity remained after 7 days at 4°C.

50

anomalous:

D. A. N. Ed/in et al.

70

60

(OC)

from 6. anomahs

where

100% activity is equivalent

to

Cu*+ all partially inhibited the enzyme activity. Fe”+, Ag+, and SDS completely inhibited the decarboxylase activity.

Substrate specificity and K,, From the 15 different substrates tested with the decarboxylase. only ferulic acid, caffeic acid, and p-coumaric acid were transformed by the purified enzyme (Table 5). It appears that the para-hydroxy group is essential for activity since neither cinnamic acid nor the ortho nor meta-hydroxy forms of coumaric acid could be decarboxylated by the enzyme. Substitution of the para-hydsoxy group with a methoxy group also inhibits the activity. Substitution of both metu positions on the aromatic ring inhibits the

Table 4 The effect of various cations and reagents (1 mM Strength) on the hydroxycinnamate decarboxylase activity

EfSects of inhibitors/activators The effects of various cations and reagents on the activity are shown in Table 4. Cr3+, EDTA, and Mgzf all enhanced the activity whereas Zn’+, Ca*+, Co*+, Mn*+, Li+, and

Table 3 Temperature boxylase activity

stability of the hydroxycinnamate

decar-

% Activity after: Storage temperature -70°C -20°C 4°C 20°C Activity (100%) is equivalent

4 days

7 days

100 100 100 49.3

100 100 46.8 0

to 566.25 nmol min-’

mg-’

protein

inhibitor/activator (1 mM) CrCI, EDTA MgCt, Control ZnCI, CaCI, COCI, MnCI, LiCl CUCI, FeCI, AgW SDS

% Relative activity 144.39 126.34 111.22 100.00 95.12 93.66 88.78 81.46 59.02 26.83 0.00 0.00 0.00

The activity assayed in the absence of inhibitor/activator (control) was set to 100%. Activity (100%) is equivalent to 3,476 nmol min-’ mg-’ protein

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237

Papers Table 5 The specificity of the purified carboxylase for various substrates Substrate

(2 mM)

Caffeic acid pCoumaric acid Ferulic acid Cinnamic acid Sinapic acid Hydrocaffeic acid o-Coumaric acid m-Coumaric acid pMethoxycinnamic acid p-Hydroxybenzoic acid iso-Ferulic acid 5-Hydroxyferulic acid 3,4-Methylenedioxycinnamic Phenylalanine Pyruvate

hydroxycinnamate

de-

% Relative activity

acid

100.0 37.5 31.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

The decarboxylation rate for caffeic acid was 7,473 nmol min-’ mg-’ protein (100%)

since neither sinapic acid nor 5-hydroxy fernlic acid were decarboxylated by the protein. The composition of the side chain also determines the substrate specificity, since activity toward phenylalanine, p-hydroxybenzoic acid, and hydrocaffeic acid could not be detected. The purified enzyme also shows no decarboxylase activity toward pyruvate. This rules out the possibility that the purified protein is pyruvate decarboxylase which is known to have a broad substrate specificity. The initial velocity for the decarboxylation of ferulic and p-coumaric acid by the purified decarboxylase was measured over the substrate concentration range of 0.1-2.0 mM in 50 mM KH2P0,/K,HP0, buffer pH 6.0 at 40°C. From the resulting Lineweaver-Burk plot, the & for p-coumaric acid was found to be 1.55 mM and for ferulic acid it was 1.15 mM. The maximal velocity (V,,,) was found to be 22,256 nmol min-’ mg-’ for p-coumaric acid and 13,494 nmol min-’ mg-’ for fernlic acid. biotransformation

optimal conditions obtained for the decarboxylase purified from B. pumilus. ” The specificity of the enzyme from B. anomalus was found to be limited to p-coumaric acid, caffeic acid, and ferulic acid. The decarboxylase from B. anomalus was found to be most active toward caffeic acid with only a third of this rate shown toward p-coumaric acid and ferulic acid. In contrast, the two decarboxylases purified from B. pumilus were found to be active toward only p-coumaric acid and ferulic acid and showed no activity toward caffeic acid. L. plan&rum is thought to possess two hydroxycinnamate decarboxylases. One is active toward p-coumaric acid and caffeic acid, and a second specific to ferulic acid. It appears that the paru-hydroxy group is essential for the activity of the hydroxycinnamate decarboxylase from B. anomalus, however, the transformation of ortho and metu substituted hydroxycinnamates is known in other microorganisms.” Similar to the enzymes purified from B. pumilus, our results indicate that the hydroxycinnamic acid decarboxylase of B. anomalus may also be a homodimer. The molecular mass of 39.8 kDa makes the yeast enzyme somewhat smaller than those from bacteria (93kDa in L. plunturum. 45kDa and 40.4 kDa in B. pumilus). The decarboxylative activity of yeasts toward hydroxycinnamic acids is well known.‘,3,‘3.‘4,22 Nonbrewing strains of S. cerevisiae which produce this decarboxylase are termed Pof+ for ‘Phenolic off flavor’ since the products from this reaction possess a strong smoky or clovelike phenolic flavor which is generally regarded as undesirable in beer and wine. Analysis of the genetics and biochemistry of this process has been studied,24 the gene from S. cerevisiae has been cloned, and the restriction map obtained.25-27 The ferulate decarboxylase gene from B. pumilus has been cloned, sequenced, and expressed in E. coli.‘* We are currently in the process of investigating the hydroxycinnamate decarboxylase gene in B. anomalus.

Acknowledgments The author thanks the B.B.S.R.C. ate research studentship.

for funding

a postgradu-

Discussion In this report, we have shown the partial purification and characterization of a hydroxycinnamate decarboxylase from B. anomalus. We found that this protein is, at low levels of activity, constitutive in this yeast since it is produced in growth medium not supplemented with phenolic substrates. The enzyme is, however, substrate inducible. The hydroxycinnamic acids are known to possess an antimicrobial activity and inhibit the growth decarof a number of organisms. “J’ Hydroxycinnamate boxylases are produced by a number of ‘wild yeasts’, and the enzyme is thought to be beneficial to the organism, since it is involved in removing the strong antimicrobial activity of the hydroxycinnamic acids by transforming them to less toxic metabolites. The protein was purified using Mono Q, Phenyl Superose, and Superose 12 column chromatography. The activity of the enzyme was found to be optimal at pH 6.0 and 40°C in 50 mM KH,PO,/&HPO, buffer. This is similar to the 238

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