Purification and properties of an alkaline proteinase of Fusarium culmorum Anja |.. To do this, Fusarium culmorum was grown in a gluten-con- taining medium from which an alkaline serine
Trang 1Purification and properties of an alkaline proteinase
of Fusarium culmorum
Anja | Pekkarinen'?, Berne L Jones? and Marja-Leena Niku-Paavola”
' Department of Agronomy, University of Wisconsin-Madison, USA; ?VTT Biotechnology, Finland;
3USDA-ARS, Cereal Crops Research Unit, Madison, WI, USA
The disease Fusarium head blight (scab) causes severe
problems for farmers and for the industries that use cereals
It is likely that the fungi that cause scab (Fusarium spp.) use
various enzymes when they invade grains We are studying
enzymes that the fungi may use to hydrolyze grain proteins
To do this, Fusarium culmorum was grown in a gluten-con-
taining medium from which an alkaline serine proteinase
with a molecular mass of 28.7 kDa was purified by size-
exclusion and cation exchange chromatographies The
enzyme was maximally active at pH 8.3-9.6 and 50 °C, but
was unstable under these conditions It hydrolyzed the syn-
thetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide
and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu
p-nitroanilide It was inhibited by phenylmethanesulfonyl
fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins Therefore, on the basis of these characteristics, the proteinase is subtilisin-like Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis The presence of extraneous protein stabilized the activity
Keywords cereal; fungus; chymotrypsin; — subtilisin; proteinase
Fusarium head blight (FHB, scab) has for many years been a
serious problem for cereal producers and for the various
cereal industries The majority of past FHB epidemics
have been caused by the fungus Fusariuwn graminearum
(Gibberella zeae), but some infestations have also been due
to F culmorum and/or F avenaceum (G avenacea), espe-
cially in Europe [1] FHB causes severe yield losses in wheat
and barley and reduces the crop quality by destroying some
of the necessary grain components and by producing
mycotoxins Fusarium contamination of malts is also
associated with ‘gushing’ problems that have sometimes
plagued the brewing industry [2-4]
The role that fungal proteinases play in the FHB
pathogenesis is not known, but there are indications that
Correspondence to A I Pekkarinen, USDA-ARS, CCRU, 501 N
Walnut St., Madison, WI 53705-2334, USA Fax: + 1 608 264 5528,
Tel.: + 1 608 262 4478, E-mail: apekkarinen@ facstaff.wisc.edu
Abbreviations: CMC, carboxymethyl cellulose; E-64, trans-Epoxy-
succinyl-1-leucylamido-(4-guanidino)butane; PMSF, phenyl-
methanesulfonyl fluoride; pAPMSF, p-amidino
phenylmethanesulfonyl fluoride; CST, chymostatin; STI, soybean
trypsin inhibitor; BBI, Bowman—Birk inhibitor; TLCK, Ne-p-tosyl-
L-lysine chloromethyl ketone; TPCK, N-tosyl-1-phenylalanine
chloromethyl ketone; SAAPFpNA, N-succinyl-Ala-Ala-Pro-Phe
p-nitroanilide; SAAPLpNA, N-succinyl-Ala-Ala-Pro-Leu p-nitro-
anilide; BVGRpNA, N-benzoyl-Val-Gly-Arg p-nitroanilide; GPpNA,
N-glutaryl-L-Phe p-nitroanilide; BApNA, Na-benzoyl-L-Arg p-nitro-
anilide
Enzymes: chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4); subtilisin
(EC 3.4.21.62); oryzin (EC 3.4.21.63)
(Received 16 August 2001, revised 20 November 2001, accepted 23
November 2001)
these enzymes may contribute to some of the problems that are associated with diseased wheat Electron microscope examinations have indicated that the wheat endosperm protein matrix disappeared when F graminearum invaded the aleurone layer [5] or the starchy endosperm tissue [5,6]
F graminearum infections also caused a decrease in the relative proportions of extractable wheat albumins and glutenins [7] When either / graminearum or F culmorum was grown on media that contained cereal proteins, it produced proteinases that had predominantly alkaline pH optima [8] An alkaline proteinase activity that was associ- ated with the breakdown of storage proteins has also been detected in FHB-diseased wheat kernels [6]
Cereal grains contain multiple proteins that inhibit the activities of microbial proteinases [9,10] and it seems likely that they make some of these inhibitors to slow or prevent the disruption of the grain proteins during fungal attacks
We are purifying, identifying and characterizing the pro- teinases that are synthesized by Fusarium fungi when they are grown on grain protein-containing media These will then be used to probe for barley inhibitors that inactivate Fusarium proteinases, to define the interactions between these enzymes and inhibitors and to ascertain whether or not they occur within infested grains In this paper we report the purification and characterization of one of the protein- ases that is produced by F culmorum
MATERIALS AND METHODS Fusarium culture
F culmorum (strain VTT-D-80148) was grown in Arm- strong medium that was modified by replacing its inorganic nitrogen salt with gluten, so that it induced the fungus to
Trang 2© FEBS 2002
produce alkaline proteinases [8] Two litres of inoculum was
grown in Armstrong mineral medium [11] and it was used to
start a 30-L culture that was identical, but contained 1 g:-L™!
(NH¿)›SO¿ This, in turn, was used to inoculate a final
270-L_ growth medium preparation The 300-L growth
medium was as described previously [8], except that it was
prepared with tap water It contained 8 g:L™! of an impure
gluten preparation (80% protein, 7% fat, Sigma #G5004,
St Louis, MO, USA) that had been dry-heat sterilized (10 h
at 160 °C) Fermentation was in a New Brunswick Scientific
IF400 fermenter
Throughout the fermentation, the pH of the culture
medium was maintained at 4.5—5.0 by adding either NaOH
or H3PO, as needed, and the temperature was maintained at
18-21 °C The fungal growth was monitored by measuring
the glucose concentration and chymotrypsin/subtilisin pro-
teinase activities of samples that were removed from the
growth medium 0, 19, 24, 27, 31 and 42 h after the culture
was started The purity of the culture was confirmed by agar
plating and by microscopic examination When ~ 50% of
the glucose had been used up (43 h), the mycelia were
separated by centrifugation with an Alfa Laval Separator
Type BPTX 205SGD-30CDP (Sweden) This step yielded
270 L of supernatant that was concentrated to 16.6 L with
four PCI modules (PCI, Whitchurch, UK) using ES625
membranes (nominal molecular weight cut-off 10 000 Da,
2.6 m each) The concentrate was divided into appropriate
aliquots, frozen, and stored at —20 °C
Analytical methods
Nonspecific proteinase assay An azogelatin assay [12] was
used for analyzing the total nonspecific proteinase activities
Each reaction was started by adding 0.5 mL of enzyme
preparation that was diluted (with 30 mm sodium citrate,
pH 6.3) to contain 1-2 pgmL™! of protein, to 2 mL of
12.5 mgmL"! azogelatin in 100 mm buffer Unless indicat-
ed otherwise, the reactions were carried out at 40 °C in
80 mm sodium citrate, pH 6.0, buffer Samples (0.5 mL
each) were removed from each reaction at appropriate times
(normally 0, 10, 30 and 60 min), mixed with 0.75 mL of
25% trichloroacetic acid, held in an ice-water bath for
20 min, and centrifuged at 10 800 g for 10 min, at room
temperature The absorbance values of the supernatants
were measured at 440 nm The enzymatic activity, in
arbitrary units (U), was the change in absorbance units
per minute multiplied by 100 Each assay was performed in
duplicate
Specific chymotrypsin/subtilisin-like activity assay The
specific chymotrypsin/subtilisin-like activities were mea-
sured with the substrate N-succinyl-Ala-Ala-Pro-Phe
p-nitroanilide (SAAPFpNA, Sigma, 5 mm) dissolved in
175 mm Tris/HCl, pH 9.0 The substrate solution (90 WL)
was heated to 28 °C, 10 uwL of appropriately diluted enzyme
was added and the change in absorbance at 405 nm was
monitored for 3 min The activities were calculated as
described earlier and are expressed as nkatmL! of sample
[8]
Protein assay For creating the purification table, the
protein concentrations of solutions were measured with the
Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA)
An alkaline Fusarium proteinase (Eur J Biochem 269) 799
[13] Bovine serum albumin (BSA, Pierce, Rockford, IL, USA) was used to prepare a standard curve In all other cases the protein contents of the enzyme solutions were calculated by measuring their absorbance at 280 nm and assuming that | mgmL~' of protein had an absorbance
of 1.0
Purification of the enzyme All purification steps were carried out at room temperature and the chromatography fractions were always collected in glass tubes that had been silanized with trimethylchloro- silane (Sigma #T-4252) For silanization, the tubes were placed in a dessicator with 5 mL of the reagent and the dessicator was evacuated and held at room temperature for
16 h, after which the derivatized tubes were rinsed thor- oughly with MilliQ-water
The concentrated culture medium was centrifuged at
1700 g for 5 min and 26.5 mL of the supernatant was applied to a 2.5 x 70cm Bio-Gel P30 (Bio-Rad) size exclusion column that was equilibrated with 20 mm NH4 acetate, pH 5.0 The column was eluted with the same buffer and 200-drop fractions were collected The absor- bance values of these and of the chromatography fractions
in subsequent steps were measured at 280 nm and their nonspecific proteinase and chymotrypsin/subtilisin-like activities were both analyzed at pH 9.0
The fractions that voided the P-30 column were com- bined and subjected to carboxymethyl cellulose (CM52, Whatman) cation exchange chromatography at pH 5.0 ona
1 x 6cm column Elution was with a 20-mm to 300 mm
NH, acetate, pH 5.0, linear gradient (45 mL of each concentration) and 2.8-mL fractions were collected The fractions having the highest proteolytic activities (19-23) were pooled and the pool was divided into 2-mL aliquots that were stored at —20 °C The enzyme was stored in this partially purified state because it was not stable when completely purified Immediately prior to using the enzyme for studies, it was subjected to a final HPLC-cation exchange purification step with a Shodex IEC CM-825,
8 x 75 mm column (Phenomenex, Torrance, CA, USA) Each thawed aliquot was filtered through an Acrodisc® 4CR PTFE 0.45 um filter (Gelman Sciences, Ann Arbor,
MI, USA), diluted fivefold with MilliQ-water, and applied
to the column, which had been equilibrated with 50 mm NH,HCO;, pH 8, buffer The loaded column was washed
at ImLmin ” with 6 mL of the pH 8 buffer and the proteins were separated with a 12.5-mL linear gradient
280 nm-absorbing fractions were collected and immediately adjusted to pH 4-5 with approximately 40 pL of 20% acetic acid The activities of the fractions were measured at pH 9.0 and the material that showed chymotrypsin/subtilisin spe- cific activity was called the ‘purified’ enzyme or ‘CM-HPLC’ fraction
To ascertain its purity, an aliquot of the CM-HPLC preparation was boiled for 1.5 min with SDS sample buffer and separated on a 12% SDS/PAGE gel [14] The gel was incubated for 45 min, with shaking, in 50% methanol/12% acetic acid It was stained in 0.1% Coomassie Brilliant Blue R-250 dissolved in 40% methanol/1% acetic acid and destained with the same solvent A Precision Protein Standard (Bio-Rad) sample was used to calibrate the gel
Trang 3Characterization of the proteinase
Effects of pH and temperature on activity The activities
of the partially purified enzyme, obtained by CMC
separation at pH 5.0, were measured by using the azogelatin
assay at 40 °C and pH 3.6, 4.1, 4.5, 5.0 and 5.5 (Na acetate),
pH 6.0, 6.5 and 6.9 (Na citrate), pH 6.9, 7.5, 7.9, 8.3, 8.7
and 9.1 (Tris/HCl) and pH 9.2, 9.6, 10.0 and 10.4 (Caps)
All buffers were 80 mm The activities of the CM-HPLC
purified proteinase were analyzed at pH 4.6, 6.0, 8.7 and 9.4
in the same buffers
The effect of temperature on the proteinase activity was
studied at 45, 50 and 56 °C at pH 6.0 (80 mm Na citrate)
and at 40, 45 and 50 °C at pH 8.7 (80 mm Tris/HCl)
Effects of pH and temperature on the enzyme stability
For measuring the stability of the proteinase at different pH
values, the purified enzyme was incubated for 90 min at
40 °C in 30 mm buffers: Na acetate, pH 4.1 and 4.9; Na
citrate, pH 5.9 and 6.4; or Tris/HCl, pH 7.7 and 8.5 The
activity of each sample was measured after 0 and 90 min of
incubation For measuring its thermal stability, the purified
proteinase was incubated in 30 mm Na citrate, pH 6.3, for
50 min, at 24, 40, 50 or 60 °C Its activity was measured
after 0 and 50 min of incubation The activity retention at
each pH or temperature was expressed as the proportion of
the initial activity that remained after the incubation
Effects of class specific inhibitors on the enzyme The
mechanistic class of the proteinase was determined by
measuring its activity at pH 6.0 in the presence of nine class-
specific protease inhibitors Samples of the semipurified
(subjected to CMC open-column chromatography) prepa-
ration were incubated on ice for 30 min with 50 HM trans-
epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64),
25 mm EDTA, 5 mm 1,10-phenanthroline in 20% dimethyl-
sulfoxide, 50 um pepstatin A in 20% methanol, 0.5 or
5.0 mm phenylmethanesulfonyl fluoride (PMSF) in 20%
isopropanol, 8.2 or 82 um chymostatin (CST) in 20%
dimethylsulfoxide, or 5.0 ttm soybean trypsin inhibitor (STI,
Type II-S, Sigma #T-9128) Samples of the purified enzyme
(about 2 ugmL~') were incubated as above in the presence
of 165 um CST, 5.0mm PMSF, or 5.0mm 4-amid-
inophenylmethanesulfonyl fluoride (pAPMSF), each of
which was dissolved in 20% dimethylsulfoxide, or in a
12.5-um solution of soybean Bowman-—Birk inhibitor (BBD),
or 5.0 um STI Control reactions were carried out with
enzyme that was preincubated in water or in 20%
dimethylsulfoxide, methanol or isopropanol, as appropri-
ate In the final reaction mixtures these enzyme-inhibitor
mixtures were all diluted fivefold with substrate solution
The effects of selected serine class proteinase inhibitors on
the hydrolysis of azogelatin at pH 6.0 by several commer-
cially available serine proteinases were examined One set of
assays was carried out with 48 pgmL (2.0 1m) bovine
a-chymotrypsin (TLCK treated, Type VII, #C-3142, EC
3.4.21.1) or 1.0 ug-mL (0.04 pm) bovine trypsin (TPCK
treated, Type XIII, #T-8642, EC 3.4.21.4) in the presence of
1.0 mm PMSF, 1.0 mm pAPMSF, 33 um CST, 1.0 um STI
or 2.5 um BBI In the other set, the effects of 1.7 um CST or
2.5 um BBI on 0.8 HgmL~” subtilisin Carlsberg (Type VIII
bacterial, Bacillus licheniformis, #P-5380, EC 3.4.21.62)
or 1.4 ngmL7! oryzin (Aspergillus oryzae protease, Type
XXIII, #P-4032, EC 3.4.21.63) were studied The PMSF, pAPMSF and CST were dissolved in 4% dimethylsulfoxide Appropriate controls were conducted All of the inhibitors and commercial enzymes were purchased from Sigma
To ascertain the effects of CST and STI on the SAAPFpNA hydrolysis activity, samples of the purified Fusarium proteinase were incubated in polypropylene tubes with 16.5 um CST in 20% dimethylsulfoxide or 10 um STI,
on ice, for 50 min and their activities were measured as described above under ‘specific chymotrypsin/subtilisin-like activity assay’ except that the substrate buffer contained 4% dimethylsulfoxide For control reactions, the enzyme was preincubated with water or 20% dimethylsulfoxide The final concentrations of STI or CST in the reaction mixtures were 1.0 or 1.7 uM, respectively
Effects of calcium on the activity and stability of the proteinase The effect of Ca acetate on the proteolytic activity was analyzed in both 80 mm Na citrate, pH 6.0 (0, 5 or 20 mm Ca**) and 80 mm NHy acetate, pH 5.4 (0, 1, 5 or 20 mm Ca*") buffers To determine the effect
of calcium on the enzyme stability, the purified enzyme was incubated for 95 min at 40 °C in 30 mm Na acetate,
pH 4.9, that contained 0 or 100 mm Ca acetate The activities of the samples were measured after 0 and 95 min
of incubation To ensure that the differing calcium levels did not affect the activity measurements, the Ca acetate concentrations of all of the reaction mixtures were adjusted to 20 mo
Effect of added protein on the proteinase stability
A solution of purified enzyme was incubated at 40 °C for 90 min with 0, 1.0, 2.5, 5.0 or 10 ugmL™ of BSA in
30 mm Na citrate, pH 6.3 The activity of the sample without BSA was measured as a control and the activities
of all of the samples were analyzed after 90 min of incubation The concentration of BSA in each of the activity analysis reaction mixtures was adjusted to
4 ugmL™ to ensure that the varying BSA levels did not affect the results
Substrate specificity and kinetic constants for SAAPFpNA
To define the substrate specificity of the enzyme, its activity was measured in duplicate with the substrates SAAPFpNA, N-succinyl-Ala-Ala-Pro-Leu pNA (SAAPLpNA), A-gluta- ryl-L-Phe pNA (GPpNA), N-benzoyl-Val-Gly-Arg pNA (BVGRpNA), or Na-benzoyl-L-Arg pNA (BApNA) The concentrations of all substrates were 5 mm The method was
as described above under ‘specific chymotrypsin/subtilisin- like activity assay’, except that all of the reactions contained 4% dimethylsulfoxide, in which the substrates were dis- solved The reactions contained 0.06 (SAAPFpNA), 0.22 (SAAPLpNA and BVGRpNA) or 1.1 (BApNA and GPpNA) ig of protein
The K,, value of the proteinase for SAAPFpNA was determined by measuring the activity of the enzyme (0.03 ug protein per reaction) with 0.13-8.0 mm concen- trations of the substrate in pH 9.0, 175 mm Tris/HCl solutions in the presence or absence of 4% dimethylsulf- oxide and at pH 6.0 in 4% dimethylsulfoxide, 175 mm Na citrate The analyses were carried out in duplicate and the kinetic constants were calculated from Lineweaver—Burk plots
Trang 4© FEBS 2002
Table 1 Purification of the F culmorum proteinase
An alkaline Fusarium proteinase (Eur J Biochem 269) 801
Activity Specific activity Purification Protein®
step (mg) (U°) (ukat”) (U-mg™') (ukatmg_ }) Yield (%) Purification (fold)
® Based on 26.5 mL of growth medium concentrate PActivities were measured with the azogelatin assay: U = A44azox 100 min Ì
© Activities were measured with N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide
Effect of azogelatin concentration on the proteolytic
activity The effect of azogelatin on the proteolytic activities
was measured with substrate concentrations between 0.5
and 10 mgmL” in solutions containing either 80 mm Na
citrate, pH 6.0, or 80 mm Tris/HCl, pH 8.7 The enzyme
concentrations of the pH 6.0 and 8.7 reaction mixtures were
0.44 and 0.22 tugmL”, respectively
Molecular mass analysis The molecular mass of the
proteinase was determined by MALDI-TOF analysis, using
a Bruker Biflex II] mass spectrometer, at the University of
Wisconsin Biotechnology Center, WI, USA
Determination of portions of the amino-acid sequence
CM-HPLC purified enzyme was freeze-dried and submitted
to the Protein Chemistry Laboratory of the University of
Texas Medical Branch Cancer Center, Galveston, USA, for
amino-acid sequence analysis The enzyme was digested
with trypsin, the resulting peptides were separated by reverse
phase-HPLC and selected peptides were subjected to amino-
acid sequence analysis using the Edman degradation
method
RESULTS AND DISCUSSION
Purification
Depending on which analysis method was used for
measuring the activities, the final yield of the proteinase
was 5.5 or 11% (Table 1) The specific activities increased
about fourfold (SAAPFpNA) or eightfold (azogelatin) as
the purification process progressed from culture medium
concentrate to CM-HPLC preparation The open column
CMC separation gave the largest single purification, but
after this step two distinct proteinases were still present To
separate these two enzymes, 1t was necessary to carry out a
final CM-HPLC separation at pH 8 (Fig 1) No separation
was obtained when the HPLC separation was carried out at
pH values lower than 8 A typical separation is shown in
Fig 1, where the enzyme of interest is indicated with an
arrow Preliminary studies established that the other major
eluting peak contained a trypsin-like proteinase This
trypsin-like enzyme 1s being studied and will be reported
elsewhere
SDS/PAGE analysis of the purified enzyme showed that
the predominant protein had a molecular mass of
~ 26.8 kDa and that a small amount of slightly faster
migrating proteins were present (Fig 2) Mass spectrometry
indicated that fresh enzyme preparations contained a
E
c
2
<
Time (min)
Fig 1 A typical cation exchange HPLC chromatogram of a F cul- morum proteinase: absorbance at 280 nm (—) The gradient (- -) was run from 50 mm to 175 mm NH,HCOs, pH 8 The proteinase that 1s described in this report is indicated with an arrow
as- 150 kDa m- 75
ww 50 we- 37
- 25
-15
—
Fig 2 SDS/PAGE pattern of the purified proteinase Lanes | and 2: 0.5 and 1.5 ug of CM-HPLC purified enzyme; lane 3: molecular mass standards
protein of mass 28 663 + 50 Da Some purified samples also included small amounts of proteins of ~ 4600, 11 180,
17 070 and 17 860 Da Apparently, contaminants were sometimes present and/or the enzyme underwent partial autolysis When the purified enzyme was subjected to a
Trang 5
100
O) © |
+> © -4 -—+ - Su - -l- - - - 4+ - —- - —- —- -l- ee eK ee He eK KKK
NO © | SO gee pT
10 11
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DH
Fig 3 The effect of pH on the proteinase activity, measured with the
azogelatin method The enzyme preparations analyzed were: open
symbols, a CMC pool or; closed symbols, enzyme purified by
CM-HPLC All buffers were 80 mm and contained: Na acetate (O,@),
Na citrate (1,9), Tris/HCl (A,A) or Caps (C,®)
second pH 8 HPLC separation, almost all of the proteinase
was lost and what remained eluted as a broad peak This
repeated HPLC separation did remove some 17.9 kDa
protein whose N-terminal amino-acid sequence was highly
homologous with that of a portion of a trypsin-like
proteinase from Fusarium oxysporum (results not shown)
Properties of the proteinase; pH optimum and effect
of temperature on activity
The proteinase hydrolyzed azogelatin between pH 4.0 and
10.5 and was optimally active at pH 8.39.6 (Fig 3) This
result was based on data obtained by analyzing the activities
of a mixture of the two Fusarium proteinases However,
when the activities of the purified enzyme were measured at
pH 4.6, 6.0, 8.7 and 9.4, essentially identical results were
obtained (Fig 3) The enzyme thus functions at pH 6, the
physiological pH of grain, but not at its maximal rate The
broad pH optimum of this proteinase resembles those of
the alkaline proteinases from some Aspergillus species
[15-18], but those of Fusarium sp *S-19-S’ [19] and
F graminearum [20] have somewhat sharper pH optima,
at approximately pH 10 A trypsin-like proteinase from
F oxysporum had a pH optimum of 8—11 [21]
At pH 8.7 (Fig 4) the initial activity of the enzyme
increased with temperature, up to 50 °C However, the
enzyme was unstable above 40 °C, so the reaction rate at
50 °C decreased with time Thus, for the purposes of this
assay method, 40 °C was the most appropriate temperature
for carrying out the analyses The proteinase was more
temperature stable when assayed at pH 6.0, but even at this
PH the activity was slightly unstable at 45 °C and it dropped
off quickly at 56 °C Several alkaline proteinases from
Aspergillus and Fusarium species have temperature optima
of +40 °C [15,16,18,20,22] The alkaline proteinase of
Fusarium sp ‘S-19-5’ was maximally active at 50 °C and
0 15 30 45 60
Reaction time (min)
Fig 4 The effects of temperature on the proteinase activities The analysis temperatures were: 40 °C (©), 45 °C (L1,M), 50 °C (A,A), or
56 °C (@) The assays were run at pH 8.7 (open symbols) or pH 6.0 (closed symbols)
pH 10.5 when its activities were determined with a 20-min assay [19]
Calcium had a negligible effect on the activity of the F culmorum proteinase in the presence of either Na citrate
or NH, acetate buffer The NHy, acetate analyses were carried out to ensure that the calcium concentration of the reaction was not affected by the presence of citrate 1on, which is a chelating agent The enzyme therefore behaved like the alkaline proteinase of Fusarium sp ‘S-19-5’, which also was not affected by calcium [19]
Factors affecting the stability of the proteinase The proteinase was heat labile and subject to inactivation
at alkaline pH When the purified proteinase was incu- bated at various temperatures at pH 6.0, the remaining activities were 88, 55, 29 and 0% after 50 min at 24, 40,
50 and 60 °C, respectively About a third of the enzyme remained active for 90 min at various pH values from 4.1
to 7.7 at 40 °C, but all of the activity was lost at pH 8.5 Calctum did not stabilize the enzyme at pH 4.9 After
95 min of incubation in the presence and absence of Ca“”, the remaining activities were 37 and 32%, respec- tively Tomoda et al [19] showed previously that calcium stabilized the alkaline proteinase of Fusarium sp ‘S-19-3’
at pH 8-9, but not at pH 5 and 40 °C However, the
‘S-19-S’ enzyme was not as sensitive to inactivation at pH
9 or at elevated temperatures as this F culmorum proteinase Bovine chymotrypsin and trypsin are stabilized
by calcitum [23,24]
The addition of 2.5 u.g-mL~' of BSA per = 1 ugmL” of proteinase was sufficient to completely maintain the proteinase activity for at least 90 min at pH 6.0 and 40 °C (Table 2), conditions under which the unprotected enzyme was almost completely inactivated BSA, and presumably other proteins, apparently protects the proteinase from autolysis, inhibits conformational changes or prevents it from binding to its containers Hence, some of the stability features of the enzyme may be affected by small amounts of contaminating proteins
Trang 6© FEBS 2002
Table 2 Stabilization of the Fusarium proteinase by BSA The samples
were incubated at 40 °C for 90 min at pH 6.0
BSA (ug-mL') Remaining activity (%)
The enzyme is a serine class proteinase
Class specific proteinase inhibitors other than those that
inactivate serine proteinases had no significant effect on the
activities of the CMC preparation (Table 3) The small
effect of soybean trypsin inhibitor (STI) was probably due
to some contaminating proteinase that was in the CMC
preparation, because the purified proteinase was not
inhibited either by it or by a Bowman-—Birk type trypsin-
chymotrypsin inhibitor (BBI) The very strong inhibitions
caused by PMSF and chymostatin (CST) indicated that the
enzyme was a serine proteinase and belonged to either the
chymotrypsin or subtilisin family Under the analysis
conditions that were used in this experiment, a TLCK-
treated bovine o-chymotrypsin was inhibited 64% by BBI
and 98% by CST Neither oryzin nor subtilisin were
affected by BBI, but they were almost totally inhibited by
CST In this aspect, the F cudmorum proteinase resembles
subtilisins However, the a-chymotrypsin was unexpectedly
inhibited by 40% in the presence of STI, showing that
Table 3 Inhibition of the Fusarium proteinase activity by various class
specific inhibitors
Concentration Inhibition Inhibition
PMSF
Chymostatin
Soybean
trypsin inhibitor
inhibitor
@ Measured with a mixture of two proteinases ° Measured with the
purified proteinase “ Measured with the substrate SAAPFpNA at
pH 9.0 All of the other measurements were made with azogelatin
at pH 6.0
An alkaline Fusarium proteinase (Eur J Biochem 269) 803
classification by ‘specific’ inhibitors is not straightforward and may depend on the analysis method
The activity of the purified F culmorum proteinase was 25% higher in the presence of STI or BBI than in the control (Table 3) This is probably not due to an activation
of the proteinase but rather to the general stabilization of the enzyme by proteins, which was mentioned earlier Both STI and BBI are proteins It is also possible that the small amount of contaminating trypsin-like proteinase caused an inactivation of the ‘subtilisin’ proteinase and, when that contaminant was inhibited by STI or BBI, the proteinase remained active STI and CST both caused similar effects when the enzymatic activities were measured with the synthetic peptide substrate (Table 3)
Amino-acid sequence studies Several attempts were made to sequence the N-terminal amino acids of the enzyme, but no data were obtained, indicating that the N-terminus was probably blocked The enzyme was therefore digested with trypsin, and the resulting peptides were separated by HPLC and some were sequenced Four of the peptides obtained had sequences of: (1) GSTSYTYDTSAGSGTYAYIVDTGIITSHN; (2) GFNWAANDIISK; (3) SYSNYGTVL and; (4) DIFAPG TSVLSS These peptides were homologous with sections
of other fungal proteinases (Table 4) Peptides 3 and 4 occupied adjacent areas of the sequences of several of these proteinases However, the peptide bond that was cleaved to separate these two peptides connected the amino acids leucine and aspartic acid Such bonds are not normally cleaved by trypsin, but are by subtilisin This indicates that these peptides were probably separated by an autolytic event rather than by trypsin hydrolysis In the subsequent discussion the peptides 3 and 4 are considered as a single peptide
Table 4 lists the corresponding amino-acid sequences of several homologous fungal proteinases The protein that showed the highest homology with all three of the F cul- morum peptides was the subtilisin-like proteinase from Cephalosporium acremonium, whose corresponding sequen- ces were 82% identical to those of F culmorum The proteinases from several Aspergillus species, from Tricho- derma harzianum, Metarhizium anisopliae, Magnaporthe poae, Tritirachium album, Yarrowia lipolytica and the Fusarium sp ‘S-19-5° and oxysporum contained sequences that were 44-76% identical Subtilisin-like proteinases from
M poae [25] and F oxysporum [26] have been detected in infected host plants, although, their roles have not been established
The proteinases that have been cloned from Fusarium sp
‘S-19-5’ and F oxysporum showed less homology to the peptides from the F culmorum proteinase than those of several other fungal species (Table 4) This was somewhat surprising, considering that most of the peptide sequences from the Aspergillus species were highly conserved Also, the proteinases K and R from T album were 85% identical to each other, showing that similar enzymes from a single species are often highly homologous However, such sequences may vary remarkably, as shown by the peptides from A niger and M anisopliae
The amino acids that comprise the catalytic triad of the serine proteinases (His, Asp and Ser) occur in different
Trang 8© FEBS 2002
orders in the primary structures of the subtilisin (Asp-His-
Ser) and chymotrypsin (His-Asp-Ser) families [27] Peptide 1
from the F culmorum proteinase contained the catalytic
Asp residue in a position that corresponded to that of the
other subtilisin-like enzymes (Table 4) This is another
strong indication that the enzyme under study 1s a subtilisin-
like, not a chymotrypsin-like, proteinase These results
therefore support the observations made with the class
specific inhibitors However, the classification of this
enzyme still needs to be confirmed by cloning its gene and
determining its entire amino-acid sequence
The hydrolytic specificity of the enzyme
When the hydrolytic activities of the proteinase were
measured at pH 9.0 with various synthetic substrates, the
results listed in Table 5 were obtained The enzyme
hydrolyzed SAAPFpNA faster than the other two chy-
motrypsin substrates, SAAPLpNA and GPpNA, indicating
that it has a preference for phenylalanine over leucine and
hydrolyzes small substrates poorly The ,, values for
SAAPFpNA were 1.1—3.1 mm, depending on the compo-
sition of the substrate buffer (Table 6) The K,, values
were scarcely affected by pH or the presence of dimethyl-
sulfoxide, but the maximal velocity (Vinax) was twice as
great at pH 9 as at 6 The alkaline proteinase from
Aspergillus fumigatus also showed this preference for the
phenylalanine substrate over the leucine one, did not
hydrolyze short substrates (succinyl-L-Phe pNA or acetyl-
DL-Phe pNA) and, in addition, had a similar K,, value
(0.62 mm) for SAAPFpNA [18] The hydrolysis of
N-benzoyl-Val-Gly-Arg pNA (BVGRpNA), a putative
trypsin substrate, may have been caused by a 17.9-kDa
trypsin-like proteinase contaminant When the activity of
a subtilisin preparation was measured using this substrate
in the presence of either STI or CST, only the STI
inhibited
Table 5 Hydrolytic activities of the Fusarium proteinase measured at
pH 9.0 with 5 mm synthetic substrates Values are shown as mean +
SD
Activity
N-Succinyl-Ala-Ala-Pro-Phe pNA 1360 + 40
N-Succinyl-Ala-Ala-Pro-Leu pNA 345 + 32
N-Glutaryl-L-Phe pNA 0.2 + 0.0
N-Benzoyl-Val-Gly-Arg pNA 145 + 5
Na-Benzoyl-L-Arg pNA 2.1 + 1.2
Table 6 A,, values and maximal velocities (V,,.,) of the Fusarium
proteinase for N-succinyl-Ala-Ala-Pro-Phe pNA at pH 6.0 and 9.0, with
or without dimethylsulfoxide
pH; Vmax
dimethylsulfoxide (%) Km (mM) (nkat-mg protein’)
An alkaline Fusarium proteinase (Eur J Biochem 269) 805
1.0 0.8 3 0.6 3 0.4 1:
0.2 1
0.0 : : : :
Azogelatin (mg/mL)
Fig 5 The effects of azogelatin concentration on the proteinase activi- ties at pH 6.0 (©) and 8.7 (@) The dashed line shows the nonlinear regression analysis curve for the pH 6.0 data
Effect of the azogelatin concentration
on the enzyme activity
As azogelatin is not a homogenous preparation containing only one protein form, but rather a mixture of proteins of varying sizes, a true K,, value cannot be calculated However, a ‘pseudo K,,’, was computed for this substrate
at pH 6.0 by nonlinear regression analysis using the Michaelis-Menten equation (Fig 5) The ‘K,, was 1.6 + 0.3 mgmL™ and the maximal activity (Vinax) Was
0.93 + 0.05 U (AAgay x 100 min) per 0.22 pg protein at
pH 6.0 The observed maximal activity was ~ 20% lower
than the calculated V4 The activities measured at pH 8.7
could not be analyzed using Michaelis-Menten kinetics, because substrate inhibition occurred at concentrations
> 3 mgmL’ (Fig 5)
General remarks
The stability of the proteinase depended on several condi- tions Either it adhered to the surfaces of containers or was inactivated by structural changes [28], because its activity was recovered much better from silanized glass tubes than from nonsilanized glass or plastic ones The purified proteinase could no longer be detected after it had been frozen at —20 °C or freeze-dried In contrast, up to 80% of the SAAPFpNA hydrolyzing activity was recovered when the purified enzyme preparation was stored on ice for
2 weeks However, dilute (less than 10 gmL~') enzyme preparations were unstable even when stored at 0 °C
CONCLUSIONS
A proteinase, whose production by F culmorum was induced/enhanced in the presence of grain protein, has been purified from growth medium and characterized The properties and the amino-acid sequence of the enzyme indicated that 1t was related to several fungal subtilisins The role this enzyme plays in FHB pathogenesis remains to be determined
Trang 9ACKNOWLEDGEMENTS
We thank the Kemira Foundation for funding the proteinase
production and Michael Bailey at VITT Biotechnology for culturing
the F culmorum The financial support of the Tor-Magnus Enari Fund,
the Raisio Group Research Foundation, the American Malting Barley
Association and the Finnish Concordia Association are also greatly
appreciated
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