Some properties of chitinase from Phycomyces blakesleeanus

Life Sciences Vol . 15, pp . 289-300 Printed in the U. 5.1~.

Pergamon Press

SOl~ PROPSRTIBS OF CHITIäA.SB FRO~t PHYCOMYCSS BLARSSLRSANDS ROBERT J. COHSN Departsknt of Biocheaistry, College of Medicine, University of Florida Gainesville, Florida 32610 (Received in final form 17 June 1974) Sill4lARY The cytosol of the aporangiophore of Phycooyces blakesleeanus has considerable chitinolytic activity . This activity ie strongly dependent on the presence of a dialysabla activator . ~=i .at activity ie achieved at pE 5 .5 ; and ionic etreagth and Cam or Mgt have little effect . Ungeroinated spores do not contribute activity . The possibility is discussed that thinness eight be involved in the growth response systaa by traasieatly loosening the rigid fraaework of chitin at specific and defined points . TNIRODIICTIOM Phycoaycas blaknelaeanus has long been studied ae a aodel sgstea for sensory traasduction .

The sporangiophore responds to light, to stretch, to

gravity and to a volatile cheaical regulator by transiently increasing its growth rata (1) .

The transient light growth responses have been characterised

poet recently by Foster and Lipsoa (2) and the behavior of several mitante by Bergman, Esldva, and Cerclé-0]sedo (3) .

The etiaulus~responee eyetes aast

involve an input, on the molecular scale, the photoreceptor for the light response, a transduciag syetea, perhaps involving several intermediates possibly including a cyclic nucleotide, and an output, the increased grrnrth rate of the sporangiophora .

This realisation has led to a search for the blue

absorbing photopigsient, so far unsuccessful, and to the elegant work of Y . N. Jan describing chitin synthetase possibly the output enzyae (4) . The actual output, hovevér, say not be so staple .

The cell wall of

Phycoayces sporangiophore ie about 40X by dry weight, in equal parts, chitin (poly-B (1 + 4) P-acetyl-D glucosaaine) and its non-scetylated form (5, and unpublished biochemical investigation of the author) . 289

The chitin is arranged

290

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Phycomycea Chitinase

as aicrofibrils embedded in an amorphous matriz and is presumed to render rigidity to the sporangiophore .

Chitin is a major component of the cell wall

of many fungi sad the thought has been resurrected many times (6) that elongation of hyphal tips is regulated via a symbiotic relationship between chitin synthetase and chitinase,

the chitinolytic enzyme .

The region of growth of

Phycamycea stage IDb sporaagiophore is not at the hyphal tip but is an area eaending from about 0.2 mm below the sporangium to about 3 mm below (the growing zone) .

Our present aim in this preliminary report ie to establish the

preaeace of chitiaase (EN 3.2 .1 .14) in the sporangiophore of Phycomyces and remark on its possible role in the regulation of the growth output . MATERIALS AND MBTHODS Isolation.

Phycomyces blakesleeaaus , NRRL1555, ie grown oa com-

mercial instant potatoes in large trays, 30 cm a 50 cm .

Synchrony of

sporangiophore development is achieved by a light-step up .

The mature stage

IVb sporangiophores (3 .5 t 0 .5 cm) are harvested in the cold, cut into 3 mm pieces into a mortar containing an equal volume of 25 mM phosphate buffer, pH 6.5, sad grinded with a pestle .

To remove the cell wall debris, the

material ie filtered through three layers of cheesecloth . grinding, and filtering are repeated .

The cutting,

The filtrate is centrifuged 10 min at

3000 z g at 4° ; the supernatant solution centrifuged at 17000 x g for 10 min and the resulting supernatant solution spun at 100,000 z g for 60 min.

The

remaining supernatant solution contains moat of the chitinolytic activity . The pellets are all resuspended in the phosphate buffer .

A portion of each is

dialyzed into the same buffer to remove gallic acid, and the dialysate assayed for protein using the Lowry method (7) or the biuret method . frozen and stored at -20° in 5 ml aliquots .

The remainder ie

Enzyme activity in the supernatant

solution remains essentially constant for aiz months . Spores .

Sporaagiophorea are grown on potato dextrose agar supplemented

by O.1X yeast extract in 10 cm petri plates .

After 6 days, spores are removed

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]91

PhyCaotgCea Chitines

by twisting the covers of the plains a~ suspended is 0 .9Z NaCl .

Spores ors

washed twice b9 alternating centrifuging 5 aia at 2000 z g on a clinical centrifuge and raeuapending in 5 ml saline .

Thn final pellet is resuspeaded

is 0.5 ml saline, and as aliquot dried under nitrogen and weighed .

Another

portion ie imndiately assayed for chitiaolytic activity . Standard assay for chitiaase.

Chitinolytic activity . is measured using

1 ng colloidal chitin in 5 .0 m1 0.05 M sodium acetate, 0.15 M NaCl, pH 5 .5 0.5 nl of the enzyme preparation ie added to initiate the reaction . of toluene is added as as aatiaeptic . 25 ° t 1.

A drop

Fach ezperineat is run is triplicate at

At 0, 4 and 8 h, the tubes are centrifuged on the clinical centri-

fuge for 5 nia at 2000 z g and two 0.5 ml aliquots of the supernatant solution reooved .

The pellets era then resuepnnded .

The aliquots are boiled 1 sin and

assayed for release of M-acetyl glucoeamina (NAG) by the Beisaig, Stroniager and Leloir method (8) .

In this procedure NAG is activated by boiling at

precisely pH 9.1 and the product reacted with p-dimathylaminobenaaldehyde to give a pink chranophore whose optical absorption ie proportional to the original MAG concentration .

The endogenous pool of NAG in the cytoaol was

about 15 yM (3 nmole/mg protein) . fit of 18 separate assags .

Each rate datum represents the best linear

Standard deviations for release never ezceeded

loz. Colloidal chitin preparation. chitin (Analabs, Inc.)

Five grass of practical grade molluscan

is stirred into 100 nl cold concentrated HC1 and the

mizture filtered through glass wool .

The filtrate is added slowly with

stirring to 4 1 H20, allowed to settle and the solvent decanted . was then repeated and the suspension allowed to settle overnight .

The process The

suspension is then centrifuged 5 nin at 1000 x g and the pellet collected . The off white chitin is soaked 4 hours in 2Z

RZEn04 and the oxidant removed

by filtration, washing with 2 1 Li ozalic acid, followed by 2 1 of H20 . chitin, which is now porn white, is dialyzed against three changes of 20

The

Phycamyces Chitinase

a92

Vol .

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No . 2

volumes of H2O to remove wall molecules and oligomers, and suspended in 2 1 H20.

An aliquot is taken, dried and weighed .

The suspension of about 2 g/1

stores indefinitely at 4° in the presence of a few drops of toluene . RESULTS 1.

Subcellular localisation .

Virtually all the chitiaolytic activity

(93X residee in the soluble fraction (Table I) and the rate of release of N-acetylglucosamine is moderately large .

Aa attempt to further purify the

material by ammonium sulfate precipitation followed by chromatography was made .

Nearly all the activity precipitated at 50X (NH4) 2504 saturation .

However, only about a quarter of the activity was recovered (Table II) . Apparently either the salt inhibits chitiaolytic activity, a very active chitobiaae activity is lost, or an activator ie lost during the precipitation. In order to ascertain which, a portion of the supernatant solution was dialysed overnight at 4° against two changes of phosphate buffer . of the activity was thereby lost .

Nearly all

Thus the cytosol suet contain a potent low

molecular weight activator .

TABLE 1 A.

Experiment

Subcellular Fractionation Specific Activity mole NAG/h/mg protein

Total Activity nmole/h

10 min, 3000 z g, pellet

2 :3

210

(0 .036)

10 nia, 17000 z g, pellet

5 .8

140

(0 .024)

60 min, 100,000 a g, pellet

2 .6

80

(0 .013)

5500

(0 .927)

5930

(1 .000)

60 min, 100,000 x g supernatant solution TOTAL

70

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293

Phycomirces Chitinasa

TABLE II A.

Specific Activity nmole NAG/h/ng Protein

Ezperiment 1.

A~wnium Sulfate Precipitation*

Ox, supernatant solution 60Z (NH4)250 4

-

1002 (NH4) 250 4 2.

Total Activity nmole/h

62

4650

(1 .00)

18

1100

(0 .24)

1.2

16

OZ, supernatant solution

80

4860

(1 .00)

40x (NH4) 2504

28

870

(0 .18)

170

(0 .035)

75Z (NH4) 250 4

5.8

100x (NH 4)250 4

27

B.

25

Effect of Dialysis

Stardard assay ündialysedt Dialysed 2.

78 .5 3.8

(1 .00) (0 .049)

41 .5 1.5

(1 .00) (0 .037)

0.0511 Trie ?faleate 0.15M NaCl, pH 4.5 Undislysedt Dialysed

To the supernatant solution left after centrifuging 60 sin at 100,000 z g, vas added (!02504 to the indicated--percentage of saturation at 25 ° . The precipitation vas tightly packed by centrifuging 5 sin at 8,000 z g and the pellet resuspended in phosphate buffer . t

The undialysed control vas left at 4° under identical conditions se the dialysed solution sad assayed si~ltaaeouely . Thus overnight incubation alone does not result in inactivation .

29 4

Phyca~myces Chinasse

2.

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Exclusion of sores as source of chitinolytic activity .

Germinating

spores presumably require chitinase to rupture their chitiaaceous cell wall . A possibility is that the chitinolytic activity originates in the spores and is somehow extracted during the preparation of crude enzyme .

The low activity

present in the low speed pellet containing mostly spores mitigates against this hypothesis . meats .

However,

the possibility is finally excluded by two ezperi-

In the first, 150 mg of intact ungerminated spores were incubated in

the usual manner with chitin . low chitinolytic activity .

The results shown in Table III indicate a very

Also the immature stage I sporangiophore contain

no spores but does yield a fairly high rate of degradation of chitin .

Thus

the chitinase activity must originate in the cytosol of the stalk of the aporangiophore . TABLE III Exclusion of Spores

Material

Specific Activity

Stage I aporangiophore

34 nmole/h/mg protein

Spores, immediately after extraction washed 2 x in 5 ml 0 .9I NaCl, ungerminated, 150 ug (16 mg protein by Lowry) Per assay.

4 .4 nmole/h/150 mg spores

3.

Partial characterisation .

FIG. 1 indicates that release of N-

acetylglucosamiae catalyzed by the soluble chitiaase is linear with time to 8 h.

After this interval the rate gradually decreases, and after 24 h the

total mount of NAG begins to decrease, presumably by catabolism .

A plot of

activity vs . pH has a somewhat broad but well defined +~=i~,+~ at pH 5 .5 (FIG . 2) .

The effect of acetate seems to iaply that acetic acid (not acetate)

enhances the activity and perhaps acts as a specific acid catalyst or activator.

Vol . 15, No . 2

PhyCO~yCes Chitinasa

295

FIG . 1 The release of N-acetylglucoeamine as a function of tine .

pH FIG . 2 Belative chitinolytic activity at various pH's . A relative activity of 1 .00 ~ 59 nmole NAG released par hour per mg proteia . ~is-Maleate, sodium phthalate both 0 .05 K in 0 .15 M NaCl, and 0 .2 N sadius acetate . The 8eieig et al . assays rare standardised using N-acetyl glucosanine in the appropriate buffer soluXions as refereaçe in these e:peri neats ae rell as those illustrated in the nee figures .

0

0

PhycomyCes Chitinaoe

296

C

~

0

Vol . 15, No . 2

I

I

100 20 0 300 [NaCI] mM I

I

40 0

T

FIG. 3 The effect of ionic strength on chitinolytic activity . The abscissa indicates the concentration of NaCl is addition to the 0 .05 M sodiua acetate buffer, pH 5.5 .

C

FIG. 4 The effect of divalent catione on the chitinolytic activity of the enzyme extracted from Phyco~ces . ~ Mgt; Cam.

Q

29 7

Phyoo~cea Chitinase

Vol . 15, No . 2

The influence of ionic strength and of the divalent metals Cam and sinisal (FIG . 3 and 4) .

MB++

is

Higher ionic strength toads to slightly elevate

activity, and calciva ion to slightly decrease activity .

liagnesius seers to

enhance chitiaase activity about 15I at 10 sN but decrease it at higher concentrations . DISCQSSION The major purpose of this report is to establish the presence of chitinase in the stalk of the sporangiophora of Phycomycea blakesleeanue .

The couplets

hydrolysis of chitin into free acetylglucosasine requires the thinness, a polysaccharidase, plus an oligosaccharidaea, chitobiase (SN 3 .2 .1 .29) .

Since

the Beisaig method caanot distinguish newly ezposed end group NAG fros free NAG, the presence or absence of chitobiase canaot be considered established . Chitinolytic enzymes are ubiquitously found in bacteria, fungi, Protosoa, Nesatoda and Arthrôpoda, and to a lesser extent among Vertebrate (9) .

In

most of these cases, one sight assign a digestive function to the chitinolytic activity so that the freed N-acetyl glucosasina could be setabolised by the organise .

However, such a function is clearly iaposaible in the giant aerial

hypha of Phycoeycae since it met derive all its necessary nutrients through the mycelia.

Thus thinness caa only be involved in the regulatioa of cell

wall growth in the eporangiophore . Dnfortunataly, enzymes which hydrolyse complex biopolysere are difficult to study in detail because activities at surfaces are not ~rAble to the usual Michaelis-Menten type analysis .

Chitiaolytic activities are especially

difficult because of the lack of well defined substrates a~ the possible presence of multiple sisilar activities .

Partly for these reasons limited

information ie available on molecular parasatere of thinness .

The chitino-

lytic syetea of Streptamyces aatibioticuB bas been most studied (10) .

It ie

camertially available and its activity ie fairly high ; the optimal pH ie 5.0, but the activity is substantially elevated by acetate at lower pH, such like

298

Phycomyces Chinasse

that of Phycomycea .

Vol . 15, No .

2

Low concentrations of Cam are necessary for activity

although higher levels of the divalent cation are inhibitory .

The active

components, all soluble enzymes, are separable by polyacrylamide gel chromatography into two chitinases and a chitobiase .

The molecular weight of the

major chitinase component is about 30,000 daltons .

Little more ie known.

Yet chitinase may perform an important general growth regulatory function Phycomycea may be a good model system .

The growing stage IVb sporangiophore

of Phycomyces elongates at about 50 u/min and spirals in a clockwise direction when viewed from above; a complete rotation takes about 20 min.

Chitin micro-

fibrils in the cell wall are highly oriented in a steep spiral in the nongrowing zone and more randomly but tending toward transverse in the growing cone (11) .

Omega, Harris and Gamow (12) have recently investigated this

spiral growth is detail using a sophisticated optical method .

They suggest

that the left-handed spiral growth pattern of stage IVb is due to the reorientation of the microfibrils from the transverse to the longitudinal direction resulting from longitudinal cell eapanaion.

This cell eapaasioa is driven

by the internal turgor pressure and the degree of reorientation toward the longitudinal is determined by the plasticity and eztensibility of the cell

wall .

Therefore, the magnitude of spiral growth will also be determined by

these rheological properties of the cell bility of the cell wall are mazimnl

wall .

The plasticity and eatenai-

in the region of the growing zone nearest

the sporangium and decrease in regions approaching the non-growing soae .

The

non-growing zone acts as an elastic, son-extensible structure compared to the cell

wall is

the growing zone .

This increase in elasticity could result from

increased bonding between chitin microfibrils or a thickening of the cell wall or both .

Omega and Gamow (13) predicted that as increase is cell

wall

eatenaion rate, such ae that which occurs during the light growth response, can be achieved by a further increase in the plasticity and ezteasibility of the cell

wall

in the growing soae .

More recently, Ortega, Gamow and Ahlquiat

Vol. 13, No . 2

29 9

Phycomycea Chitinase

(saauscript in preparation) have demonstrated that an increase in cell wall eztensibility does occur after a step-up light stimulus (the physiological response is a transient increase in growth rate) .

Thus, it seems reasonable

to suggest that a light stimulus results in a softening of the cell wall, which, in turn, results in an increased growth rate . Chitin, itself is a notably rigid nonplastic biomaterial .

Light stimu-

lated chitinolytic activity could result in the softening of the cell wall, thus rendering it more eaensible .

Specific partial hydrolysis by chitinase

could reduce bonding between adjacent microfibrils by attacking chitin molecules which croselink neighboring microfibrile or by shortening the microfibrils themselves, also resulting in the loosening of the cell wall .

Dnder

the constant large turgor pressure (2~3 atm) a alight increase in chitinolytic activity in the growing call wall could result in the increased ezteneion rate observed during the photoresponsee of Phycomycee . ACKNOÜLSDGMBlIT I wish to thank Drs. J . R. S . Omeg a and R. I. Gamow for discussions concerning their model, Mr . Louie Allea for technical assistance, and !lies Sharon Brysnt for typing .

This work was supported by NIH grant GM 19390 . REFSRSNCSS

Bergman, R., Burke, P ., Carda-Olmedo, S., David; D . M., Delbruck, M., Foster, It. W., Goodell, S. W., Heisenberg, M., Meissner, G., Zalokar, M ., Denai.son, D. S ., and Shropshire, J . W ., Bacteriol . Rev. 33 :99-157 (1969) . 2.

Foster, R . W. and Lipeon, 8. D., J. Gen . Physiol . 62 : 590-617 (1973) .

3.

Bergman, R., Selava, A . P ., and Carda-0lmedo, S., Molec . Gen. Genet . 123 : 1-16 (1973) .

4.

Jas, Y. N ., J . Biol . Chew . 249 : 1973-1979 (1974) .

5.

Rreger, D. R., Biochee. Biophye . Acta . 13 : 1 (1954) .

300

6.

Phyco~myces Chitinase

Vol . 15, No . 2

Bartnicki.-Garcia, S ., and Lippman, E ., J . Gen. Microbiol . 73 :487-500 (1972) .

7.

Lowry, 0 . H., Roeebrough, N . J., Farr, A. L. and Randall, R . J., J. Biol . Chem . 193:265 (1961) .

8.

Reiaeig, J . L., Stromanger, J. L. and Leloir, L . F., J. Biol . Chem . 217 : 959 (1955) .

9.

Jeuaiaua, C., Biochemical evolution and the origin of life, ed . E . Shoffeaiels, North-Holland Publishing Co ., 1971, pp 304-313 .

10 .

Skujans, J., Pukite, A. and McLaren, A. D ., Enzymologia 39 :353-370 (1970) .

11 .

Middlebrook, M. J. and Preston, R. D., Biochem. Biophys . Acta 9 :32-48 (1972) ; Roelofsen, P . A., Biocham . Biophys. Acta 6:340-356 (1950) ; Roelofsen, P . A., Biocham . Biophys . Acta 6:357-373 (1951) ; Roelofsen, P . A., Adv . Bot. Res . 69 :59-149 (1965) ; Heyn, A . N. J., Protoplasma 25 :372 (1936) ; and Fred-Wyesling, A. and Muhlethaler, R ., Vierteljahrber . Naturforsch. Gemeinach. Zurich 95 :45-52

12 .

(1950) .

Ortega, J . R. E ., Harris, J. F . and Gamow, R. I ., Plant Physiol . 53 :485490 (1974) .

13 .

Ortega, J . R. 8. and Gamow, R. I ., J . Theor . Biol ., in press .