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March 2000

- ''ir, ..'C* ‘-.'-•■'•v-»

r o : : - . - - — '

DIAS

r e D o r t No. T I • Plant Production

Henrik Brinch-Pedersen

Phytate and Phytase in Plants

(Review)

M in istry o f Food, A g ric u ltu re and Fisheries

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Phytate and Phytase in Plants

(Review)

H en rik B rin c h -P e d e rse n D e p a r tm e n t o f P la n t B iology R esearch C e n tre F la k k e b je rg DK-4200 S la g e lse

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DIAS r e p o r t P la n t P ro d u c tio n n o . 27 • M arch 20 0 0

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Preface

Being the major storage compound o f phosphorus in plants, phytic acid (phytate, m^o-inositol 1,2,3,4,5,6- h ex a^5ph0sphate) is o f special importance in plant biology. Plant tissues with storage function such as seeds, tubers, pollen and roots contain large amounts o f this acid.

The enzymes responsible for the initial steps in the degradation o f phytic acid are referred to as phytases. They comprise a special class o f phosphatases that catalyse a sequential hydrolysis o f phytic acid to lower inositol phosphates and, in some cases, wyo-inositol.

The present review describes the present state o f knowledge within the field o f phytic acid biosynthesis and deposition, phytase enzymes from different organisms, the degradation o f phytic acid mediated by phytase, the role o f phytate and phytase in nutrition, and the available evidence on the performance and biological significance o f phytase as an additive to animal feed or as a heterologous enzyme in transgenic plants.

Dr. Preben B. Holm, Department for Plant Biology, is acknowledged for finitful discussions and for helpful suggestions to the manuscript.

Henrik Brinch-Pedersen Research Centre Flakkebjerg February 2000

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C o n t e n t s

T he storage of phosphorus in p la n ts ...6

Deposition and accumulation o f phytin in the seed ...6

Biosynthesis of phytic a c id ...8

A/3;o-inositol...8

Phosphorylation o f »13/0-in0sit0l ...10

CompartmentaHsation o f phytic acid synthesis... 13

P h y ta se ...14

Classification o f phytases... 14

Active site determ ination... 14

Plant phytase... 15

Microbial phytase...15

Regulation o f phytase synthesis and activity... 16

The action o f phytase... 17

Role of phytin and phytase in n u tritio n ...21

The nutritional aspect o f feeding non-ruminants with high phytin d iets... 21

Application o f phytase to fe e d ...22

Increasing the phytase activity of plants by tran sfo rm atio n ...24

Transformation o f plants with phytase genes for improved growth performance o f non-ruminants...24

Effect o f transgenic phytase seeds on phosphorus excretion from non-ruminants... 25

Heat stability...27

R eferences... 28

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P h y tic acid (p h y tate, /«>'o-inositol 1,2,3,4,5,6-hexaÄ isphosphate) (F ig u re lA ) is th e m ain sto rag e form o f p h o sp h o ru s in p lan ts. It is d e p o site d as p h y tin (F ig u re IB ), a m ix e d salt w ith co u n terio n s in c lu d in g Fe^^, Mg^*, an d (L o tt, 1984). In leav e s p h y tin is a tem p o rary p h o sp h ate re s e rv e an d th e d ep o sits in the g rain s e n su re th a t the d e v e lo p in g se e d lin g s can be p ro v id e d w ith a c o n tin u o u s su p p ly o f p h o sp h a te an d m in erals th a t are lib e ra te d fro m th e m o lecu le d u rin g g erm in atio n . R em o v al o f th e first p h o sp h a te g ro u p s fro m p h y tin re q u ire s a specific en zy m e , p h y tase, w h ile th e re m a in in g p h o sp h a te g ro u p s m ay b e c le a v e d o f f b y p h o sp h atases w ith b ro a d su b strate sp ecific ities. P h y tin m ay also serv e as an e n e rg y re so u rc e an d th e lo w er /n ^ o -in o sito l p h o sp h ates ap p e a r to b e essen tial in sig n al tra n sd u c tio n p a th w a y s (R ab o y an d G erb asi, 1996). T h e c o n te n t o f p h y tin in seed s m ay acc o u n t fo r 0 .4 % to 5% o f the seed d ry w e ig h t in leg u m es an d o ilseed s and 0 .5 % to 1.9% in c ereals (R e d d y et al., 1982). U p to 8 7% o f th e to tal p h o sp h o ru s in th e seed m ay b e p re s e n t as p h y tin b o u n d p h o sp h a te e.g. in so y b ean an d w h e a t 5 3 % a n d 7 2 % o f th e to tal p h o sp h o ru s is p re s e n t in p h y tin (L o las et al., 1976).

The Storage of Phosphorus in Plants

Deposition and accumulation of phytin in the seed

P h y tin in seed s are d ep o site d in sin g le-m em b ran e sto rag e m ic ro b o d ie s (p ro te in b o d ies), m ain ly as d is c re te in c lu sio n s (g lo b o id s) (L o tt, 1984). G lo b o id c ry s ta ls c o n sist p re d o m in a n tly

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o f p h y tin salt c o m p le x e s o f p rim a rily p o ta s siu m an d m a g n esiu m , a lth o u g h m in o r a m o u n ts o f o th e r m etals su c h as zin c, iron, m an g an e se, c o p p e r an d c a lc iu m h a v e b e e n re p o rte d (O 'D e ll et al., 1972). A d d itio n a lly , so m e p ro te in -p h y tin -m in e ra l co m p le x e s h a v e also b e e n rep o rte d to b e p re se n t in p ro te in b o d ie s in b arley , ry e, w h e a t a n d so y b ean (L o tt, 1984).

P h y tin a c cu m u lates ra p id ly d u rin g seed d ev elo p m en t, an d is in m o n o c o ts p re d o m in a n tly d e p o site d in th e o u te r lay ers (a le u ro n e a n d p e ric a rp ) an d th e g erm , w h e re a s leg im ies and o ilseed s h o ld th e ir p h y tin in th e e n d o sp erm a n d c o ty le d o n s (O 'D e ll et al., 1972; P e m o lle t, 1978). In rice {O ryza sativa L .) an d w h e a t {Triticum aestivum L .) 8 0% a n d 87% o f th e p h y tin is lo calised in th e o u te r lay ers, w h ile 7 .6 % an d 12.9% is fo u n d in th e g erm (s c u te llu m and em b ry o ) (O 'D e ll et al., 1972). M aize is an e x c ep tio n to th e ty p ic a lly lo c a lisa tio n p a tte rn seen in the o th e r c ereals sin ce 8 8% o f th e p h y tin is co n fin e d to th e g erm (O 'D e ll et al., 1972).

In d ico t sp ecies, p h y tin is d e p o site d in th e en d o sp erm , th e em b ry o , an d th e c o ty le d o n s. In som e cases th e p h y tin is lo c a lise d to sp ecific tissu es. In th e y ello w lu p in e (Lupinus luteus L .) p h y tin in c lu sio n s are th u s foim d in th e five to te n su b ep id erm a l c e ll-la y e rs o f th e co ty le d o n (S o b o lev et al., 1976), w h e re a s p h y tin d ep o sits in so y b ean (Glycine max L .) a re d istrib u te d ev e n ly th ro u g h o u t th e co ty le d o n tissu e s (L o tt, 1984). T h e c a sto r b e a n e n d o sp e rm co n ta in s 9 7 % to 9 8% o f th e p h y tin w h ile th e re m a in in g 3% p rim a rily are c o n fin e d to th e c o ty le d o n s (G reen w o o d et a l , 1984).

P h y tin is also p re s e n t in th e p o lle n o f sev eral p la n t sp ecies an d th e c o n c e n tra tio n h a s b e e n su g g ested to re la te to th e sty le len g th (Ja c k so n et al., 1982; H e lsp e r et al., 1984). A n an aly sis o f 25 an g io sp erm sp ecies re v e a le d th a t sp ecies w ith a m e a n sty le len g th lo n g e r th a n 5 m m also had a sig n ific a n t a m o u n t o f p h y tin in th e p o lle n (0 .0 5 % to 2 .1 % b y d ry w e ig h t). P o lle n o f Triticum aestivum L , Hordeum bulbosum L. a n d Secale cereale L., all h a v in g a m e a n sty le len g th o f a b o u t 2 m m , lack e d p h y tin . In co n trast, p o lle n o f Zea m ays L ., w ith a m e a n sty le len g th o f 2 50 m m , c o n ta in e d 0 .2 6 % p h y tin b y d ry w eig h t. T h is c o rre la tio n d id , h o w e v e r, n o t h o ld fo r th ree g y m n o s p e rm sp ecies th a t c o n tain ed 0.1 % to 5 .9 % p h y tin b y d ry w e ig h t in the p o llen irresp ectiv e o f th a t th ese sp ecies o n ly fo rm sh o rt p o lle n tubes. T h u s th e fu n ctio n o f p h y tin m a y n o t o n ly b e re stric te d to p o lle n tu b e d ev e lo p m e n t b u t m a y in g y m n o s p e rm s also b e im p o rtan t fo r p o lle n d ev elo p m en t.

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Biosynthesis of Phytic Acid

M y o -in o sito l

F ree /w>'o-inositol is g e n erated from D -g lu co se b y th re e en zy m a tic steps: A ) h e x o k in a s e (E C 2 .7.1.1); B ) IL-TMj^o-inositol 1-p h o sp h ate sy n th ase (E C 5. 5. 1. 4 ) a n d C ) w3/0 -in 0sit0 l 1- p h o sp h ate p h o sp h a ta se (E C 3.1 .3 .2 5 ), (F ig u re 2).

Figure 2. T h e c o n v e rs io n o f (1) D -g lu co se to (4 ) free /w>'o-inositol v ia (2) D -g lu c o s e 6- p h o sp h ate an d (3 ) IL -m j'o -in o sito l 1-p h o sp h ate c a ta ly se d b y (A ) h e x o k in a se , (B ) \L-m yo- in o sito l 1-p h o sp h ate sy n th ase, an d (C ) w j^o-inositol m o n o p h o sp h a ta se (L o ew u s, 1990)

A fter p h o sp h o ry la tio n o f D -g lu co se to D -g lu co se 6-p h o sp h ate b y h ex o k in ase, th e n e x t step in v o lv es /n y o -in o sito l 1-p h o sp h ate sy n th ase. In th e reactio n d e sc rib e d in fig u re 3 th e b o n d th a t links p h o sp h a te to c a rb o n re m a in s u n d istu rb ed . T h e in term ed iates (w ith in b ra c k e ts) are tig h tly b o u n d to th e en z y m e as re v e a le d b y stu d ies u sin g iso to p ic a lly la b elled su b stra te s as w ell as partial re a c tio n seq u e n c e s (L o ew u s, el al., 1984). T h e en z y m e e x h ib its an ab so lu te req u irem en t fo r N A D ^, sin ce th e first p ro d u c t I L -7n3;o-inositol 1-p h o sp h ate, is g e n e ra te d b y an N A D ^ c a ta ly se d o x id a tio n o f D -g lu co se 6 -p h o sp h a te a t c a rb o n 5 to 5 -k e to -D -g lu c o se 6- p h o sp h ate (M a e d a an d E isen b erg , 1980; M au ck et al., 1980). A fte r th e o x id a tio n , th e rem o v ed h y d ro g en is tra n sfe rre d as a h y d rid e ion to th e B p o sitio n o f c arb o n 4 o n th e n ic o tin a m id e m o iety o f NAD'^ (B ry u n an d Je n n e ss, 1981; Y o u , 1985).

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p h o sp h ate b y lL -m > 'o-inositol 1-p h o sp h ate sy n th ase (L o ew u s et al., 1984).

C ycH sation o f D -g lu c o se 6 -p h o sp h ate is re q u ire d fo r th e fo rm atio n o f 1 L -m y o -in o sito l 1- p h o sp h ate. D u rin g th is a sp ecific h y d ro g en is lo st from p o sitio n 6 o f 5 -k e to -D -g lu c o se 6- p h o sp h ate. T h e en z y m e re m o v e s th e 6 -R h y d ro g e n w h e re a s th e 6-S h y d ro g e n is re ta in e d in th e p ro d u c t (L o e w u s et al., 1980). T h e red u ctiv e p a rtia l re a c tio n b y e n z y m e -b o u n d N A D H o f th e seco n d p u ta tiv e in te rm e d ia te w y o -in o so se-2 1-p h o sp h ate (D -2 ,4 ,6 /3 ,5 -p en tah y d ro x y - c y clo h e x an o n e 2 -p h o sp h a te ) tran sfers a h y d rid e fi-om th e B p o sitio n o f c a rb o n 4 o n the n ico tin am id e rin g o f N A D H to th e c arb o n y l g ro u p to g e n e ra te lL -w > 'o -in o sito l 1-p h o sp h ate (B ry u n a n d Jetm ess, 1981; L o e w u s et al., 1982).

T h e sy n th esis o f 1 L -w jo -in o s ito l 1-p h o sp h ate b y /n y o-inositol 1-p h o sp h a te sy n th ase is a critical p o in t as it is th e so le b io sy n th etic ro u te to w j^o-inositol. T h e en z y m e ex ists in a cy to p lasm atic fo rm in a w id e ran g e o f p la n ts, an im a ls a n d flingi. It h a s also b e e n o b serv ed in sev eral b a c te ria a n d a c h lo ro p la st form is p re s e n t in a lg a an d h ig h e r p la n ts (M a ju m d e r et al., 1997). N o rth e rn an aly sis a n d in situ h y b rid isa tio n o f a re c e n tly iso la te d ric e c D N A w ith h ig h h o m o lo g y to th e /nj^o-inositol 1-p h o sp h ate sy n th ase o f y e a st an d o th e r p la n ts sh o w e d th a t tran scrip ts a c c u m u la te d to h ig h lev els in e m b ry o s b u t w as u n d e te c ta b le in sh o o ts, ro o ts, an d flow ers. S tro n g sig n a ls w e re d etec ted in th e sc u tellu m a n d a leu ro n e a t 4 d a y s an d in c re a se d un til 7 d ay s a fte r a n th esis w h e re a fte r it g rad u ally d ecreased . P h y tin c o n ta in in g g lo b o id s ap p eared 4 d ay s a fte r a n th esis in th e scu tellu m an d aleu ro n e, c o in c id in g w ith th e p re s e n c e o f the TTzj^o-inositol 1-p h o sp h ate sy n th ase tran scrip ts. T h e te m p o ra l an d p a rtia l p attern s o f accu m u latio n o f th e tra n sc rip ts a n d g lo b o id s su g g est th a t th is g en e d ire c ts p h y tin b io sy n th e sis in rice seeds (Y o sh id a et al., 1999).

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C o n v ersio n o f 1 L-w j^o-inositol 1-p h o sp h ate to free /w3^0-in o sito l is cata ly sed b y m3^0-in0sit0 l m o n o p h o sp h atase (F ig u re 2, en z y m e C). T h e en zy m e is a d e p e n d e n t a lk a h n e p h o sp h atase w ith h ig h affin ity fo r b o th ID - an d 1 L-m>^o-inositol 1-p h o sp h ate. Its h a s a low affin ity fo r w ^'o-inositol 2-p h o sp h ate and little o r no a ffin ity for h ex o se p h o sp h ates o r the m o d el p h o sp h a ta se su b stra te p -n itro p h e n y l p h o sp h a te (E ise n b e rg an d P a rth a sa ra th y , 1987). In to m ato w3^0-in 0 sit0l m o n o p h o sp h atase appears to b e en co d ed b y a sm all g en e fam ily and th ree d ifferen t c D N A 's h a v e b e e n ch a ra c te rise d e n c o d in g d istin c t b u t h ig h ly co n serv ed en zy m e s (G illa sp y et al., 1995). In p articu lar, c e lls asso ciate d w ith th e v a sc u la tu re e x p ressed h ig h lev els o f th e p ro te in , su g g estin g a c o -o rd in ated re g u la tio n b e tw e e n p h lo e m tra n sp o rt and sy n th esis o f in ositol.

Phosphorylation o f myo-inositol

P h y tic acid a p p ears to b e fo rm ed b y a series o f seq u en tial p h o sp h o ry la tio n s o f m3/0 -in0 sit0 l b y a /M>'o-inositol k in ase. E a rly stu d ies sh o w ed th a t free /n>'o-inositol in g e rm in a tin g m u n g b e a n s is p h o sp h o ry la te d b y a Mg^* A T P d e p e n d e n t k in a se (M a ju m d e r et al., 1972). T h e p ro d u ct, m;/0 -in0sit0 l- l- p h 0sp h ate, is iden tical to th at p ro d u ced b y w>’o -in o sito l 1-p h o sp h ate syn th ase, h o w ev er th e k in a s e p h o sp h o ry la te s m yo-inositol m o re efficien t th an g lu co se. L ik ew ise, a step w ise ad d itio n o f p h o sp h a te resid u es w as rep o rte d in g e rm in a tin g m u n g b e a n s u sin g th e sam e Mg^'" A T P d e p e n d e n t k in a se g en eratin g m3;o-inositol 1,3,4,5,6 penta^z'sphosphate (C h ak rab arti a n d M aju m d er, 1978). T h e ste re o sp e c ific ity o f th e p o sitio n a l 7w>’o -in o sito l p o ly p h o sp h ates o f th e in term ed iates w as n o t d eterm in ed . A d d itio n a l d a ta fro m m u n g b ean describ ed a p h o sp h o ry la tio n o f m y o -inositol 1 ,3 ,4 ,5 ,6 -p e n ta ^ ijp h o sp h a te to m y o -inositol 1,2,3,4 ,5,6-hexa)b'5p h o sp h a te b y a m yo-inositol h ex a^ zjp h o sp h ate-ad en o sin e d ip h o sp h ate p h o sp h o tra n sfe ra se (B isw a s et al., 1978). F ro m im m atu re so y b ean se e d s m y o -inositol 1,3,4,5,6 p en ta/3jp h 0sp h ate 2 -k in ase has b een p u rified (P h illip p y et al., 1994). T h e k in ase sp ecific ally p h o sp h o ry la te d th e 2 -p o sitio n o n th e m y o -inositol 1 ,3 ,4 ,5 ,6 -p e n ta /a sp h o sp h a te in o sito l rin g , b u t c o u ld also u tilise m y o -inositol 1 ,4 ,5 ,6 -te tra ^ jp o s p h a te as a su b strate.

M o re c o m p re h e n siv e d a ta from th e slim e m o u ld Dictyostelium illu s tra te th a t th e c o n c e p t o f a seq u en tial p h o sp h o ry la tio n to p e n ta k isp h o sp h a te b y a sin g le k in a s e d e sc rib e d fo r m u n g b ean is p ro b a b ly m isle a d in g . In stead , th e seq u en tial p h o sp h o ry la tio n is m e d ia te d b y a se rie s o f k in ases w ith d iffe re n t p re fe re n c e s an d sp ecific ities (S tep h en s a n d Irv in e, 1990; K a a y et al..

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1995). T h e se q u e n tia l p h o sp h o ry la tio n o f in o sito l ap p ears to o c c u r v ia m y o -in o sito l 3- ph o sp h ate, w h e re a fte r w ^'o-inositol 3 ,6 -d ip h o sp h ate, w >'o-inositol 3 ,4 ,6 -trip h o sp h a te , myo­

inositol 1,3,4,6- tetraA ijphosphate, an d /n>'o-inositol 1,3,4,5,6-pentaA z.sphosphate are g en erated (F ig u re 4). T h e p h o sp h a te s in p o sitio n 3 an d 5 o f m >'o-inositol 1,2,3,4,5,6-hexaÄ j.sphosphate ad d itio n ally ta k e s p a rt in tw o fiitile c y cle s in w h ic h w >'o-inositol 1 ,2 ,4 ,5 ,6 -p en ta^j5 p h o sp h ate an d /w>'o-inositol l,2,3,4,6-pentaÅ 75phosphate are ra p id ly fo rm ed b y d e p h o sp h o ry la tio n o f m_yo-inositol 1 ,2 ,3 ,4 ,5 ,6-hexafc'5phosphate, o n ly to b e re p h o s p h o ry la te d to y ie ld th e ir precursor.

C M P , P A

P td ln s - ► P td ln s(4 )P i - ► P td ln s(4 ,5 )P 2

E n v iro n m e n t • Ins In s(l,4 )P 2

I t ^ ^ ^

G lu co se-6 P —► In s(3 )P i I n s ( 4 ) P i - ^ I n s ( 4 ,5 ) P 2 - ^ In s (l,4 ,5 )P 3 + d ia c y lg ly c e ro l

I

Ins(3,6)P2

I

Ins(3,4,6)P3

I

In s(l,3 ,4 ,6 )P 4

?

I

In s (l,3 ,4 ,5 ,6 )P 5

I

InsPö I n s ( l,2 ,4 ,5 ,6)Ps

I t

In s (l,2 ,3 ,4 ,6 )P 5

Figure 4. T h e in o sito l m e ta b o lism in Dictyostelium. A b b re v ia tio n s: P td ln s, p h o sp h a tid y lin o sito l; C M P , P A , c y stid in e m o n o p h o sp h a te p h o sp h a tid ic acid , (S te p h e n s and Irv in e, 1990).

A lso in p la n ts, th e in v o lv e m e n t o f m u ltip le k in ases in /w yo-inositol 1,2,3,4,5,6- hexaÄ zsphosphate sy n th e sis is su g g ested . T h ree en zy m e fra c tio n s is o la te d fro m Lemna gibba.

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c o u ld to g e th e r c o n v e rt w j^o-inositol to /«3/0-in0sit0 l 1 ,2 ,3 ,4 ,5 ,6 -h ex a^ jjp h o sp h ate. O ne fraction co n v e rte d w j'o -in o sito l to »i>'o-inositol m o n o p h o sp h ate, a seco n d to trip h o sp h a te , and a th ird to w y o -in o sito l h e x a ^ jjp h o sp h a te (B o llm a n et al., 1980). M o re o v e r, M u rth y (19 9 6 ) pro p o sed tw o p a th w a y s fo r p h y tic acid sy n th esis (F ig u re 5). P ath w ay 1 sh a re d m y o -inositol 3- p h o sp h ate and w j^o-inositol 1,3,4,5,6-pentaÄi.yphosphate w ith th e D ictyostelium p a th w a y , h o w ev er th e p a th w a y in clu d ed /n>>o-inositol 1,2 ,3 ,5,6 -penta/:i5p h o sp h a te and d iffe re n t di-, tri- and te trak isp h o sp h ates. T h e seco n d p ath w ay sta rte d fro m m3'o -in o sito l 2 -p h o sp h ate an d ended w ith w j'o -in o sito l 2 ,3 ,4 ,5 ,6 -p en ta^ j'jp h o sp h ate in v o lv in g in term ed iates all b e in g d iffe re n t from p ath w ay 1 an d th e D ictyostelium p ath w ay .

In s(3 )P i -► I n s ( l,3)?2 - > I n s ( l,3 ,5)?3 -► I n s ( l,3 ,5,6)P 4

I n s ( l,2 ,3,5,6)P s-i-Ins(l,3,4,5,6)P 5 -► In sP 6

In s(2 )P i -► In s(2,6)P 2 -► Ins(2 ,5,6)P 3 -► In s(2 ,3,5,6)P 4

In s(2 ,3 ,4 ,5 ,6 )P s -► In sP6

Figure 5. T h e p la n t b io sy n th e sis o f m_yo-inositol l ,2,3,4 ,5,6-hexaÅi'5p h o sp h a te (InsPö) from

»i_vo-inositol 3 -p h o sp h a te (In s(3 )P i an d w3/o-inositol 2 -p h o sp h ate (In s(2 )P i) (M u rth y , 1996).

So far o n ly o n e g e n e e n co d in g a k in ase in v o lv ed in w3/0-in 0sit0 l p h o sp h o ry la tio n h as been iso lated from p lan ts. A p artial cD N A from A ra b id o p sis (W ilso n an d M a je ru s, 1997) e n c o d in g a p u ta tiv e m >'o-inositol 1 ,3 ,4 -trip h o sp h ate 5/6 k in a se w as e x p ressed in E. coli a n d sh o w n to p ro d u ce in o sito l 1 ,3 ,4 ,6 -tetra k isp h o sp h a te and in o sito l 1 ,3 ,4 ,5 -tetra k isp h o sp h a te in a ratio o f 1:3 w h en o ffe re d in o sito l 1 ,3 ,4 -trip h o sp h ate as a substrate.

T he m u tag en esis a p p ro ach c u rre n tly u n d ertak en in m a iz e an d b a rle y m ig h t p ro v e to be e ssen tial for the id e n tific a tio n o f th e in d iv id u al g en es in v o lv ed in p h y tic ac id b io sy n th e sis. In b o th m aize an d b a rle y tw o loci h a v e b e e n id en tified th a t w h en m u ta te d re s u lt in a d ra stic decrease in th e c o n c e n tra tio n o f p h y tic acid and a c o rre sp o n d in g in c re a se in free p h o sp h a te (R ab o y an d G erb asi, 1996; E rtl et al., 1998; L arso n et al., 1998; R a b o y , 1998). M u tan ts h o m o zy g o u s fo r th e m u ta te d Ipal locus g iv e rise to an alle le sp e c ific d e c re a se in p h y tic acid

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a m o u n tin g to 5 0 -9 5 % w ith n o a c c u m u latio n o f th e lo w e r in o sito l p h o sp h a te s. In m a iz e , the lpa\ m u ta tio n m a p s to th e p o sitio n o f th e lo cu s fo r w > 'o -in o sito l-l-p h o sp h a te sy n th ase. In barley, Ipal is k n o w n to re sid e o n ch ro m o so m e 2 H b u t it rem ain s to b e p ro v e n i f th is locus co d es fo r m > 'o -in o sito l-l-p h o sp h ate synthase. T h e o th er, an d less fre q u e n t m u ta tio n , is referred to as lpa2. In c o n trast to th e situ atio n fo r th e lp a \ m u ta tio n s, th e re d u c tio n in p h y tic acid is in m a iz e a c c o m p a n ie d b y th e acc u m u la tio n o f lo w er w >/o-inositol p h o sp h a te s, in p a rtic u la r /w yo-inositol 1,2,4,5,6-pentaÅ zsphosphate an d /w>/o-inositol 2 ,3 ,4 ,5 ,6 - p en ta/a^p h o sp h ate w h ile in b a rle y th e p ro m in e n t form is w j^o-inositol 1,2,3,4,6- pentaA ziphosphate. A t R isø a sim ila r m u ta g e n e sis p ro g ra m h as also lead to th e id e n tific a tio n o f tw o m u ta n ts in th e p h y tic ac id b io sy n th e sis o f b a rle y an d it re m a in s to b e se e n i f th e tw o m u tatio n s are in th e sam e loci, lp a \ an d lpa2 as d esc rib e d fo r m a iz e an d b arley .

Compartmentalisation of phytic acid synthesis

It h as b e e n su g g e ste d o n th e b asis o f stu d ies in d ev e lo p in g seed s th a t p h y tic a c id sy n th esis tak es p lace in th e c y to p la sm p rio r to its d e p o sitio n w ith in p ro te in b o d ie s (G re e n w o o d an d B ew ley , 1984). T h e c o u rse o f ev en ts b e g in s w ith p h y tin sy n th esis in c iste rn a l en d o p lasm ic reticu lu m , fo llo w ed b y m ig ra tio n o f p h y tin b e a rin g v esicles to w a rd s th e p ro te in b o d y . A fte r d isch arg e o f th e v e sic u la r co n te n ts w ith in th e p ro te in b o d y , p h y tin ric h p a rtic le s are accu m u lated as a g lo b o id . T h u s in Dictyostelium ly sates, a c y to so lic ro u te e x h ib itin g fo rm atio n o f /w>'o-inositol l,2,3,4,5,6-hexaA j'5phosphate b y seq u en tial p h o sp h o ry la tio n o f in o sito l (F ig u re 4 ) h as b e e n rep o rte d (S tep h en s an d Irv in e, 1990).

H o w ev er n u c le u s a sso c ia te d p h o sp h o ry la tio n c o u ld tak e p la c e in p lan ts. S tu d ies in Dictyostelium in d icate th a t th e step w ise p h o sp h o ry la tio n o f in o sito l to p h y tic acid , c a n o c c u r v ia n u cleu s a sso c ia te d p h o sp h o ry la tio n (K a a y et al., 1995). H P L C a n a ly sis in d icated th a t 77ij/0-inositol 1 ,4 ,5 -p h o sp h ate in th e n u c le u s is co n v e rte d in to p h y tic a c id v ia seq u en tial p h o sp h o ry la tio n a t th e 3-, 6- an d 2 -p o sitio n s.

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Phytase

T he en zy m e s re sp o n sib le fo r th e in itial step s in th e d eg rad atio n o f p h y tic acid are re fe rre d to as p h y tases. T h e y c o m p rise a sp ecial class o f p h o sp h a ta se s th a t c a ta ly se a seq u en tial h y d ro ly sis o f p h y tic ac id to lo w er in o sito l p h o sp h a te s and, in so m e ca se s, to in o sito l. A rich d iv ersity o f p h y ta s e s o c c u rs in a v ariety o f o rg a n ism s in clu d in g p la n ts a n d m ic ro -o rg a n ism s (Irving, 1980a; W o d zin sk i an d U llah , 1996). D ifferen ces in p H o p tim a , su b stra te sp ecific ity an d sp ecific ity o f h y d ro ly sis h a v e b e e n identified.

P h y tase are o fte n d e sc rib e d a cco rd in g to th e ir p H o p tim u m as ac id p h y ta s e o r alk alin e p h y tase, also re fe rre d to as the pH 5 an d th e pH 8 p h y ta se re s p ectiv ely . B a se d o n th e sp ecific ity o f th e in itial h y d ro ly sis, the In tern atio n al U n io n o f B io c h e m istry (1 9 7 9 ) re c o g n ise s tw o c lasses o f a c id p h y tase, th e 6 -p h y tases (E C 3 .1 .3 .2 6 ), an d th e 3 -p h y ta se s (E C 3 .1.3.8).

T h e 6 -p h y tase h y d ro ly se s th e p h o sp h a te e ste r a t th e L -6 (o r D -4 ) p o sitio n o f p h y tic acid , and th e 3 -p h y tase h y d ro ly se s th e p h o sp h a te e ste r a t th e D -3 p o sitio n . L a te r re s u lts h a v e sh o w n th at th is d e fin itio n m a y b e to o rig o ro u s as p h y ta se s m ay d iv e rg e from th e 6-, 3 -p h y tase te rm in o lo g y in th e ir a ctio n o n p h y tic acid.

C lassification o f phytases

Active site determination

P h y tases b e lo n g to th e fam ily o f h istid in e p h o sp h a ta se s, a su b class o f p h o sp h a te m e ta b o lisin g e n zy m es, all u tilisin g a p h o sp h o h istid in e in te rm e d ia te in th e ir p h o sp h o ry l tra n sfe r re actio n (E tten, 1982). A n a ly se s fo r co n serv ed m o tifs in th e seq u en ces o f d iv e rse p h o sp h a te lib eratin g en zy m e s h a v e led to th e id e n tificatio n o f th e ac tiv e site c o m p risin g a trip e p tid ic re g io n in A rg -H is-G ly , th e N -te rm in a l seg m e n t o f th e p ro tein . T h e A rg -H is-G ly m o tif is h ig h ly c o n serv ed in p h o sp h a ta se s from o rg a n ism s such as Escherichia coli, Aspergillus niger, Saccharomyces cerevisia, Schizosaccharomyces pom be, Homo sapiens, rat an d m o u se. T h e h istid in e cla ss p h o sp h a ta se s u ses th e p o sitiv e ch a rg e o f th e g u an id o g ro u p o f a rg in in e fo r th e reco g n itio n a n d a n c h o rin g o f th e n e g a tiv e ly c h arg ed p h o sp h a te g ro u p . T h e p h o sp h a te g ro u p is

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tran sien tly tra n sfe rre d to th e h istid in e gro u p to form an u n sta b le p h o sp h o e n z y m e c o m p lex b efo re h y d ro ly tic cle a v a g e to fo rm o rth o p h o sp h ate (E tten , 1982).

P la n t p h y ta s e

A cid ic p h y ta se sh o w s a b ro ad affin ity fo r v ario u s p h o sp h o ry la te d su b strates. T h u s th e w h e a t b ran pH 5 p h y ta s e ca ta ly se s th e h y d ro ly sis o f sev eral in te rm e d ia te fo rm s o f p h o sp h o ry la te d m yo-inositol, sp an n in g from m>/o-inositol 1 ,2 ,3 ,4 ,5 ,6 -h e x a ^ 'ip h o sp h a te to /n ^ o -in o sito l 2- p h o sp h ate (F ig u re 6) ( U m an d T ate, 1971;1973). M u n g b e a n co ty le d o n p h y ta s e , in sp ite o f a pH o p tim u m o f 7 .5, e x h ib its th e en zy m a tic p ro p e rtie s o f th e g ro u p o f a c id ic p h y ta s e s (M aiti and B isw as, 1979). U n lik e th e p H 5 p h y tases, th e p H 8 p h y ta se s are h ig h ly sp e c ific fo r p h y tic acid, and n o n e o f th e w >'o-inositol p h o sp h ates co n ta in in g th ree o r few er e ste r g ro u p s can act as su b strate (B ald i et a l, 1988; B arrien to s et al., 1994). A lk a lin e p h y ta s e h a s b e e n id e n tifie d in h ly p o llen an d in a v a rie ty o f leg u m es (S co tt an d L o ew u s, 1986b; B ald i et al., 1988; Scott, 1991; B arrien to s et al., 1994).

A lth o u g h p la n t p h y ta s e activ ity h as b e e n d e sc rib e d in n u m ero u s ca se s, o n ly p h y ta s e s fro m a few so u rces h a v e b e e n p u rifie d to h o m o g en eity . T h e m o st n o ta b le are fro m m u n g b ean co ty led o n (M aiti a n d B isw as, 1979), so y b ean co ty le d o n (G ib so n a n d U llah , 1988), m aize seed lin g s an d ro o ts (L ab o u re et al., 1993; H ü b el an d B eck , 1996), sc a llio n leav e s (P h illip p y , 1998), an d ry e (G re in e r et al., 1998). H o w ev er, so far it h as o n ly b e e n p o ss ib le to iso la te a c D N A fo r m a iz e p h y ta s e s (M a u g e n e st et a l, 1997; M a u g e n e st et al., 1999).

M ic r o b ia l p h y ta s e

T h e n u m b e r o f m ic ro -o rg a n ism s screen ed fo r p h y ta se p ro d u c tio n is e n o rm o u s. O f all th e org an ism s su rv ey ed th e Aspergilli fim gi p ro d u c e s th e m o s t ac tiv e e x tra c e llu la r p h y ta se (W o d zin sk i an d U lla h , 1996). C o n seq u en tly , a n u m b e r o f p h y ta se s fro m th is o rg a n ism h av e b een p u rified to h o m o g e n e ity an d th e g en es isolated. T h u s, a p h y ta s e w ith a p H o p tim u m o f 5.0 an d 2.5 w a s iso la te d fro m a c u ltu re filtrate o f Aspergillus niger, b io c h e m ic a lly ch aracterised , p a rtia lly se q u en ced an d th e p h yA g en e fo r th e en z y m e c lo n e d (U lla h an d D isch in g er, 1992; P id d in g to n et al., 1993; H a rtin g sv e ld t et al., 1993). A se c o n d e n z y m e from

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Aspergillus niger en co d ed b y th e phyiQ g en e w ith pH o p tim u m 2.5 w as in itia lly c o n sid e re d to be an a c id p h o sp h a ta se (E rlich et al., 1993; P id d in g to n et al., 1993). H o w e v e r, ad d itio n al stu d ies h av e sh o w n th a t it is ab le to h y d ro ly se p h y tic acid at pH 2.5 b u t n o t a t p H 5 (U llah and P h illip p y , 1994). A lso th e A spergillus niger phyB g e n e h as b e e n clo n e d (E h rlic h et al., 1993).

F ro m Aspergillus fum igatus th e ph yK g ene h as b e e n clo n e d an d fo u n d to en c o d e a n en zy m e w ith a pH ra n g e o f 2.5 to 7.5 (P asam o n tes et al., 1997). A phyA. g e n e fro m Aspergillus terreus has b e e n iso la te d a n d th e re su ltin g p h y ta se en z y m e w ith a p H o p tim u m aro u n d 5.5 d e sc rib e d (M itch ell e / a / . , 1997).

D etailed c h a ra c te risa tio n s h av e also b e e n p e rfo rm e d o n m icro b ial p h y ta s e s su c h as Bacillus subtilis (P o w a r an d Jag a n n a th a n , 1982; S h im izu , 1992; K ero v u o et al., 1998), Escherichia coli (G rein er et al., 1993), M yceliophthora thermophila (M itch ell et al., 1997), Klebsiella terrigena (G re in e r et al., 1997), Thermomyces lanuginosus (In te rn a tio n a l p a te n t a p p licatio n n um ber: P C T /U S 9 7 /0 4 5 5 9 ; B erk a et al., 1998), Em ericella nidulans an d Talaromyces thermophillus (W y ss et al., 1999).

Regulation of phytase synthesis and activity

A lth o u g h a sm all a m o u n t o f e n d o g en o u s p h y ta se activ ity h as b e e n d e te c te d in u n g e rm in a te d and d ry seeds, e.g. p e a s (G ib so n an d U llah , 1990), a larg e in crease in p h y ta s e a c tiv ity an d a d ecrease in p h y tic acid c o n te n t can be o b serv ed w h e n seed s are g e rm in a tin g o r ev e n sh o rtly after w a te r im b ib itio n . T h u s, a 2 -fo ld in crease in p h y ta s e a c tiv ity w as o b se rv e d in m aize du rin g g e rm in a tio n a n d sev eral v arieties o f p e a s e x h ib ited an 8- to 3 0 -fo ld in c re a se in p h y tase activ ity from d ay 1 to d ay 5 o f g e rm in a tio n (G ib so n an d U llah , 1990). In so y b e a n , an ap p ro x im ately 10-fold in crease in p h y ta se a c tiv ity w as o b serv ed , w ith a m a x im u m lev el o f activ ity at d a y s 8 to 10 a fter g e rm in a tio n (G ib so n an d U llah , 1988). A 24 h rs in c u b a tio n o f w h eat, rye, an d h u lle d an d d eh u lled b arley seed s w ith w ater, re su lte d in a 4 6 -7 7 % re d u c tio n o f p h y tate c o n ten t, in d ic a tin g a h ig h p h y ta s e activ ity (F red lu n d et al., 1997).

T h e in crease in p h y ta s e a c tiv ity d u rin g g e rm in a tio n can b e a re s u lt o f e ith e r an a c tiv a tio n o f a p re -e x istin g p h y ta se o r a de novo sy n th esis. In ad d itio n to th e low p h y ta s e a c tiv ity p re s e n t in d o rm an t seed s o f p e a s, tw o a d d itio n al fo rm s are p re s e n t e x c lu siv e ly in g e rm in a tin g seeds in d icatin g a de novo sy n th e sis (G ib so n and U llah , 1990). In so y b ean s, p h y ta s e a c tiv ity is

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o b serv ed in u n g e rm in a te d seeds, h o w e v e r in b o th so y b ean an d m a iz e , a n in c re a se in im m u n o sp ecific stain in g o n w e ste rn b lo t ap p ears d u rin g g e rm in a tio n , in d ic a tin g a de novo sy n th e sis o f th e en z y m e (G ib so n an d U llah , 1988; L ab o u re et al., 1993). In m aize, no tran scrip t w as p re s e n t in d ry seeds, h o w e v e r m R N A ac c u m u la te d d u rin g th e first d ay o f g e rm in atio n , to re a c h a m a x im u m a fte r 2 d ay s, an d th e n d e c re a s e d in y o u n g seed lin g s (M au g en est et al., 1997).

P h y tase a c tiv ity is re g u la te d b y th e lev el o f in o rg an ic p h o sp h a te in se e d s o f Phaseolus vulgaris (C h an g a n d S ch w im m er, 1977; L o las an d M ark ak is, 1977) in w h e a t b ra n (L im and T ate, 1 9 71;1973), in so y b ean c o ty le d o n s (G ib so n an d U llah , 1988), in Lilium longißorum p o lle n (S co tt an d L o ew u s, 1986a; L in et al., 1987), an d in Petunia hybrida p o lle n (Jack so n an d L in sk en s, 1982). A lso , th e E. coli p h y ta s e activ ity is in h ib ited b y in o rg a n ic p h o sp h a te (G rein er et al., 1993). In co n trast, low p h o sp h a te lev els ap p e a r to in d u c e p h y ta s e gene ex p ressio n . T h u s, in c e ll su sp en sio n cu ltu res o f so y b ean , p h y ta s e a n d ac id p h o sp h a ta se ex cretio n w a s in d u c e d a fte r 3 to 4 d ay s o f g ro w th u n d e r p h o sp h a te d e fic ie n t c o n d itio n s (G ib so n an d C h risten , 1987). L ik ew ise, in Aspergillus niger, the p h y ta s e e n c o d in g p h yA g en e is tra n sc rip tio n a lly u p re g u la te d u n d e r low p h o sp h a te c o n d itio n s (H a rtin g sv e ld t et al., 1993).

In su m m ary , p h y ta s e g e n e e x p re ssio n m a y b e u p reg u lated at th e tra n sc rip tio n a l lev el b y low in o rg an ic p h o sp h a te lev els w h ile th e p h y ta se en zy m e ap p ears to b e in h ib ite d b y h ig h lev els o f in o rg an ic p h o sp h a te , i.e., a feed b a c k in h ib itio n .

The action of plant phytase

T he seq u en ce o f h y d ro ly sis o f p h o sp h a te g ro u p s h as b e e n in v e stig a te d in w h e a t (L im and T ate, 1971; 1973), m u n g b e a n (M aiti an d B isw as, 1979), an d lily p o lle n (B ald i et a l, 1988;

B arrien to s et al., 1994). A d d itio n ally , th e »i;;o-inositol co m p o sitio n in b a rle y a le u ro n e cells h as b e e n c h a ra c te rise d in vitro as b ein g ste re o c h e m ic a lly v ery m u c h sim ila r to th e d eg ratio n pro d u cts o f p h y tic ac id a fte r th e actio n o f w h e a t b ran p h y ta se (B re a rle y an d H a n k e , 1996).

A cid p lan t p h y ta s e m e d ia te d d eg ra d a tio n o f p h y tic acid to m ^ o -in o sito l ap p e a rs to b e rath er co m p lex an d h as b e e n stu d ie d in m o s t d etail fo r th e w h eat b ra n p h y ta s e (F ig u re 6). T hus, T o m lin so n a n d B a llo u re p o rte d th e su b strate sp e c ific ity o f a c ru d e w h e a t b ra n p h y tase

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p rep aratio n a lread y in 1961 an d 1962. F u rth e r c h a ra c te risa tio n w as p e rfo rm e d and su b seq u en tly th e w h e a t b ran p h y ta se w as re so lv e d into tw o fra c tio n s n a m e d F l an d F2 (Jo h n so n an d T ate, 1969; U m an d T ate , 1971; 1973). T h e c ru d e e x tra c t c h a ra c te rise d b y T o m lin so n an d B a llo u an d th e later F l fractio n y ield ed th e sam e p ro d u c ts from p h y tic acid, p erh ap s in d icatin g th a t th e F 2 fractio n w as a re la tiv e ly m in o r co m p o n en t.

Ins(l,5,6)P3 +

F l : InsP -► In s( 1,2,3,5,6)Ps -► Ins( 1,2 ,5 ,6 )? 4 -N n s ( 1,2,6)P3->-Ins( 1,2)P2 -► Ins(2)Pi - > I n s

■ ^-► 1 n s(l,2 ,3 ,6 )P 4 -^ In s(l,2 ,3 )P 3 -M n s(2 ,3 )P 2

In s(l,3 ,4 ,5 ,6 )P v

"'"-A F 2: 1nsP - ► In s (l ,2,3,5,6)Ps -► In s( 1,2 ,3 ,6 )P 4 -N n s(l ,2 ,3 )P 3 - ^ In s (l ,2 ) P 2 - > I n s ( 2 ) P i- ^ I n s

I n s ( l , 2 , 3 , 4 , 6 ) P 5 ^ ^ ^ Ins(2,3)P2

Figure 6. T h e h y d ro ly sis o f p h y tic acid (InsPe) to m>'o-inositol (In s), c a ta ly se d b y w h e a t bran p h y ta se fractio n 1 ( F l ) and fractio n 2 (F 2 ) resp ectiv ely .

A s illu strated in F ig u re 6, th e p h y tase activ ity o f F l in itia lly re m o v e s th e D -4 p h o sp h ate, y ield in g /wjyo-inositol 1 ,2 ,3 ,5 ,6 -p en ta/s5 p h o sp h ate. D u rin g th e su b s e q u e n t F l m e d ia te d step, tw o d iffe re n t te tra ^ /jp h o sp h a te s are g en erated , n a m e ly a m in o r co m p o n e n t, m ,yo-inositol

l,2 ,3 ,6 -tetra/a'5 p h o sp h ate an d a m a jo r p ro d u ct, w j^o-inositol 1,2,5,6-tetra/:i5phosphate. F u rth e r d e p h o sp h o ry la tio n y ie ld s w >'o-inositol 1 ,2 ,3 -trip h o sp h ate an d m j^o-inositol 1 ,2 ,6 -trip h o sp h ate (T o m lin so n an d B allo u , 1962). A^yo-inositol 1 ,2 -d ip h o sp h ate an d /o r m >'o-inositol 2 ,3- d ip h o sp h ate w e re id en tified w h en tre a tin g m^^o-inositol I,2 ,3 ,4 ,5 ,6 -h ex a/:i5 p h o sp h ate w ith th e c ru d e p h y ta se , an d it h as b e e n sh o w n th a t cru d e w h e a t bran p h y ta s e y ie ld s o n ly a sin g le myo- inositol m o n o p h o sp h a te , w j^o-inositol 2 -p h o sp h ate (Irv in g , 1980b). In a stu d y u sin g a co m m ercial a v a ila b le p re p a ra tio n o f w h eat p h y tase, m u ch o f th e ab o v e c o n c lu s io n s w as

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co n firm e d b y N M R , b u t ad d itio n a lly th e fo rm atio n o f /«;yo-inositol 1 ,5 ,6 -trip h o sp h ate from w y o -in o sito l 1 ,2 ,5 ,6 -te tra ^ jp h o s p h a te w a s rep o rte d (PhiU ippy, 1989). O n ly tw o m3'o -in o sito l te tra k isp h o sp h a te p ro d u c ts w e re c h aracterised , sin c e m y o-inositol 1,2,6-trip h o sp h ate an d myo­

inositol 1 ,2 ,3 -trip h o sp h ate w ere id en tified as p ro d u cts fo llo w in g th e a c tio n o f th e c ru d e ph y tase o n wi>'o-inositol 1,2,3,4,5,6-hexaÄ w phosphate as w ell (T o m lin so n an d B a llo u , 1962).

In c o n tra st to th e re s u lts o b ta in e d w ith the F l fractio n , th e F 2 fractio n o f w h e a t b ra n p h y ta s e gen erates th ree c h ro m a to g ra p h ic a lly d istin ct p eak s o f m jo -in o s ito l p e n ta ^5p h 0sp h ates (L im an d T ate, 1971; 1973), i.e., /wj^o-inositol 1 ,2 ,3,5,6-pentaÄ jjphosphate as th e m a jo r p ro d u c t an d sm aller a m o u n ts o f /M j'o-inositol 1,3,4,5 ,6 -p e n ta ^ jjp h o sp h a te an d » ij'o -in o sito l 1,2,3,4,6- p e n ta /n jp h o sp h a te . F u rth e r c a ta b o lism o f th e 1,2,3,5,6 an d 1,2,3,4,6 is o m e rs y ie ld e d th e sam e pro d u ct, w jvo-inositol 1 ,2 ,3 ,6 -te tra ^ ip h o s p h a te , w h ic h is th e sa m e is o m e r as th e m in o r m yo­

in o sito l tetraÅi5p h o sp h a te co m p o n en t p ro d u ced b y c ru d e w h e a t b ra n p h y tase. A sin g le trip h o sp h ate w as id e n tifie d as /wjvo-inositol 1,2 ,3 -trip h o sp h ate. F u rth e r b re a k d o w n y ie ld e d a m ix tu re o f m >'o-inositol 2 ,3 -d ip h o sp h a te an d /nj^o-inositol 1 ,2 -d ip h o sp h ate, w ith th e latter d o m in atin g , a n d fin a lly m >'o-inositol 2-m o n o p h o sp h ate and wi>'o-inositol.

T he b ro ad su b stra te affin ity o f th e acid p h y ta se s is c o n firm e d w h e n c o m p a rin g th e w h e a t b ran /b arley a leu ro n e in term ed iates w ith th e in term ed iates id e n tifie d in o th e r p la n t sp ecies. N o clea r co n sen su s fo r th e p a tte rn o f th e p en ta^ \y p h o sp h ates is p re s e n t in w h e a t, b a rle y , m u n g b ean an d so y b ean . In so y b ean , m yo-inositol 1 ,3 ,4 ,5,6-pentaA ijphosphate is re p re se n te d as in w h e a t and b a rle y , b u t a lo n g w ith m>’o -in o sito l 1 ,2 ,3 ,4 ,6 -p en ta^ isp h o sp h ate, it is as a m in o r co m p o n en t c o m p ared w ith m >'o-inositol 1,2,4,5,6-p enta^\yphosphate (P h illip p y an d B land, 1988). In m u n g b e a n seed lin g s, 1,2,4,5,6; 1,2,3,4,5; 1,3,4,5,6 a n d m3'o -in o sito l 1,2,3,4,6- pentaÄ i^phosphate w e re all id en tified , b u t th e latter tw o re p re se n te d less th a n 4 % to ta l m yo­

inositol pentaA zjphosphate in [^H] m yo-inositol o r [^^P]Pi- la b e lle d p re p a ra tio n s (S tep h en s, 1990; S tep h en s et al., 1991). In b a rle y m y o -in o sito l 1,2 ,3 ,4 ,5 - a n d -1 ,2 ,3 ,4 ,6 p entaÄ jjphosphate are th e d o m in a tin g p e n ta ^ ijp h o s p h a te w h ile m y o -in o sito l 1,2,3,4,6 p en ta^ i5phosphate is o n ly p re s e n t in sm all a m o u n ts in so y b ean an d m u n g b e a n seed lin g s c o m p ared to m y o -in o sito l 1,2,4,5,6 p en ta^7jp h o s p h a te an d m y o -in o sito l 1,2,3,4,5 pentaAi5p h o sp h ate.

T o g eth er, th ese d a ta in d icate lim ite d su b strate sp ecific ity fo r th e p h y ta s e s d u rin g th e in itial h y d ro ly tic step. T h is m a y also b e th e c ase fo r th e su b seq u en t steps. A lth o u g h m y o -inositol

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1,2,3,4 tetra/3 sp h o sp h ate an d m y o -inositol 1,2,5,6 tetraA i^phosphate are th e p re d o m in a n t fo rm s in b a rle y an d m u n g b ean , o th e r ty p e s o f tetra^j,yphosphates are g e n e ra te d in so y b ean seed lin g s an d w h eat. I f fo cu sin g o n w h eat an d b arley , m >'o-inositol 1,2,3 trip h o sp h a te and w>'o-inositol 1,2,6 trip h o sp h a te are h y d ro ly se d to th e d ip h o sp h a te s w >/o-inositol 1,2 d ip h o sp h ate an d » ij'o -in o sito l 2,3 d ip h o sp h a te an d fin ally to w iyo-inositol 2 p h o sp h a te (w h eat and b arley ) o r w y o -in o sito l 3 p h o sp h a te (barley).

A n alk alin e pH 8.0 p h y ta se activ ity h as b een id en tified an d e x te n siv e ly stu d ie d in p o lle n o f Lilium longiflorum (B ald i et al., 1988; B arrien to s, 1994). T h e in itial h y d ro ly sis o f the p h o sp h a te e ste r b y a lk a lin e p h y ta se o ccu rs at th e D -5 p o sitio n o f p h y tic a c id to y ield myo- inositol 1,2 ,3 ,4 ,6 -p e n ta /:jjp h o sp h a te (B a rrie n to s et al., 1994). T h e tw o su b seq u en t d ep h o sp h o ry la tio n s o c c u r ad ja c e n t to th e D -5 h y d ro x y l g ro u p to y ie ld w>>o-inositol 1,2,3- trip h o sp h ate as th e final p ro d u ct. T h e p h y sio lo g ic a l sig n ifican ce o f a lk a lin e p h y ta se s in relatio n to acid ic p h y ta s e s rem ain s to b e elu cid ated . L ik ew ise, it is n o t k n o w n i f m j^o-inositol 1 ,2 ,3 -trip h o sp h ate h as a b io lo g ical role.

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Role of Phytin and Phytase in Nutrition

The nutritional aspect o f feeding non-ruminants with high phytin diets

In the d ry seed a n d in th e d ig estiv e tract o f n o n -ru m in a n ts an im a ls th e re is o n ly a lim ited p h y tase activ ity (G ib so n an d U llah , 1990; L an tzsc h et al., 1992; U sa y ra n an d B a ln a v e , 1995).

R u m in an ts re a d ily u tilise th e p h y ta te p h o sp h o ru s o f seed s b e c a u se o f th e p h y ta s e p ro d u c e d b y ru m en m ic ro -o rg a n ism s. In co n trast, d ig estib le p h o sp h a te co m p o im d s h a v e to b e a d d ed to seed b ased feed fo r p ig s a n d p o u ltry to c o m p en sate fo r th e low d ig e s tib ility o f p h y tin . L arg e am oim ts o f p h y tin p h o sp h o ru s p ass th ro u g h th e g a stro in testin al tra c t an d o n ly 3 0 % o f th e ph o sp h o ru s eate n b y p ig s are d ep o site d d u rin g g row th. O f th e re m a in in g p h o sp h o ru s, 5 0 % is ex creted w ith th e m a n u re an d 2 0 % w ith th e u rin e, re fle c tin g u n u tilise d p h y ta te -P and su p p lem en tal in o rg an ic-P re sp e c tiv e ly (P o u lsen , 1996). T h e u n d ig e ste d p h y tin cau ses en v iro n m en tal p h o sp h o ru s p o llu tio n w h en th e m a n u re is u se d as fe rtilise r to cro p lan d s.

T h e stro n g a sso c ia tio n b e tw e e n p h y tate an d im p o rtan t d ietary ca tio n s re d u c e s th e ab so rp tio n o f th ese ele m e n ts in th e d ig estiv e tract o f n o n -ru m in a n ts (H u rrell et al., 1992). T h is is a m a jo r p ro b le m in th e d e v e lo p in g w o rld in p o p u latio n s feed in g p rim a rily o n c e re a ls w h e re iron, zin c an d p h o sp h a te d efic ie n c ie s c au se serio u s h e a lth p ro b lem s, in c lu d in g a n a e m ia ’s, rick ets, o steo p o ro sis an d b o n e d e fo rm itie s (L o tt, 1984). S ev eral stu d ies h a v e d o c u m e n te d th a t an en h an cem en t o f p h y tin p h o sp h o ru s u tilisa tio n also in creases th e a b so rp tio n o f i.e. zinc, m a g n e siu m a n d iro n (R o b e n so n an d E d w ard s, 1994; M u rry , 1995; S an d b e rg et a l , 1996).

S trateg ies fo r im p ro v in g th e b io a v a ila b ility o f p h o sp h o ru s fo r an im a l feed are a c c o rd in g ly also v ery re le v a n t fo r th e g en eratio n o f m in eral d en se food fo r h im ian c o n su m p tio n .

P h y tate h as fu rth er b e e n sh o w n to in h ib it p ep tic d ig e stio n o f p ro te in s a n d to in te rfe re w ith the a ctiv ity o f p ro te o ly tic en zy m e s, p ro b a b ly as a re s u lt o f th e fo rm atio n o f p ro te in /p h y tin c o m p lex es (L o tt, 1984).

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It h as b e e n k n o w n fo r sev eral y ears th a t a su p p lem en tatio n o f c e re a l d ie ts w ith p h y ta se o f Aspergillus niger N R R L 3135 en h an ces th e re le a se o f p h o sp h a te from p h y tin and c o n seq u en tly re d u c e s th e p h o sp h a te e x cretio n from n o n -ru m in a n ts (N e lso n et al., 1968;

1971). L a te r stu d ies o f b ro ile rs h a v e fu rth e r d em o n stra te d th a t th e a d d itio n o f e n z y m e even im p ro v es th e p e rfo rm a n c e c o m p ared to th a t attain ed b y a d d in g su p p lem en tal in o rg an ic p h o sp h ate (S im m o n s et al., 1990; Jo n g b lo ed et al., 1992).

T h e e n v iro n m e n ta l lo ad im p o sed b y th e ex cess p h o sp h a te in an im al m a n u re h as b e e n o n e o f th e d riv in g fo rces in th e d e v e lo p m e n t o f p h y ta s e as a feed ad d itiv e. E u ro p e a n c o u n trie s su ch as D en m ark , T h e N e th e rla n d s an d G erm an y , th a t all h av e larg e scale an im a l h u sb a n d ry , are n o w v ia le g islativ e m e a su re s try in g to b ala n c e th e n itro g e n an d p h o sp h a te in p u t w ith th at req u ired b y th e cro p s. F o r th e last 50 y ears m o re p h o sp h a te h a s b e e n ad d e d to th e c u ltiv ated so ils in D eru n ark th a n h as b e e n rem o v ed b y th e crops. F o rm erly , th is w as c o n sid e re d a m in o r p ro b lem sin ce p h o sp h a te is firm ly b o u n d in th e so il in an o rg an ic o r in o rg a n ic form . It is ap p aren t, h o w e v e r, th a t w a te r an d w in d d rain ag e and e ro sio n as w ell as d ra in a g e in p o o r soils resu lts in a sig n ific a n t tra n sfe r o f p h o sp h a te to sw eet w a te r as w ell as co astal areas, cau sin g m assiv e a lg a g ro w th an d a n a ero b ic co n ditions.

A p p licatio n o f p h y ta s e to an im al feed c a n d ra stic a lly re d u c e th e re q u ire m e n ts fo r a su p p lem en tatio n w ith d ietary p h o sp h a te and red u c e the p h o sp h a te ex cretio n . It h a s b een estim ated (T ab le 1) th a t i f p h y ta se w as u sed as a feed in g red ien t in th e d ie t o f all the m o m o g astric an im a ls in th e U n ited S tates, th e a m o u n t o f e x creted p h o sp h o ru s c o u ld be red u ced w ith a to ta l o f 8 .2 3 * 1 0 ’ k g p e r y e a r (W o d zin sk i an d U llah , 1996). T h e to ta l v a lu e o f th e p h y tin p h o sp h o ru s in th e an im al feed h a s b een estim a te d to a m o u n t 1.68*10* $ p e r y ear (d ata fo r 1993, W o d z in sk i an d U llah , 1996).

Application o f phytase to feed

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Table 1. E ffe c t o f u s e o f p h y ta se o n a b a te m e n t o f p h y ta te p o llu tio n (W o d z in sk i an d U llah , 1996).

Animal No. in US in 1992

Average live wt. (kg)

kg o f feed animal

g P e.xcieted animal if supplemented

w ith P

g P e.xcieted animal if supplemented

with pliytase

kg P year not excreted

Broilers 6.14*10’ 2.01 3.8 14.5 8.4 3.75*10’

Layers 3.64*10* 36.4/year 139/year 80.5/year 2.20*10’

Ducks 1.8*10’ 2.95 7.08 27 (estimate) 15.6/year 2.81*10*

(estimate)

Turkeys 2.89*10* 9.91 26.4 101 (estimate) 58.5 (estimate) 1.69*10’

Pigs 5.78*10’ 80.4 265 271 (estimate) 177 5.62*10‘

Total - - - 8.23*10’

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Increasing the Phytase Activity of Plants by Transformation

Transformation o f plants with phytase genes for improved growth performance of non­

ruminants

T he su ccess o f u sin g p h y ta s e as a feed a d d itiv e h as stim u lated rese a rc h to g e n e ra te tra n sg e n ic p lan ts w ith a h ig h e r p h y ta s e p o ten tial in th e m atu re seed. A tra n sfo rm a tio n stra te g y ad d ressed to the o v e re x p re ssio n o f a h o m o lo g o u s o r h e te ro lo g o u s p h y ta se g en e in p la n t seed s c o u ld lead to an in crease in th e b io a v a ila b ility o f th e p h o sp h o ru s an d essen tial m in e ra ls o f th e seed.

R esu lts o b tain ed in to b a c c o an d w h eat in d icates th a t tra n sfo rm a tio n w ith th e A spergillus niger p h y ta se e n co d in g g e n e ph yA , d riv e n b y a co n stitu tiv e p ro m o te r c a n in c re a se th e to tal p h y tase a ctiv ity in th e seed s (P en et a i , 1993; B rin c h -P e d e rse n et al., 2 0 0 0 a). S eed s o f tra n sg e n ic p lan ts e x h ib ite d n o rm a l p h e n o ty p e ev en w h e n th e p h y ta s e en z y m e ac c im iu la te d in a m o u n t up to 1% o f th e so lu b le p ro te in in th e seed. T h e en zy m e sh o w e d to b e sta b le sin c e sto ra g e o f tran sg en ic seed s, in tact o r m illed , fo r up to 12 m o n th s at 4°C o r ro o m te m p e ra tu re c a u se d no sig n ifican t d e c re a se in p h y ta s e activ ity level. In a b ro ile r feed in g trial, th e p h y ta se a ccu m u lated in to b a c c o seed s p ro v ed to b e effectiv e fo r in creasin g th e b io a v a ila b ility o f p h o sp h ate (P en et al., 1993). T h e ad d itio n o f m ille d tra n sg e n ic seed s, A spergillus niger p h y tase o r in o rg a n ic p h o sp h a te h a d co m p a ra b le effe c ts o n th e g ro w th rate (T a b le 2). In tran sg en ic w h e a t g rain s, in vitro stu d ies h av e showTi th a t th e h e te ro lo g o u s p h y ta s e is cap ab le o f red u cin g th e a m o u n ts o f p h y tic acid and lo w er w j^o-inositol p h o sp h ates. C o n se q u e n tly the a m o u n t o f in o rg a n ic p h o sp h a te is in creased sig n ific a n tly (B rin c h -P e d e rse n et al., 2 0 0 0 b ).

R ecen t stu d ies h a v e fu rth er d o c u m en ted th a t a fully fu n ctio n al A spergillus niger p h y ta s e can be accu m u lated in tra n sg e n ic a lfa lfa seed s an d so y b e a n s (B e u d e k e r an d P en , 1994; D e n b o w et al., 1998). In ad d itio n , Aspergillus niger p h y ta se h as b e e n sy n th esised a n d sto red in an active form in to b acc o an d c a n o la leav e s and in so y b ean c e ll su sp en sio n c u ltu re s (V e rw o e rd et al., 1995; K o eg el et al., 1997; Li et a l , 1997).

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Table 2. E ffe c t o f p h y ta se -tra n sg e n ic seeds on p erio d (P en et al., 1993).

th e g ro w th rate o f b ro ile rs o v e r a 4 -w e e k

D ietary Supplem entation'

Phosphate" Phytase Acti\ ity.’ (F' (g leg diet) diet)

r u kg

0-2

Growth (g) Age (weeks)

2-4 0-4

None 5.0

_

203‘ 492'» 695'

Inorganic P 6.5 - 314“ 729» 1043“

A. niger pbytase 5.0 202 276'’ 665* 941'’

A. niger phytase 5.0 404 293’" 692“ 985*'’

C ontrol seeds'* 5.0 - 193' 515'’ 708'

T ransgenic seeds'*"* 5.0 295 299* 693* 992*'’

S tat. signiflcance o f difference: P< 0.001 0.001 0.001

s e d‘ 13.2 34.8 44.7

'Differences in the amount o f diet, caused by the supplementation was not compensated. The highest dietary supplementation’s were 33 g/kg feed (diets 5 and 6), while maximally 8 g/kg was added in the other diets.

^The basal diet contains 3.3 g/kg plant phytate and 1.7 g/kg o f available phosphorus.

^Phytase activity was determined in the supplement prior to addition to the diet.

^Seeds were milled and added as such.

’Seeds o f this line had an expression level o f 0.4% o f soluble protein.

‘SED, standard error o f differences between two means.

“■'’■‘Values within a column having identical superscript letter are not significantly different.

Effect o f transgenic phytase seeds on phosphorus excretion from non-ruminants

T he effect o n p h o sp h o ru s e x c re tio n w as asse sse d in a feed ex p e rim e n t w h e re b ro ile rs w e re fed tran sg en ic so y b e a n s co n ta in in g p h y ta s e (D en b o w et al., 1998). T h u s, 1 200U p h y ta s e a c tiv ity p e r k ilo o f tra n sg e n ic so y b e a n s c au sed a b o u t 5 0% re d u c tio n in th e p h o sp h o ru s ex cretio n , w h en co m p a re d to a d ie t su p p le m e n te d w ith an in term ed iate lev el (0 .1 6 % ) o f d ie ta ry n o n p h y tate p h o sp h o ru s (T a b le 3). In a d d itio n , the e x cretio n o f p h o sp h o ru s w a s in th e exp erim en ts w ith th e tran sg en ic so y b ean s re d u c e d o n av e ra g e 11% w h e n c o m p a re d to exp erim en ts w h e re feed b a se d o n w ild ty p e so y b ean w as ad d e d a c o m m e rc ia l m icro b ial d eriv ed p h y ta se (N atu p h o s). T h e re d u c e d e x cretio n reflects an a p p ro x im a te ly 10% in c re a se d

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phosphorus digestibility o f the feed with the endogenous phytase compared with feed supplemented with phytase. The results illustrate that diets low in non phytate phosphorus but with endogenous phytase activity ensure growth rates similar to that o f diets supplemented with inorganic phosphorus. Moreover the bioavailability o f phosphate in the former type o f feed was higher than when using the same feed supplemented with exogenous commercially available phytase from Aspergillus niger phytase (Natuphos).

Table 3. Effect o f feeding phytase transformed soybean (TSB) or Natuphos phytase on growth, phosphorus digestibility and excretion (Adapted from: Denbow et al., 1998).

Diets BW'' yaiii week

2 to

Feed intake week 2 to 3

Gain: Teed week 2 to 3

Pliosplioriis Diyeslibility

Phosphonis excretion

(g) (g) (g/kg) (% o f intake) (g/kg DM*’ intake)

Basal 407 618 658 47.2 2.56

B4<).08P 436 669 650 42.8 3.01

B+0.16P 487 739 659 44.0 3.56

B+0.24P 488 749 649 40.0 3.90

B+400U phytase TSB

435 643 678 55.8 2.15

B+800U phytase TSB

451 707 638 60.4 1.98

B+1200U phytase TSB

474 720 658 61.6 1.82

B+400U phytase N atuphos

416 616 677 50.6 2.34

B+800U phytase N atuphos

440 686 641 53.3 2.24

B+1200U phytase Natuphos

454 710 639 55.1 2.10

*BW, body weight

d m, diy matter

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Heat stability

Currently available industrial phytases all originate from Aspergillus niger. The temperature profile o f the enzyme is asymmetric. It is sluggish from 20°C to 30®C and displays its highest activity at 58®C. The activity rapidly declines at 65®C and at 6 8®C only 2% o f its activity is retained (Gibson and Ullah, 1990; Engelen et al., 1994). The thermal inactivation appears however, to be partly transient since a subjection to 6 8®C for 10 min and subsequent transfer to 58®C resulted in regaining o f 40% o f the original activity (Gibson and Ullah, 1990). The limited thermostability is a drawback o f the phytases originating from A spergillus niger since modem feed-pelleting processes utilise a heat treatment (Gibson, 1995).

However, in plan ta synthesised Aspergillus niger phytase shows altered thermal stability when compared to the isolated fimgal derived enzyme (Li et al., 1997). After preincubation at 63®C for 10 min, 90% o f the activity remained for the recombinant phytase, whereas only 75% remained for the fungal phytase. Above 63®C, activity declined rapidly for the fimgal and the recombinant enzyme. At 70®C, the recombinant enzyme still retained about 20% o f the activity while the fimgal enzyme was completely inactivated. Prolonged incubation (63°C for 1 hr), resulted in a 60% loss o f activity for the fimgal enzyme compared to 2 0% loss of activity for recombinant phytase. It was suggested that the changes in thermal stability o f the recombinant enzyme might be attributed to the addition o f two amino acids at the translational fiision site or differences in glycosylation between phytases synthesised in plan ta and in fimgi (Li et al., 1997). Further studies may reveal the mechanisms determining the improved thermostability o f the plant derived phytase.

Phytases with superior heat stability has recently been described from Aspergillus fum igatus and Thermomyces lanuginosus. The A. fum igatus enzyme was able to withstand temperatures up to 100°C for 20 min while loosing only 10% o f the initial enzymatic activity (Pasamontes et al., 1997). In addition, the enzyme showed high activity at a pH range o f 2.5 to 7.5. The high thermostability o f this enzyme is remarkable and similar phytases or acid phosphatases have so far not been described. Thus, Thermomyces phytase retained activity at assay temperatures only up to 75°C (Berka et al., 1998).

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Baldi, B.G. Scott, J.J., Everand, J.D. and Loewus, F.A. (1988). Localization o f constitutive phytases in lily pollen and properties o f the pH 8 form. Plant Sei., 56, 137-147.

Barrientos, L., Scott, J. J, and Murthy, P.P.N. (1994). Specificity o f phytic acid by alkaline phytase fi-om lily pollen. Plant Physiol., 106, 1489-1495.

Berka, R. M., Rey, M. W., Brown, K. M., Byun, T. and Klotz, A. V. (1998). Molecular characterization and expression o f a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl. Environ. Microbiol., 64, 4423-4427.

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