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Indian Journal of Expcrimcntal Biology Vol. 40, Dcccmber 2002. pp . 1393- 1398
Purification and some important characters of extracellular inulinase of Alternaria alternata (Fr.) Keissler
Hossam S Hamdy
Biolog ical Scicnces & Geology Departmcnt, Facul ty of Education. A in Shams Uni vcrsity, Roxy, Heliopoli s, Ca iro, 11 757. Egypt
E-mail : hossamhamdy @yahoo.com
Neceil'ed 28 Mav 2001; rel'ised 9 Seplelllber 2002
Pro tcin prcc ipitatc of cc ll - frcc dialysatc of cx traccllular inulinase (2, I -~- fru c t an fructanohyd rolasc, EC 3.2. 1.7) of A. a/lem a/a was max imall y obta incd by mcthanol. Such protcin was fractionatcd by using 2-step column chromatography on Scphadcx G 1511 and DEAE-cc ll ulose. The part iall y puri ficd enzymc had acti vity of 8 1 x 103 Ullllg protcin , with a y icld of 69% of thc ori ginal acti vity and th c fold of purificati on was 62. Opt imum tcmpcralll re and pH for the act i vity of thc purificd cnzymc wcrc found 10 be 55°C and 4.5 , respcc ti vely . Thc cnzyme was found to bc stablc up to 55°C and in p H rangc of 4 to 5. 8 } + and Ca~+ wcrc found to sti mulatc thc cnzymc activi ty while Cu~+ , FcJ+, Hg~+ , and iodoaectatc werc rccordcd as strong inhibitors. T lI~ of thc cn zymc was esti lllatcd to bc two wecks and its apparcnt K ill was calculatcd to bc 0.066 M. Thc cnzymc rccordcd hydrolyzing acti vit y against sucrosc and raffinosc rccord ing I/S rat io of 0.50. M olecular mass of the cn
zymc preparati on was cs timatcd by gc l f i ltrat ion and found to bc 11 5 ± 5 kDa .
Mi crobi al inulinases are an important cl ass of industri al enzymes th at have gained much attention recently. Inulinase ( 2, 1- ~ - fru ctan fructanohydrol ases, EC 3.2.1.7) hydrolyze inulin to fructose in a single stepl. It finds several appli ca tions, fo r exa mple, in the production of hi gh fructose sy ru p, alcohol, ace tone and butanol2 ; as a tool in diagnosing kidney problems3
, for the sacchari fication of fructans of pl ant origin (Jer llsa l elll a rticli oke, Chi cory)" and for the preparation of fructose syru p fro m inulins.
Di fferent fungal species have been reported as extrace llular inulinase producers i. e. A sp erg iffll s .fiCIlIIIIl
1 1 f 6 7 A fi ' 8 A . 9· tt - a t lermoto erant "LlIlgus " ' . III1 /lgat lls; . IIIger ;
A. O/yzae l2 ; A. vers icolo r l3· l ~ Cladosp o rilll1l clado-. ./ 15 F' 16 j:J . '11' . SPOI"IO/G CS , - [(sa 1"//(11 I oxysp o 1"1 II 11 ; CIlICI (LIlli j a llC-1 . . 5 P ~ d P u' 1, .. 17 ze lVSt\( , . pU l jJ lIrogen[(1Il an .Ir zc IllSt\ /1 .
The present study was undertaken to puri fy and study some important charac ters of ex tracellul ar inulinase of A ltem aria a l l em ara (Fr. ) Keissler which has not been reported earli er as an inulinase producer.
Materials and Methods
O rgan islIl and lIlediulIl
A ltem aria a lt em ara (Fr. ) Keissler used in thi s work is an Egypti an soil iso late prev iously identi fied by the authority of the Co mmonwealth Mycological Institute. It was maintained on Czapek' s-agar medium and the spore suspension was prepared in 10 ml of 0.85 sa line from 7-days-old cultures. One ml contain-
ing 34 x 1 O~ spores was used to inocul ate a tri plicate set of 250-Erlenmeyer fl as ks, each containing 50 ml of the fo llowing fermentat ion medium (gil 00 ml): sucrose, 1.6; NaNOJ , 1.8%; KH 2PO~ , 0.1; KC1, 0.05 ; MgS04.7H20 , 0.05 and initi ally adj usted to pH 5.5. Submerged incubati on (1 50 rev - I) last for 108 hr at 30°C.
EnZYlll e assay Ex tracellul ar inulinase was assayed as follows IS: to
2 ml of 0.2% inulin suspension (i nulin fro m Dahlia tubers, Sigma, Germany) and 2 ml of 0.2 M acetate buffer (pH 4.6), 0.5 ml of appropri ately diluted crude enzyme preparati on (culture filtrate) was added and incubated at 50°C for 20 min . The tubes were then boiled for 10 min to in ac ti vate the enzy me, cooled, centrifuged and assayed fo r reducing sugars as fructose by the dini trosalicy lic ac id method llJ using fructose as standard . One unit of inulinase (U) was defined as the amount of enzy me that produced 1l1mol ' min·1 of fructose under the assay co nditi ons. Sucrose replaced inulin in case of assay ing in vertase act ivity. One unit of in vertase was considered as the amoun t of enzy me which produces 1 I1mol min·1 of reducing sugar as an equimolecul ar mi xture of glucose and fructose that was uti 1 ized as standard .
Protein assay
Protein content was assayed by the method of Bradford2o.
1394 INDIAN J J::XP BIOL. DECEMBER 2002
EII ZVllle pllrificaliol/ al/d cllOl"Ocleri ;:.a liol/
Aliquots of ce ll-free dial ysa te (C FD) were sepal"a tely treated with ammonium sulfate using range of saturation from 0.5 to 0.9; methanol or iso-propano l in a rati o of 1:1,2: 1,3 :1 ,4: 1 or 5: 1 (a lcohol : CFD). All samples were left overnight at 4°C and the preci pitates were collected by centri fugat ion at J 2 x 103 g for 15 min., dissolved in 10 ml acetate buffer (0.0 1 M, pH, 4.6) and dialyzed overni ght against the same bu ffer.
Protein precipitate obtained from methanol I: I, co ntaining most of the enzy me activity, was then fractionated on Sephadex G 150 (Phannac ia product). Sephadex was suspended to swell in di still ed water ror 24 hr. During thi s time, it was stirred gentl y and the fine parti cles, if any, were removed by decantation. The water phase was then removed and the gel was suspended in 0.2 M acetate bu ffer (p H, 4.6). The gel suspension was carefully poured into a col umn (2.5 x 82 em) of Fraction Co ll ector (Fra- IOO, Pharmac ia-Fin Chemica ls) to avo id formation of air bubbles, then all owed to settle while slow fl ow rate of the buffe r was maintained through the co lumn. Two ml of the enzy me preparati on obtained from methanol ( I: I) was cautiously app li ed to the co lu mn. Ac ti ve fract ions (5 ml each) were poo led, lyophilized and subj ected fo r further purifi cation on DEAE-cellulose co lu mn (Diethylaminoethyl-cellu lose, fast flow, fibrous fo rm - Sigma product) prepared according to the procedure of Peterson and Sober"', in whi ch: DEDEcellulose was thoroughl y washed with di stilled water fo ll owed by washing with I N HCI and water till pH of the suspension was about 6.0. It was then washed several times with 0.5 N NaOH untill no more co lour was removed from the ge l. After the last alkaline wash, the res in was rinsed with di stilled water un ti l it was alkali-free. The ge l was allowed to settle and the fine partic les were removed. The column was packed
with the ge l and injected with the enzyme preparation as in sephadex.
The enzyme was eluted with a linear grad ient of NaCI concentration (0.0 to 0.5 M ) prepared in 0.2 M acetate buffer (pH , 4.6). pH va lue should be kept at
thi s level or lower because inulinase bound firmly wi th DEAE-cellulose at pH SA and cou ld not be elu ted even on rai sing NaC I concentration up to I M I2
.22
. Acti ve fractions were pooled, di alyzed, lyophili zed and kept cool for subsequent work .
The molec ular mass (Mr) of the enzy me preparati on was es timated by gel filtrati on::3, in whi ch a superose 12 co lumn was ca librated \\' ith standard proteins: carboni c anhydrase, 29 kD("!; ovalbumin , 43 kDa; phosphorylase, 94 kDa ; alcoholi c dehydrogenase, J SO kDa and catalase 232 kDa. Elution takes place with 0.2M aceta te buffer (p H 4.6) and a standard calibration graph was constructed represent ing the relation between eluti on vo lume against Mr of the standard proteins. The Mr of the enzy me was est imated by reference to the calibrat ion graph.
Results and Discussion Steps of the procedure used for puri fication of ex
tracellul ar inulinase from A. a /lerlia/a have been summarized in Tab le 1. The 1'1 step of purifi cati on was the di alysis of cell-free filtrate (CFF) aga inst 0.2 M acetate buffe r (p H 4.6). The result s show a decrease of inulinase activity by 90.5 %. This can be attributed to the loss of certain ac ti vati ng ions during the dial ys is. The results show the superiority of methanol ( 1:1) in obtaining protein frac tion hav ing the hi ghest total enzy me ac ti vi ty (- 135 x 103 U/m l) with a yie ld of 8 1.6% of the original ac tivity. Its specific activity was calculated to be 11.6 x 103 U/mg prote in . This finding confirmed that thi s enzyme has a partic ul ar structure which makes it res ist the known denaturing effect of organic so lven ts. Suitability of
Table I - Su llllll ary of treallllents used lhroughout purify ing ex trace ll ular inul inase of A/lemari(/ a/lemata
Step TOlal activity Total protcin (U x 10' ) (Illg)
Cel l- frec fi l trale (CFF) 165.00 127.00
Ccll-free dialy~ate
CFD 149.33 127.00
Prolc in prccipitatc of Illcth:.lIlol 134.7 1 11.60
1:1
Gcl fil l ral ion 115.50 3.5 1
Ion cxchange chrolll'llOgraphy 114 1.4 1
Total vo lulllc Ll sed was 500 IllI or CFF
Spccific aClivity (Ullllg x 103
)
1.3
1.2
11.6
32.9
80.7
Yield (%)
100
90.5
81.6
70.0
69. 1
Purifi calion fold(s)
0.90
9
25
62
HAMDY : EXTRACELL ULAR INU LI ASE OF ALTERNARIA ALTERNIITA 1395
organic so lvents in thi s regard was prev iously recorded by Vullo et al?4 and Vi swanathan et a/ 25
.
The purificati on was foll owed by gel-fil tration through Sephadex G150. The results obta ined indicated the presence of two peaks. Only the major one (fracti ons 6 to 10, containing -33 x L03 U/mg protein with 25-fold puri fication) was subjected to ion exchange chromatography. This resulted in single sharp peak possess ing about 8 1 x 103 U/mg protein with 62-fold puri fication (Table I ) . This enzy me preparation was diluted to a final concentrati on of 100 U/ml to fac ilitate presentati on of results, kept at 4°C and used in the subsequent experi ments.
Electrophoretic pattern of the enzy me preparati on that was carried out using sodium dodecy l sul fatepolyacrylamide gel electrophoresis (S DS-PAGE) revealed that the enzyme was not puri fied to electrophoretic homogeneity since a minor band in addit ion to a broad one were detected. Yet, puri ficati on to electrophoreti c homogeneity is not requi red for industrial
? 5 purposes-" .
Effect of temperature on enzyme activity and stability The puri fied enzy me preparation was subjected to
di ffe rent temperatures and the results were graphi call y represented in Fig. I . It is apparent that the max imum enzyme acti vity was observed in the narrow range of 50° to 60°C with its max imum at 55°C. Thi s is in complete agreement with the results obtained for other inulinases from A. oryzae l2
; P. janczeIVskii5 and P. t rzebil1ski i 17
. The noticed behavior of the enzyme acti vity in response to temperature, obviously obeyed
100
80 E -... :::l 60 c "> 40 u <t:
20
0
/. /.
/. ..... 25 30 35 40 45 50 55 60 65 70 75 80
Temp. C
100 ~
80 e.-.c .:;
60 TI (\l
(])
40 > .~
20 Qj IT:
0
Fig. I-Effec i of temperature on the ac ti vi ty (- + - ) and stab ili ty (- l - ) of purified inulinase enzyme. The reaction mixture contained 2 ml of 0.2% in ulin suspension, 2 ml of 0.2 M acetate buffer (pH, 4.6) and 0.5 ml of the enzyme preparation he ld at the ind icated temperature for 20 min fo r ac ti vity and fo r I hI' in case of stab ility
the kinetic theory. Enzy me preparati on was incubated at the indicated
temperature for 1 hr and the residual acti vity was measured. The enzyme appears to be completely stable up to 30°C, and also show hi gh stabi lity up to 50°C, after whi ch there is a gradual loss in enzyme act ivity. In thi s respec t, inulinases fro m A. j i/l l/ igatlls and P. pllrpurogenuI1l8 and from A. oryzae l 2 MTCC-152 show higher thermal stabil ity.
T II2 of the puri fied enzy me was found to be two weeks when kept in 0.2 M acetate bu ffe r, pH 4.6, at either - 15°C or at 4°C.
Effect of pH on the ell zyme activity and stability pH optimum fo r activity was found to be 4.5
(Fig. 2), identi cal to that recorded fo r inulinase from A. niger-24526
. It li es in acidic range as those recorded fo r A. niger, 5.027
, P. janczewskii , 5.05 and A. versicolor, 5.5 13
. As p H value di verged fro m the the opti mum level, the efficient functioning of the enzy me affec ted, most probabl y, due to change in ac ti ve site confo rmation which is determined, in part , by ioni c and hydrogen bonding which can be affected by pH.
The pH stab ility of the enzy me was stud ied in the pH range of 3.0 to 9.0 after I hr ex posure to the indicated p H. The enzyme was stable in the ac idi c range and sharp decrease was observed at pH values hi gher than 5. It is also clear that at 4.5 (the optimum value for ac tivity) the enzy me retained only 90% of its original acti vi ty. This may be fo r the absence of its substrate during the time of exposure that may affect its behavi or.
120 100 ~ ~
'E 1oo ~
80 c :3 80 '5
60 'D c 60 co '5 40 OJ
'D 40 > <t: ·til
20 20 w a:
0 0
"," 1>" ,,;" Ie" f\" 00" pH value
Fig. 2- EfTcc t of p H on the ac ti vity (-+ -) and stabilit y (-r-~- ) of puri fied inulinase enzyme. The enzyme preparation was held at the indicated pH for 20 min fo r measuring the act ivity and for I hI' in case of stab ility. BufTer solutions used fo r cont roll ing p H's were: 0.05 M citrate fo r p H 3.0-3.5; 0.01 M acetate fo r p H 4-5 .5: 0 .1 M phospliate fo r p H 6.0-7.0: Tris-HCI buffer for 7.0-9.0 and temperature was adj usted to 55°C Other reac ti on conditions arc samc as ill lega nd to Fig. I.
1396 IND IAN J EXP BIOL. DECEMBER 2002
"'x I'") Ongen-8aysa l el 0 /.- and Gupta el a /. - reported
the industri al importance of microbial inulinases that possess high ac ti vity and stability at relatively high temperatures and low pH becau se these conditions increase so lubility of the substrate and help in avoidance of microbial contamination. As a conclu sion, w ith its therma l and ac idic stability and high temperature and ac idi c pH optima, inulinase from A. a/lem ala
appears suitable fo r use in industrial applications.
Effecl of lllelO / ions and inll ibifors on illulinase aCli l' ily With regard to the examined metal ions and inhibi
tors on inulinase act ivity, Tabl e 2 demonstrates the riuctuating effects of these compounds. K+ and Na+ had insigni ficant effec t whil e Hg2+, Fe'+ or Cu2+ ex
hibited strong inhibiti on espec iall y at the higher concentrations. On the other hand, 8a2+ and Ca2+ exhibited a highl y significant stimultory effect. Simil ar re-
I f· I f'f' f' C "+ F 1+ H "+ suits were reportee or t l e e ect 0 u-, e', g-, 8 }+ and Ca2+ on the activity of inulinases from A. lIiger , A. oryzae and A. I'ersic% r I2.13 .1 5
The inhibition caused by Hg2+ substantiates the view that an SH group parti cipates in the enzyme structure. The inhibition effect of arsenate and arsenite in additi on to the great inhibiti on due to the presence of the more spec if ic sullhydry l inhibitor i .e. iodoacetic acid were additional evidences for thi s poss ible participati on. EDT A at 5 and 10 mM retarded the enzyme acti v ity mostly due to binding wi th 8 a2+ and/or Ca"' . Nakamura ef a l. lo also found that EDTA cause sli ght reduction in acti vity of inulinase fr III A. lIiger.
O fll er enZYlIlalic properlies Relat ion between inulin concentration and apparent
activity of inulinase was also studi ed and LineweaverBurk plot of the reciprocals of ini tial ve loc ities and inulin concentrat ions was constructed and the apparent Km value of this enzyme was found to be 0.066 M.
Free fructose was the on I y product released from inu l in (as ev idenced by adopting paper chromatography technique) indicating that this enzy me di splays exo-inulinase ac ti vity. Thi s f inding is in comp lete ac-
Table 2 - EITcc l o r l11 etal ions and enzyme inhibi to rs on re la li vc ac tivity or the purifi cd in ulinase
I Dala rcpresc nt the mea n o r 3 rcad ings approx im ated to thc neares t intcger numberl
Mctal ions*
g}+
C}+
Co2+
Cu2+
Fc"+
J-Ig2+
K+
M h " e
a+
EnzYl11 c inhi bito r
EDTA
lodoacclatc
Sod iulll arscnatc
Sodiulll arscn itc
Rc lati vc ac ti vit y as arfected by thc eonccntra li on of
ImM 5 111M 10 mM
11 8 ± 5.44"
11 8 ± 7.44"
83 ± 4.9S"
77 ± 5.39"
8 1 ± 2.S4"
74±5.I S'*
99 ± 396"
1I 0±7.70'
109 ± 4.25 '
100 ± 6.00"
78 ± 6.24**
" 97 ± 3.SH
62 ± 5.5H"
SO ± 5.60"
72 ± 3.60"
132 ± 1.32"
108 ± 2. 16'
6 1 ± 3.66**
13 ± 0.65"
40 ± 2.40"
43± 1.72"
" 10 1 ± 5.05
102 ± 3.00"
99 ± 6.93"
102 ± 5. 1"
6 1 ± 3.90"
9 1 ±2 .73"
43 ± 0 .g6"
62 ± 4 .96"
59 ± 2.30"
140 ± 6.60"
100 ±AOO"
30 ± 0 .60"
o ± 0 .00"
14 ± 0.98*'
15 ± 0.60"
100 ± 0.00"
95 ± 8 .55"
80 ± 5.20"
100±7.0S"
34 ± 1.36"
90 ± 5.40'
13 ± 0.65"
50 ± 1.50"
42 ± 2 .52*'
':'Thc in vcs ti ga tcd mctal ion as chl o ride was added at the indi catcd eoncc ntrat ion was incu bated with
the cnzy mc 1'01' 30 minat 30°C before add ing substratc. Activity of thc enzy me in compl ctc ab~ence
of such cO l11pou nds served as cont rol ( 100% ac ti vity) to whi ch o ther data wcrc stati stically compa rcd accmding to thc mathcmatica l princ iplcs dcse ribcd by G lantz .. 2 Thc res ult s wcrc considc rcd non s ignifican t whe n J> is grcatc r than 0.05 , signi ficant when Pis Icss than 0.05 and highl y s ignifica nt whc n P is less than 0.0 I and exprcssed as " = NO li s ig lli ri can t, • = Signifi ca nt and . , = Il igh ly sig ni fican l, rcspcc ti vcly.
HAMDY : EXTRACELLUL A R I ULI ASE OF A LTERNARIA ALTERNXrA 1397
cordance w ith th at of Ohta el a l. ~ who stated that mi crobial inulinases are usuall y exo-acting.
Microbial inul inases were also reported to have the b'I' f' h I I ' ff" d (, 10'6 capa I Ity 0 yc ro yZlIlg ra l nose an sucrose' '-.
[n thi s regard , inulinase of A. allem alO recorded simi lar capability aga inst sucrose ( 198 U/ml) and raffinose (73 U/ml). Such substances were separately added under conditi ons completely simil ar to th ose described for inulinase assay, but inulin was replaced with the substances under in vesti gati on.
M olecular mass o f the enzy me was es timated by gel filtrat ion and was found to be II S±S. Guptal :> stated that the molecular mess of inulinases is dependent upon the source o f the enzyme. for example 38 kDa was recorded for the inulinase from A. ory::.ae, 34-8 1 kDa for inulinases from d i fferent Aspergillus species and 300 kDa for inulinases from A. lIigt!r and F. OX I'SI )() n (11/ .
As many mi crobial preparati ons or inulinase possess remarkab le in vertase ac ti vity (S) accompany ing the inulinase act i vity (I ). their ca taly ti c ac ti v it y is described in terms of liS or S/l rati os:>' :><J . liS o f th is enzyme preparati on was found to be = O.S (equa ls S/l o f 1.9). liS rati os ror other inulinases were in the range of 0.3 1-0.39: CladosporiulII cladosporioides' 5
; A. lIige/o; 0.62S , A. .fiCIIIII// " and 0.7 , A. l'ersicolor'1,. Despite the above considerati ons, Uhm el al. ? determined a rati o of in finity for endo-inulinase from A. fiCIIU Ii/. 1 {1I11i ng of B-fructosidase as an inulinase or in vertase is based on such hydroly ti c capacity for sucrose and inulin (S/l ratio) w here true inve rtase has S/I ratio of more than 1000 12 and there is no microbial inulinase preparati on comp letely free from in ve rtase acti vity has so far been reported I:> . M oreover, during rhe puri f ication of inulinase. the sucrose and inulin hyd rolyz ing ac ti v ities are never separated2.1 . From th at, thi s enzyme preparat ion could sa rely be named inulinase.
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