Ms. 4434 ASSAY AND PROPERTIES OF ALGINATE LYASE AND l, 3-13-GLUCANASE IN INTERTIDAL SANDS by M. WAINWRIGHT Department of Microbiology, University of Shefh'eld, SIO 2TN, U.K. KEY WORDS Accumulated enzymes Alginate degradation Laminarin Soil enzymes SUMMARY Alginate lyase and 1, 3-13-glucanase activity were detected in intertidal sands below decomposing seaweeds (Fucus sp. and Laminaria sp). Linear relationships between activity and sand w eight; ength of incubati on and substrate concentration, were established for both enzymes. O ther properties of these enzymes in intertidal sands are reported. INTRODUCTION Little is know n about the factors influencing the degradation of seaweeds, even though large amounts are deposited on beaches throughout the world. Seaweeds contain numerous polysaccharides such as alginates and laminarin which are enzymatically degraded by micro-organisms s. Alginic acid and its salts are linear block copolymers composed of D-man- nuronic and L-guluronic acid s, which function as food reserves in brown seaweeds, comprising between 14-40~o of their weight. They are depolymerized by the enzyme alginate lyase which occurs in bacteria 1~ in the hepatopancreas of abalone v, and in brown seaweeds 6. Alginate lyase has not previously been assayed and characterized in soils or marine environments. Laminarin, a water soluble [3-glucan, also occurs as a reserve material in brown algae, but generally in larger am ounts than alginates s. It is hydrolized by the enzyme complex laminarinase (laminarase), which comprises exo and endohyd- rolytic 1, 3-[3-glucanase and [3-glucosidase 2. Laminarinases occur in a wide range of organism s including algae; bacteria; fungi; higher plants and molluscs 2 and have also been found in soils 4. This paper reports investigations to determine if sands on beaches where brown algae are decomposing, possess accumulated alginate lyase and 1, 3-[3- 8 3 Plant and Soil 59, 83-89 (1981). 0032-079X/81/0591-0083501.05.
Transcript
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
A S S A Y A N D P R O P E R T I E S O F A L G I N A T E L Y A S E A N D
l , 3 -1 3 - G L U C A N A S E I N I N T E R T I D A L S A N D S
b y M . W A I N W R I G H T
Department of Microbiology, University of Shefh'eld, SIO 2TN , U .K.
K E Y W O R D S
A c c u m u l a t e d e n z y m e s A l g i n a te d e g r a d a t i o n L a m i n a r i n S o il e n z y m e s
S U M M A R Y
Alginate ly ase and 1, 3-13-glucanase activity w ere detected in intertidal sand s bel ow decom posingseaweeds (Fucus sp. and Laminaria sp). Linear relationships between activity and sand w eight; engthof incubation and substrate concentration, w ere established for both enzym es. O ther properties ofthese enzyme s in intertidal sand s are reported.
I N T R O D U C T I O N
L i t tl e is k n o w n a b o u t t h e f a c t o r s in f l u e n c i n g t h e d e g r a d a t i o n o f s e a w e e d s , e v e n
t h o u g h l a r ge a m o u n t s a r e d e p o s i t e d o n b e ac h e s t h r o u g h o u t t h e w o r l d . S e aw e e d s
c o n t a i n n u m e r o u s p o l y s a c c h a r i d e s s u c h a s a l g in a t es a n d l a m i n a r i n w h i c h ar e
e n z y m a t i c a l ly d e g r a d e d b y m i c r o - o r g a n i s m s s.
A l g i n ic a c id a n d i ts sa lt s a re l in e a r b l o c k c o p o l y m e r s c o m p o s e d o f D - m a n -
n u r o n i c a n d L - g u l u r o n i c a c i ds , w h i c h f u n c t i o n a s f o o d r es e rv e s in b r o w n s e a w e e ds ,
c o m p r i s i n g b e t w e e n 1 4 -4 0 ~ o o f t h e ir w e i g ht . T h e y a r e d e p o l y m e r i z e d b y t he
e n z y m e a l g i n a t e l y a se w h i c h o c c u r s i n b a c t e r ia 1~ i n t h e h e p a t o p a n c r e a s o f
a b a l o n e v, a n d i n b r o w n s e a w e e d s 6.
A l g i n a t e l y a se h a s n o t p r e v i o u s l y b e e n a s s a y e d a n d c h a r a c t e r i z e d i n s oi ls o r
m a r i n e e n v i r o n m e n t s .
L a m i n a r i n , a w a t e r s o l u b l e [ 3 -g lu c an , a l s o o c c u r s a s a r e s e rv e m a t e r i a l i n b r o w n
a l g a e , b u t g e n e r a l l y i n l a r g e r a m o u n t s t h a n a l g i n a t e s s . I t is h y d r o l i z e d b y t h e
e n z y m e c o m p l e x l a m i n a r i n a s e ( l am i n a r as e ), w h i c h c o m p r i s e s e x o a n d e n d o h y d -
r o l y t i c 1 , 3 - [ 3 - g lu c a n a s e a n d [ 3 - g lu c o s i d a se 2 . L a m i n a r i n a s e s o c c u r i n a w i d e r a n g e
o f o r g a n i s m s i n c l u d i n g a l g a e ; b a c t e r i a ; f un g i ; h i g h e r p l a n t s a n d m o l l u s c s 2 a n d
h a v e a l s o b e e n f o u n d i n s o il s 4.
T h i s p a p e r r e p o r t s i n v e s t ig a t i o n s t o d e t e r m i n e i f s a n d s o n b e a c h e s w h e r e
b r o w n a l g a e a r e d e c o m p o s i n g , p o s s e s s a c c u m u l a t e d a l g i n a t e l y as e a n d 1, 3 -[3-
8 3
Plant and S o i l 5 9 , 8 3 - 8 9 ( 1 98 1 ). 0 0 3 2 - 0 7 9 X / 8 1 / 0 5 9 1 - 0 0 8 3 5 0 1 . 0 5 .
9 Ma rtinus N ijho ff/D r. W. Ju nk b.v. Publishers , The Hague, Printed in Th e Netherlands.
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
g l u c a n a s e a c t iv i t y , a n d i f s o , t o c o m p a r e t h e i r p r o p e r t i e s w i t h t h o s e o f t h e s a m ee n z y m e s d e r i v e d f r o m o t h e r s o u rc e s .
M E T H O D S
Surface samples (top I0 cm) of sand w ere taken f rom bene ath d ecaying seaweeds at Red W harfe Bay,
Anglesey, U.K. (Fucus spiralis L.) dur ing Oc tob er 1979 and f rom G ibral ta r Po int , L incolnshire , U.K.
(Fucus spiralis an d Iaminaria digitata Huds . La mo ur) dur ing Jan uary 1980. The Anglesey sand was
gritty in textu re, 15.6% w/w calcareou s, with a pH of 7.6 (glass electrod e, 1:10 sand: wate r ratio). The
Gi bral tar Poi nt sand had a much smaller par t ic le s ize ( < 1 ram) than the Anglesey sand, was 9;5% w/w
ca lcareous and had a pH of 7 .0 .
E n z y m e a s s a y s
The < 1 mm fract ion of the Anglesey sand was used for the develo pm ent of the alginate lyase assay.
Sand (5 g) was weighed into 20 ml U niversa l bot t les and 2 ml of 0 .1% w /v sodium azide was added (as
bacter ios tat) . After 15 mins preincu bat ion, the enz yme react ion was s tar ted b y adding 1 ml of sodium
alginate (contain ing 501a moles uron ic acid equivalen ts /ml. l~ 1 ml KC I (0 .01M) and 5 ml of Tr is
ma leate (0 .1 M) buffer (pH 7.0) . Co ntro l react ions lac ked subs trate or w ere stopped imm ediately af ter
subs trate was added. The react ion mixtures were incub ated for 5 h at 35~ when the react ion was
s topped by add ing 10 ml t r ichlora cet ic acid (10% w/v). F i l t rate (1 ml) was then reacted with H IO 4
(1.25 ml) and assayed for 13 formyl pyru vate us ing the th ioba rbi tur ic ad d assay x0, exqept th at 2.5 mls
of sodium arseni te and 10 mls of th iob arbi tur ic acid were used.1 , 3-13-glucanase act iv i tyw as assayed us ing the m eth od of Lethbr idge et al.4, except that g lucose
was determ ined us ing glucose oxidase -peroxida se reagent 5 , and the react ion mixtures were no t
shaken. Pho spha te buffer (0.01 M) was used to d etermin e the pH act ivi ty curve of the enzyme. Tr i-
p l i ca tes were used th rou ghou t .
R E S U L T S A N D D I S C U S S I O N
A l g i n a t e s c o n s i s t o f [3-1, 4 - D m a n n u r o n a n a n d m - l, 4 - g u l u r o n a n , w h i c h a r e
d e p o l y m e r i s e d b y a l g i n a t e l y a s e t o f o r m o l i g o s a c c h a r i d e s c o n t a i n i n g a n u n -
s a t u r a t e d u r o n i c a c i d a t t h e n o n - r e d u c i n g e n d , a n d 4 - d e o x y - L - e r y t h r o - 5 - h e x o s e -u l o s e u r o n i c a c i d . T h e b a s is o f t h e a s s a y u s e d h e r e t o m e a s u r e a l g i n a t e l y a s e i s
t h a t t h e p r o d u c t s o f t h e d e p o l y m e r i s a t i o n , b u t n o t s u b s t r a t e , r e a c t w i t h H I O 4 t o
f o r m [3 f o r m y l p y r u v a t e w h i c h is t h e n m e a s u r e d c o l o r i m e t r i c a l l y .
A l g i n a t e l y a s e a c t i v i t y w a s d e t e c t e d i n s a n d s f r o m A n g l e s e y b u t n o t G i b r a l t a r
P o i n t . S y n t h e s i s o f t h e e n z y m e d i d o c c u r , h o w e v e r , w h e n s a m p l e s o f t h e l a t t e r
s a n d w e r e a m e n d e d w i t h 1~o w / v a l g i n i c a c i d a n d i n c u b a t e d a t 25 0 C fo r 1 0 d a y s .
L i n e a r r e l a t i o n s h i p s b e t w e e n a l g i n a t e l y a s e a c t i v i t y a n d w e i g h t o f s a n d ( u p to
8 g) ; l e n g t h o f i n c u b a t i o n ( u p t o 8h ) a n d s u b s t r a t e c o n c e n t r a t i o n ( u p t o 1 0011 m o l e s
u r o n i c a c i d e q u i v a l e n t s / m l ) w a s d e m o n s t r a t e d i n t h e A n g l e s e y s a n d ( F ig . 1)s h o w i n g t h a t t h e a s s a y is r e p r o d u c i b l e .
T h e o p t i m u m p H f o r c e l l -f r e e a l g i n a t e l y a s e i n s a n d w a s 7 .0 ( F i g . 2 ), t h e s a m e a s
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
E N Z Y M E S A N D S E A W E E D D E G R A D A T I O N 85
4 8
4 0 Q
32
> 2 4
1 6
o ~ ~ ~ ~ T i m eI
2 ~ 5 '0 7 ~ 1 ~ 0 w t s a . d
Subst rate
F i g . 1 . E f f e ct o f l e n g t h o f i n c u b a t i o n ( h ), ( [ ] ) ; w e i g h t o f s a n d ( g), ( 9 a n d s u b s t r a t e c o n c e n t r a t i o n
( ~ tm o l es u r o n i c a c i d e q u i v a l e n t s a d d e d / m l ) , ( 9 o n a l g i n a t e l y a s e in A n g l e s e y s a n d ( A c t i v i ty e x p r e s s e d
n m o l e s 13 f o r m y l p y r u v a t e f o r m e d / g / h ) .
t h a t q u o t e d f o r a lg i n a t e l y as e f r o m b a c t e r i a a n d s e a u r c h i n s 8. T h e o p t i m u m
t e m pe ra t u re fo r a c t i v i t y w a s be t w e e n 30 -35 ~ (F ig . 2 ), w h i c h is s i mi l a r t o t he
o p t i m u m r e p o r t e d f o r th e e n z y m e in m a r i n e p s e u d o m o n a d s 3.
A l g i na t e l ya s e i n s a nd w a s a c t i va t e d b y K C 1 up t o 50 m M (F i g . 2 ), t ha t i s t he
s a m e c o n c e n t r a t i o n r e q u i r ed f o r m a x i m u m a c ti v it y o f a b a l o n e h e p a t o p a n c r e a s
a n d m a r i ne ba c t e r i a l a l g i na t e l ya s e s 7 ,1~ H i g h i on i c s t r e ng t hs o f K C 1 a r e a p -
p a r e n t l y r e q u i re d f o r m a x i m u m a c t iv i ty o f a l g in a t e l ya s e f r o m a b a l o n e h e p a t o -
p a n c r e a s b e c a u s e t h e y r e m o v e b o u n d w a t e r f r o m a l g in a t e a n d n e u t ra l is e n e g a ti v ec h a r g e s o n t h e p o l y a n i o n , r e n d e r i n g th e m o l e c u l a r c o n f i g u r a t i o n o f t h e s u b s t r a t e
s u i t ab l e f o r e n z y m e a t t a c k ~. C o n t i n u a l b a t h i n g o f s e a w e e d a n d u n d e r l y i n g s a n d
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
Tem perature, (0 ); pH -activity curves, (9 and activation of alginate lyase by KC1 (Activityexpressed n m ole 13 form yl pyruvate formed/g/h).
b y s e a w a t e r, p r e s u m a b l y s a t u r a t e s b o t h e n z y m e a n d s u b s t r a te , w i t h a s o l u t io n o f
s u ff ic ie n t s t r e n g t h t o a c t i v a t e t h e a c c u m u l a t e d e n z y m e .
1 , 3 - [ 3 - g l u c a n a s e a c t i v i t y w a s d e t e c t e d i n G i b r a l t a r P o i n t , b u t n o t A n g l e s e y
s a n d s . L i n e a r i t y w a s e s t a b l is h e d b e t w e e n e n z y m e a c t iv i t y a n d w e i g h t o f s a m e ( u pt o 7 g); l e n g t h o f i n c u b a t i o n ( u p t o 5 h ) a n d s u b s t r a t e c o n c e n t r a t i o n u p t o 1 00
m g / m l ) ( F i g . 3 ) .
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
Fig. 3. Ef fectof length of incubation (h), ([]); weight of sand (9), (9 and substrate co ncentration(mg/m l laminarin), (11) on 1, 3-13-glucanase activity in G ibraltar Po int sand (Activity expressed ~t
mo les x 10 2 glucose released/g/h).
T h e o p t i m u m p H f o r 1 , 3 - 1 3 -g l u c an a s e a c t i v i t y i n s a n d w a s 5 . 0 ( F i g . 4 ), t h e s a m e
a s t h a t q u o t e d f o r e x o - 1 , 3 - [ ~ - g lu c a n a s e in f u n g i a n d t h e e n z y m e i n s o il 2 '4 . T h e
t e m p e r a t u r e o p t i m u m f o r t h e 1, 3 - 13 - gl u ca n as e a c t i v i ty in s a n d w a s 4 5 ~ ( F ig . 4),
w h i c h i s l o w e r t h a n t h a t q u o t e d f o r so il 4, b u t s i m i l a r t o t h a t o f f u n g a l e n d o - 1 , 3 -[3-g l u e a n a s e 2.
T h e e n e r g y o f a c t i v a t i o n o f 1, 3 - 13 - gl uc a n as e i n s a n d w a s c a l c u l a t e d u s i n g t h e
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
Te m per a t u re ( e ) an d pH ac t i v i ty cu rve , (A ) fo r 1 , 3 -!3-gl ucanase ac t i v i ty i n G i b ra l t a r P o i n t
s and (Ac t i v it y exp res s ed g m o l es x 10 -2 g l ucos e r e l eas ed / g /h ) .
A r r h e n i u s e q u a t i o n a n d w a s f o u n d t o b e 5 9 k J / m o l , w h i c h i s h i g h e r th a n t h e 4 9
k J / m o l r e p o r t e d b y L e t h b r i d g e e t a I . 4 f o r t h e s a m e e n z y m e in s o il .
S h a k i n g G i b r a l t a r P o i n t s a n d w i t h b u f f e r ( 10 g s a n d t o 2 0 m l b u f f e r , s h a k e n f o r
1 5 m i n , 1 00 t h r o w s m i n ) r e m o v e d t h e e n z y m e f r o m t h e s an d . L i n e a r i t y b e t w e e n
v o l u m e o f b u f f e r e x t r a c t a n d e n z y m e a c ti v i ty w a s t h e n d e m o n s t r a t e d .1 - 3 - [3 - g l u c an a s e a c t i v i t y w a s f o u n d i n a , w i d e r a n g e o f sa n d s , w h i l e a l g i n a t e
l y a s e a c t i v i t y w a s d e t e c t e d o n l y in t h e s a n d s f r o m R e d W h a r f e B a y , A n g l e s e y.
S i m i l a r l y , 1 , 3 - [ 3 -g l u c an a s e a c t i v i t y c o u l d b e d e t e c t e d i n a w i d e r a n g e o f t e r r e s t r i a l
s o i l s , w h i l e a l g i n a t e l y a s e c o u l d n o t . T h i s r e f l e c t s t h e w i d e a v a i l a b i l i t y o f 1 3 -
g l u c a n s b u t p a u c i t y o f a l g i n a t e s i n s o il s, d e s p it e t h e f a c t t h a t t h e s e p o l y s a c -
c h a r i d e s a r e s y n t h e s i s e d b y b o t h s o il a n d m a r i n e b a c t e r i a 9.
A p p r e c i a b l e a c t i v it y o f t h e t w o e n z y m e s w a s f o u n d o n l y i n s a n d s o v e r l a i n w i t h
d e c o m p o s i n g s e aw e e d , s o t h a t t h e m a i n s o u r c e s o f t h e e n z y m e s a re p r o b a b l y
m i c r o - o r g a n i s m s T M , o r d e c a y i n g s e a w e e d 6 '8 . O n c e c e ll -f r ee , t h e e n z y m e s a r el i k e ly to b e a d s o r b e d o n t o t h e s u r f a c e o f s a n d , w h e r e t h e y c a n b e a c t i v e i n
d e g r a d i n g t h e s u b s t r a t e s . E x p e r i m e n t s w i t h b u f f e r a s a n e x t r a c t a n t o f 1 , 3 - [ 3 -
8/8/2019 Wainwright_Assay and Properties of Alginate Lyase
g l uc a na s e sugge s t , ho w e v e r , t h a t su c h a d so r p t i o n i s l i ke ly t o be r e l a ti ve l y l oose . Itis a l s o o f i n t e r e st t h a t m a r i n e s a n d s h a v e b e e n e v a l u a t e d a s s u p p o r t s f o r a v a r i e ty
o f e n ym e s i m m ob i l i z e d f o r i ndu s t r i a l p u r po se s 1. Bo t h a l g i na t e s a nd 1, 3-13-
g l u c a n s a r e w a t e r s o l u b l e a n d a r e l ik e l y t o b e l e a c h e d f r o m s e a w e e d s b y s e a w a t e r
i n t o un de r l y i n g sa nds . H e r e t he e n z ym e s a l g i na t e l ya se a n d l , 3 -13- gluca na se c a n
p a r t i c i p a t e i n t h e d e g r a d a t i o n o f th e s e p o l y s a c c h a r i d e s , r e le a s in g o l i g o s a c c h a -
r id e s a n d g l u c o s e i n t o s o l u t io n . A l g i n a t e g el s m a y p l a y a r o l e i n t h e b i n d i n g o f
m i c r o - a g g r e g a t e s o f ' m a r i n e s a n d s , t h e r e b y h e l p i n g t o s t a b i li z e b e a c h es . A l g i n a t e
l y as e b y b r e a k i n g d o w n s u c h g els , m a y h i n d e r m i c r o - a g g re g a t e f o r m a t i o n a n d
r e d u c e t h e s t a b i li t y o f t h e s y s t em .
Received 28 May 1980. Revised September 1980.
REFERENCES
1 Brotherton, J., Emery, A. and Rothwell, V.W. 1970 Characterisation of sand as a support for
2 Bull, A. T. and Chesters, C. S. C. 1966 The biochemistry of laminarin and the nature oflaminarinase. Adv. Enzymol. 28, 325-364.
3 Davidson, I. W., Sutherland, I. W. and Lawson, C. J. 1976 Purification and properties ofalginate lyase from marine bacteria. Biochem. J. 159, 707-713.
4 Lethbridge, G., Bull, A. T. and Burns, R.G . 1978 Assay and properties of 1, 3-13-glucanase in
soil. Soil Biol. Biochem. 10, 389-391.
5 Lloyd, J. B. and Whelan, W.J. 1969 An improved method of enzymic determination of glucose
in the presence of maltose. Anal. Biochem. 30, 467470.
6 Madwick, J., Haug, A. and Larsen, B. 1973 Alginate lyase in the brown alga. L a mi n a r i a
d ig i ta ta (Huds) Lamour. Acta Chemica. Scand_ 27, 711-712.
7 Nakada, H. 1. and Sweeney, P.C. 1967 Alginic acid degradation by eliminases from abalone
hepatopancreas. J. Biol. Chem. 242, 845-857.
8 Percival, E. and McDowell, R. H. 1967 Chemistry and Enzymology of Marine Algal Polysac-
charides. Academic Press, London, 219 p.
9 Pinder, D. F. and Bucke, C. 1978 The biosynthesis of alginic acid by Azo toba cter v ine land ii .Biochem. J. 152, 617-622.
10 Preis, J. and Ashwell, G. 1962 Alginic acid metabolism in bacteria. J. Biol. Chem. 237, 309 316.
11 Wainwright, M. 1980 Alginate degradation by the marine fungus Dendryphiel la sal ina. Mar.Biol. Lett. In Press .
