3.1 Introduction
14
A s s ays fo r t h e arg i n i n e d e i m i n ase (A D I ) pathway e nzymes, a rg i ni n e deiminase (AD I ) , ornithine transcarbamylase (OTC) and carbamate kinase (CK), are available for some microorganis ms. These assays may n ot be appropriate, however, for the three enzymes from wine lactic acid bacteria (LAB). This is because these enzyme assays were developed under specific conditions using one particular species of microorganism. lt is incorrect to ass u m e t h at s u c h a n assay can be u se d , p e r s e, to analyse fo r that
e nzymatic activity i n a differe nt m icroorg anism, si nce e nzyme p roperties such as pH optima and kinetic parameters vary appreciably from species to species (Dobrogosz, 1 981 ).
For the purpose of this project, it was necessary to establish enzyme assay conditions appropriate for the win e LAB being i nvestigated. Therefore, the effect of facto rs such as substrate and e nzym e concentration and pH on e nzyme activity was investigated in extracts of the wine LAB strains used in the present study. lt is essential that the concentrations of substrates used are saturating, that the enzyme activity is li nearly dependent on the amount of e nzyme added and that the e nzyme assays are conducted at their pH o pti m a . The g o a l h e re was to e st ab l i s h sta n d ard assay co n d iti o ns appropriate for wine LAB, so that these assays could be used routin ely for subsequent experiments.
3.2 M aterials and methods
3.2.1 Lactic acid bacterial strains
Strains of wine LAB used in this investigation included strains MCW, OENO and Er1 a of L euconostoc oenos, Lactobacillus brevis 250 and Lactobacillus buchn eri CUC-3. All strai ns are h eteroferme ntative and were ammonia
positive when tested using Nessler's reage nt (see Section 8.2). The original sources of these strains are listed in Table 4. 1 , Chapter 4.
15
3.2.2 Med i u m p reparation, culturing and h arvesting of w i ne LAB
The basal medium (pH 5.5) for culturing win e LAB consisted of the following com ponents in deionised ( D I ) water per litre: tryptone, 5 g ; peptone, 5 g; yeast extract, 5 g ; Tween 80 (5% aqueous solution), 1 mL; MgS04.7H20, 0 . 2 g ; M n C I2 . 4 H20, 0 . 0 5 g and clarified vegetable juice, 200 ml. T h e c l arified j u i ce was p re p a red fro m C a m p b e l l's V B V e g etable Juice TM (Campbell's Soups, Australia) with no added sugar. The juice was filtered through Whatman No. 1 filter paper and diatomaceous earth filter aid (Witco Corp . , Ke n ite D i at o m ite 3000). This m ed i u m co ntai n ed t h re e p o ss i b l e sou rces o f L-arg inine (yeast extract, tryptone and peptone) at a basal level of approxi mately 0.5 g/L and was further supplemented with L-arginine and 0-glucose to a fin al concentration of about 5 g/L each. Solutions of arginine and glucose (pH 5.5, 1 0 g/L each) were first aseptically membrane-filtered (0.45 11m po re-size), then added to equal volumes of the autoclaved basal medium (2-fold conce ntrated). The supplemented medium was referred to as To mato Juice B roth -Arg i n i n e-G lucose (T J BA G ) . Before i nocu lati ng T J BAG, wine LAB were precultured in vegetable juice or apple juice M RS medium (VJ MRS or AJMRS, pH 5.5) usi ng Difco Lactobacilli M RS B roth or Oxoid M. R.S. Broth supplemented with clarified vegetable juice (prepared as above) o r app l e ju i ce ( 2 0 0 m LIL) . A p p l e j u ice was p re pared fro m a concentrate (Fresh-up ™, the New Ze aland A pple and P ear Marketing Board) by diluting to normal strength with Dl water.
Actively growing wine LAB in VJMRS or AJMRS were used to inoculate ( 1 %, v/v) TJ B A G m e d i a , w h i c h were t h e n i n cu bated at 3oec. C e l l s w e re harvested betwee n the late exponential phase and early stationary phase, as i ndicated by o bserving the initial settli ng of cells. Cell h arvesti ng was achieved by centrifugation at 7000 X g for 1 0 min at sec in a Sorvall RC-58 refrigerated ce ntrifuge. The harvested cells (fro m 300 mL cu ltu re ) were divided into three portions, each containing approximately 300 mg wet cells. The wet cells were washed by resuspending in 0.01 M potassium phosphate buffer (pH 7.0) and ce ntrifuging at 7000 x g for 1 0 m in at sec. This was repeated two more times, then the washed cell pellets were stored frozen (-1 3eC) u ntil used for assay. The phosphate buffer (0.01 M) was prepared by dissolving 0.871 g K2Po4 in 500 mL D l water, then adjusting to p H 7 with 30% H3P04.
16
3.2.3 Preparation of cel l-free extracts
F r o z e n ce l l p e l l e t s ( ab o u t 3 0 0 m g w e t w e i g h t ) w e re t h aw e d a n d resuspended i n 5 m l of 0.01 M potassium phosphate buffer (pH 7.0). Cell disintegratio n was achieved by two passages through a Fre nch pressure cell (Ami nco, Maryland, U SA) at a p ressure of 7 , 6 0 0 PS I . Cell d e b ri s was re m oved by centrifugat i o n at 1 4 , 5 0 0 x g fo r 1 0 m i n at 5°C a n d t h e supernatant extract was used to assay for t h e three enzymes o f the arginine deiminase pathway (AO I, OTC and CK). Enzyme assays were carried out on the day of extract preparation.
3.2.4 Analytical methods
This section describes common analytical methods employed in these and subsequent experiments. Other specific methods are given in Chapters 4, 6, 7, 8 and 9.
3.2.4.1 Determinatio n of protein concentratio n
Protein co nce ntratio n was dete rmi ned usi ng the dye bi ndi ng m ethod of Sedmak and Grossberg (1 977). This method is sensitive, fast, and at least as accu rate as the Lowry method (Sco pes, 1 9 87) . lt i s based o n t h e conversion o f Coomassie brilliant blue G250 in <;lilute acid from a brownish o range co l o u r to an intense blue co lour u po n the binding of the dye to proteins.
The Coomassie blue G250 dye (BOH) was prepared as a 0.06% solution in 3% (w/v) perchloric acid and filtered through Whatman No. 1 filter paper. The assay involved mixing 1 .0 ml sample, 1 .5 ml deio nized water
(01
water) and 2.5 ml dye solution and measuring the absorbance at 595 nm at room temperature, using a Bausch and Lamb Spectronic-20 Spectrophotometer. A mixture of 2.5 ml each of dye solution and01
water was used as a blank to zero the instrument. A standard solution (20 �g/ml) of bovine serum albumin (Fraction V, Sig ma) was used to prepare the standard curve. This was made with 0.5-2.5 ml of protein standard solution and01
water to make up to 2.5ml and dye solution (2.5 ml).
3.2.4.2 Determinatio n o f arginine concentratio n
Argi nine was determined colorimetrically based o n the Sakaguchi reaction using the method of Gilboe and Wi lliams (1 956).
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1 ) U rea (40%): 200 g urea dissolved in a small amount of
01
water first and made up to 500 ml with01
water.2) 8-Hydroxylquinolin e (0.02%) : 0.2 g 8-Hydroxylquinoline dissolved i n 1 00 m l ethanol (95%) first and made up to 1 000 ml with
01
water.3) Sodium hydroxide ( 1 0%) : 50 g N aOH in 500 ml
0 1
water. 4) Sodium hydroxide (5%): 25 g N aOH in 500 ml0 1
water.5) Sodium hypobromite ( 1 %) : 0.68 ml liquid bromine in 200 ml 5% NaOH. 6) A rg i n i n e standard stock solutio n (500 J.Lg/m l) : 60.50 mg argin i ne . HCI (Sigma) i n 1 00 ml
01
water.7) Arginine standard working solution (1 0 J.Lg/ml) : 50-fold dilution of argi nine standard stock solution in
01
water.Procedure
To a 5 ml sample, 1 m l 0. 02% 8-hydroxylquinoline and 1 ml 1 0% NaOH were added. The solution was mixed thoroughly and placed in an ice bath for 2 m i n . Th e n 0 . 2 m l 1 % sodi u m hypobro mite was added rapidly to develop colou r. After m ixing and within 1 5 seconds, 1 ml 40% urea was added to destroy excess hypobro mite and prevent colour fading. One min after addi n g hypobro m ite, 5 m l i ce-co ld water was added, the solutio n mixed and the absorbance read within 5 m i n at 500 nm using a Bausch and La m b S pectro nic-20 Spectrophoto meter. A m i xtu re of 5 ml wat e r and reagents was u sed as the b lan k to zero t h e i n stru m e nt. The a rg i n i n e standard curve was prepared with the arginine standard working solutio n ( 1 -4 ml), with
01
water to make up to 5 ml.3 .2.4.3 Determinatio n of citru l line concentration
Citrulline was determined using the method of Archibald (1 944). This method is based on the fo rmatio n of a co lou red reactio n product with d iacetyl monoxime in acid solution.
1 ) Sulphuric-phosphoric acid mixture : 1 volume of concentrated sulphuric acid plus 3 volumes of concentrated phosphoric acid (85%).
2) Oiacetyl man oxime (3%): 3 g diacetyl monoxime in 1 00 ml
01
water. 3) Citrulline stock standard ( 1 00 mg/L) : 1 0.0 mg citru lline (Sigma) in 1 00 ml18
4) Citrulline working standard (1 0 11-g/ml) : 1 ml stock standard i n 1 0 m l D l water.
Procedure
To a 1 m l sample, 3 ml D l water, 2 m l acid mixture and 0.25 ml diacetyl m o n ox i m e reag e nt w e re add e d . A b l a n k co ntai ni ng 4 ml wat e r p l u s reagents was used t o zero t h e i n stru m e nt. T h e test tubes were cap p ed, m ixed thoroug h ly and heated i n a covered boiling water bath for 3 0 m i n (Spector and Jones, 1 963). After cooling for about 1 0 m in, t h e absorbance was read at 490 n m . The standard cu rve was prepared with the citrulline working standard (0.5-4.0 ml), with Dl water to make up to 4.0 ml.
A h eating period of 30 min was employed instead of the 1 0 m in suggested by Archibald ( 1 944) . This is necessary for full colour development, since colour development is still proceedi ng at a li near rate after 1 0 min (Spector and J o n es, 1 963) . Both L- and D-cit ru l l i n e isomers react with d i acetyl monoxime while arginine does not give a colour reaction (Oginsky, 1 957). H oweve r, u re a re acts with d i acetyl m o n o x i m e to p roduce a co l o u r (Arch ibald, 1 944). The possibi lity of u re a interfe re nce i n the citru l l i n e analysis i s considered i n Section 5.4, Chapter 5.
3.2.4.4 Determination of o rn ithine concentration
The concentration of ornithine was determined using an amino acid analyzer (Pharmacia LKB alpha plus). These determinations were carried out by the D epartment of C h e mistry and Biochemistry, M assey U niversity. In some experi me nts citru lline was also determ in ed using the amino acid analyzer. Ami no acids were separated by ion-exchange and estimated by post-column derivatization with ninhydrin.
3.2.4.5 Analysis o f ammon ia, u rea, L-mal i c acid, g lucose a n d fructose
These compounds were analysed enzymatically using e nzyme kits supplied by Boehringer Mannheim, Germany.
3.2.4.6 Measu rements of cell mass
Bacterial cell mass was measured as optical density (OD) at 600 nm using a Bausch and Lo mb Spectronic-20 Spectrophotometer. When OD readings were above 0 . 6 , samples were d i luted with the same but uni nocu lated media.
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3 .2.4.7 Measu rement of pH
pH was measured using an Orion Research pH meter (Digital lonalyzer/501 ) calibrated against pH 7.0 and 4.0 buffers.
3.2.5 Assay for a rg i nine deiminase
Arginine deiminase (AD I) catalyses the hydrolysis of arginine to citrulline and a m m o n i a. Thus, the activity of AD I can be assayed by measure m e nt of either end product, citrulline or ammo nia. For this project, AD I activity was m e as u red by dete rm i n i n g t h e rate of citru l l i n e fo rm ati o n , s i n ce t h e determination of citrulline by the procedure o f Archibald ( 1 944) as m odified by Spector and Jones (1 963) is inexpensive, sensitive, and relatively simple. The measu rement of ADI activity adopted here is based on the method of Oginsky (1 955) with modifications.
1 ) 0.2 M L-arginine (pH 5.8) : 1 .742 g L-arginine (free base, Sigma) i n 50 ml Dl water, adjusted to pH 5.8 with cone. HCI.
2) 0.2 M phosphate buffer (pH 5.8) : 3.484 g K2H P04 in 1 00 ml D l water, adjusted to pH 5.8 with 30% H3P04.
3) Enzyme extract: prepared fresh ; undi luted o r di luted with 0.01 M pH 7 phosphate buffer.
Arginine and buffer solutions were stored at 4°C and used within two weeks. Procedure
T h e standard assay mixtu re (total 6 . 1 m l) consisted of 4.0 m l 0 . 2 M potassium phosphate buffer (pH 5.8), 1 .6 ml 0.2 M L-arginine (pH 5.8) and 0.5 m l u ndi luted or di luted extract. The enzymatic reaction (carried out in duplicate) was started by the addition of e nzyme extract to a preincubated (approximately 1 0 min) reaction mixture containing phosphate buffer and arginine held in a water bath at 37°C. At 1 0 m in or 1 5 min intervals, a 1 .4 ml aliquot of the reaction mixture was re moved and added to 0 . 1 ml ice-cold 7 0 % p e rc h l o ric acid and m ixed to stop t h e react i o n . O n e m l of the supe rnatant (top layer) of the mixture was then analysed for citru lline. One unit of enzymic activity was defined as that amount of enzyme catalysing the format i o n of o n e ).lm o l e of cit ru l l i n e p e r m i n . S pecific activiti e s we re expressed as units per mg extract protein (U/mg).
2 0
3.2.6 Assay f o r o rn ithine transcarbamylase
The in vivo physiological role of catabolic ornithine transcarbamylase (OTC)
is to catalyse the conve rsion of citru lline (resulting from the hyd ro lysis of a rg i n i n e by A D I ) to o r n ith i n e and carbamyl p h osphate. H oweve r, t h e equilibrium is much i n favour o f the formation o f citrulline in vitro (Stalon et al. , 1 967). Hence, the activity of OTC is most easily measured by the rate of
citru llin e synthesis from ornithi ne and carbamyl phosphate. Although two s e p a rat e OTC e n zy m e s ( o n e c at ab o l i c a n d o n e a n a b o lic) e xi st i n
Pseudomonas (Stalon et al. , 1 967), appare ntly only one OTC e nzyme with
two functions (anabolic and catabolic) is present in Lb. buchneri NCDO 1 1 0
(Manca de Nadra et al. , 1 981 ). To date" there is no evidence for the p resence
of two separate OTC enzymes in any LAB. With physiologically reve rsible e nzymes, e nzymic activities are often similar in both forward and reverse d i rections (Scopes, 1 987). Therefo re , for this project, OTC activity was measu red in the direction of citrulline synthesis from ornithine and carbamyl phosphate. The procedure described below is a modification of the m ethod of Jones (1 962).
1 ) 1 M Tris.HCI (pH 8.2) : 7.88 g Tris. HCI (Boehringer Mannheim) i n 50 ml D l water, adjusted to pH 8.2 with KOH pellets and 30% KOH.
2) 0.2 M L-ornithine. HCI (pH 8.2) : 1 .686 g L-ornithine.HCI (Sigma) in 50 ml D l water, adjusted to pH 8.2 with 30% KOH.
3) 0.2 M carbamyl phosphate : 0 . 1 22 g di-lit h i u m carbamyl p h os p h ate (Sigma) in 4 ml D l water; prepared fresh.
4) Enzyme extract : prepared fresh ; u ndi luted or appropriately diluted with 0.01 M pH 7 phosphate buffer.
Buffer and ornithine solutions were stored at 4°C and used within two weeks. Procedure
The standard assay mixture (5.0 ml) consisted of 2.3 ml 1 M Tris. HCI (pH 8 . 2 ) , 1 . 2 ml 0 . 2 M L-ornith i n e . H C I ( p H 8 . 2 ) , 1 . 0 ml 0 . 2 M c a rbamyl phosphate and 0.5 ml diluted extract. The reaction was initiated with the addition of carbamyl phosphate, followed by addi ng enzyme extract to the p re-i ncu bated (3rC) mixture of Tris . H CI and L-ornithine.HCI. At 3-min i ntervals, a 1 .2 ml aliquot of the reaction mixture was removed and added to 0. 1 ml ice cold 70% perchloric acid and mixed to stop the reaction. One ml of the supernatant (top layer) of the mixture was analysed for citrullin e . One
2 1 u nit of enzyme activity was defined as that amount of enzyme catalysing the f o r m ati o n of o n e ).lm o l e of citru l l i n e p e r m i n . S pecific activitie s w e re expressed as units per mg extract protein (U/mg).
Control for OTC
S ma l l a m o u nts of citru l l i n e can be synthesised n on-enzymatically fro m o rnithine and carbamyl phosphate at 37°C (Jones, 1 962). An OTC assay mixture without enzyme extract represents this n on-enzymatic synthesis of citrulline. lt was experi me ntally demonstrated du ring this investigation that the non-enzymatic synthesis of citrulline was directly proportional to h eating time and substrate concentrations. Citru lline synthesised non-enzymatically can result i n i nterference i n the assay of OTC using the standard assay mixt u re u nder co nditions of prolo nged heating (> 1 2 m i n ) or when using h ig h e r su bstrate co nce ntrations (60 m M o r h i g h e r fo r e ach s u b strate). Vari ation of pH in the range of 7.6-9 . 1 did not affect the non-enzymatic synth esis of citru lline (data not shown). Very little citrulline is synthesised non-enzymatically u nder the standard assay conditions described above. B l a n ks lacking enzyme extracts we re ofte n i ncluded, particu larly when e nzyme activities we re weak. The amount of citru l li n e synthesised non e nz y m atically was subtracted fro m the total a m o u nt of citru l l i n e . This correction was, however, relatively small.
3.2.7 Assay for carbamate kinase
Carbamate kinase (CK) catalyses the reve rsible conversion of carbamyl phosphate and ADP to ATP, NH3 and C02. The equilibrium favours ATP