The :<::ffects of '3inc on Chitinase Activity
An Honors Thesis (TO
499)
by
Gretchen L. Billman
Dr. Carli;. 'darnes
Ball 3tate University
:Viuncie, Indiana
Hay
1986
Graduation 3ummer
1987
TM
~~FF}:;;GT3
OF ZDV! ON" GHITINASE AC7IVITY
Chitinase, an en7.yme important in the degradation of chitin,
is uroducecl by the
:~F-l.l-a
bacterium.
Heavy concentrations of
zir:c were found to inhibit en?yme activity.
This decrease in
activity was more urominent with the harvested crude enzyme
than with the puri:ied rl ialysate.
One reason for this differ-
ence could be that chitin chelates the zinc, inhibitinG its
cleg;radation and decreasing substrate availability.
Because of
lov;-er protein concentration in the dialysate, excess chitin is
uresent.
This excess chitin is degraded by chitinase and T)ro-
ducts of enzyme activity reflain constant.
Another reason for
the difference could be that a dual enzyme system is present in
the crude.
One of these enzymes is capable of degradinG chitin
which is chelated to zinc 'vfhile the other enzyme cannot.
The
dialysate may contain only the enzyme that does degrade chitin
which is chelated to zinc, having been purified through affinity
chromato,graphy.
Therefore, the zinc does not interfere with
degradation, and the chitin does react with the chitinase.
=~nzyme
acti vi ty remains hieh and does not decline.
INTRODUCTION
,ath current interest in the use of chitin in medicine,
agriculture, and industry, the chitin-chitina:se enzyme system is
of great importance (l).:::hitin is a polymer of B-l,4-N-acetyl.J-glucosamine and is Hidely found in the aquatic environment.
Here it is imnortant as it nrovides carbon and nitrogen for
nutrient regeneration (9).
The purpose of this study was to determine the effects of
different concentration::. of zinc on the activity of a chitinase
enz.yme system.
Various enzyme fractions of supernates of
cultures of EF-ha were to be analyzed and compared with rer:ards
to incubation with zinc.
Both soluble products of hydrolysis
and identification of products by HPLC were to be performed.
Although several microorganisms have been found to be
chitinolytic, the organism used in this study, the
not been Hidely used in research.
EF-L~a,
has
Serratia and yibrio cpp. have
often been studied for chitinolytic activity (17, 12).
Zinc is a heavy metal that is found naturally in the aquatic
In amounts greater than 5 mg/ 1 it is considered
environ!"lent.
to be toxic (5).
3ince heavy metals are known toxic pollutants,
their presence in the aquatic environment is a major concern
(7).
:::ell numbers or growth rates have often been used to measure
toxic effects
(3. 13).
long experimental tirles.
Unfortunately, these methods require
Neasurements of the metabolic activity
of organisms give a more immediate indication of the toxic
effects of heavy metals (2, 6).
ORGANISIU.
A culture of chitinolytic
~F'-4a
was used for this
study.
It was grown on chitin agar made according to Skerman (15).
'·SJIA.
:::hi tin substrate "as prepared by a modified method of
I·iolano, et. al. (8).
:Jhi tosan was acetylated with acetic an-
hyC:.ride in methanol, and the chitin was dialyzed to neutrality.
The cells were grown up in a. chitin broth (Table 1) made
up accordin~ to 3mucker (16).
Cultured supernates were harvested
by centrifuga.tion cwd concentrated by ul tran.l tration uslng a YI·1
10 filter with 10,000 r.r. ! cutoff.
Affinity "hTomatography was
performed on concentrated fractions according to the methods of
Ro1)erts and Cabib (11+).
p~WrTi::I~l
A33.\YS.
Protein determination was made by the Bio-Rad
dye-binding assay described by Bradford (1+).
Absorbance was read
on a Sequoia-Turner Hodel 320 Spectrophotometer at
Protein was also checked by
~I
595 nm.
absorption at 280 nm using a
Pe:c-kin-Elmer dual beam spectrophotometer (model 121+).
CHITINASE AS3AY.
~hitinase
activity was determined by monitoring
release of JH-soluble products from 3H-labeled chitin.
This
method used was a modification of :lo1ano, et. al. (8).
1i::ach
rea.ction mixture contained 20 u1 of 3H-colloidal chi tin,
.5 ml
of enz,yme solution, and .25 ml of ZnS01.s. solutions of concentra.tions ranging from 0.0008 mg Zn/ml to 0.32 mg Zn/m1.
Zn30
The
4 solutions were made up fresh each time the assay was per-
formed.
The mixture was incubated at 37 0 C for one hour, and
the reaction stopped with .2 ml of
10~
TeA.
The mixture was
filtered and collected ln scintillation vials containing 9 fill
of scirtillation fluid.
The Beckman model
1,:3
3801 scintillation
counter was used for counting.
HPU::.
lm attempt was made to show product variation wlth the
use of HPLC.
HOH8ver, because of instrument malfuntions, this
was not accomplished.
Ri~3U1T3
Colorimetric protnin determinations were nerformed on both
the crude supernate and the
dialysat~
of affinity chromatography.
Concentrations of protein standards (gamma globulin) were plotted
and used each time protein was analyzed.
read from this curve.
Unknowns were then
(:!rude and dialysate concentrations are
shown in Table 2 along with tN absorntions.
The chitinase assay was nerformed in duplicate on both the
crude and dialysate.
.\ d i. stinct decrease in enzyme acti vi ty was
seen with the crude, but little or none was seen with the dialysate.
The sudden drop in activity was seen between 0.008 mg '''n/ml
and 0.0162 mg Zn/ml.
The zinc concentrations were nlotted against
CPV's (Figures 1-4).
DI3GU33ION
There are several Dossibilities for the difference in
results from the cruae and purified dialysate.
consideration that chitin is a chelating agent.
the zinc out of solution and binds to it.
1
One takes into
The chitin pulls
Then this happens,
the chitin may be incapable of reacting with the chitinase
em;yme.
Therefore, enzyme acti vi ty decreases.
~!hen
the crude
em.yme is reacted with chi tin in the presence of zinc, there is
a limited amount of chi tin available.
'tihen small concentrations
of zinc are used, not enough chitin is bound up to cause a
decrease in enzyme activity.
However, as zinc concentrations are
increased, more chitin is required to bina with zinc, and sub-
st:rate availability decreases.
~;Jhen
the dialysate is reacted with chitin in the presence
of zinc, there may be an excess of chitin present.
This is
beeause protein levels aTe lower in the dialysate.
As zinc
concentrations are increased, more chitin is llsed to chelate to
the zinc.
;~nzyme
activity does not decrease, however, because
there is excess chitin available to react with the chitinase.
Another possibility for the difference in results is a
dual enzyme system.
ei~~her
Two enzymes could be present in the crude,
a chi tinase and a chi tobiase or two chi tinase enzymes.
One of these enzymes is capable of degrading chitin whlch is
chelated to zinc while the other is not.
been purified, contains both eD7;ymes.
The cTude, having not
',{hen the crude is reacted
with chitin in the presence of small concentrations of zinc, the
zinc is chelated by the chitin, but enough chitin remains for
de e7adation by one of the chitinases.
hi[;h.
~nzyme
activity remains
'Ji th increasing concentrations of zinc, however, more
chitin is needed for chelation and less is available for degradation.
Enzyme activity will then drop.
The purified dialysate, having been purified by affinity
chromatography, contai:1s only one chitinase enzyme.
The enzyme
it contains is the one capable of degrading chitin which is
chelated to zinc.
'.Ihen the dialysate is reacted Hi th chi tin in
the presence of zinc, the chitin is degraded by chHinase with
no interference from zinc.
This causes enzyme activity to remain
stable even when high concentrations of zinc are added.
The level at which chitinase activity was found to decrease
in the crude was at 0.008 mg
7.'1 / ,,:1
or 8 mg/l.
This is above the
concentration designated as toxic or 5 mg/l (5).
thG~t
It Hould seem
the addition of zinc resulting in concentrations greater
then 3 mg/l into the aquatic environment could greatly upset the
balance of this environment.
Because chitin could be chelated
Hith zinc, the ability of chitin to be degraded biologically
(er:.zymatically) Hould be diminished.
JJess carbon and nitrogen
wov.ld then be provided for nutrient regeneration.
This could
then result in a subsequent loss of food in the environment.
~mphasis
to chelation.
has focused thus far on substrate modification due
~Ii th
known chelatine; ability of chitin, this Hould
seem most likely (10, 11).
However, another aspect yet to be
examined is direct interaction of zinc on the enzyme(s) in
question. This has not yet been resolved.
Future experimenta-
tion should be directed at resolving this issue.
-
Table 1.
Ingredients and amounts per 1 liter of 3mucker's chitin
broth
MgS0 4 •
FeS04·
H2 0
7 tf 20
9
10 mil
300 mil
CaC' 2
ZnS04-
7H 2 0
MnS04 -
H20
I mil
10 mil
CuS04 - 5 H2 0
0.0 I mil
CoS0 4 - 5 H 2 0
0.0 I mil
K 2 HP0 4
134 mg
NaC'
0.5 mg
CHIT I N
I
YEAST
-
I
EXTRACT
9
10 mg
Table 2.
Protein concentrations and
~l
absorption (280 nm) for
crude supernate and purified dialysate of
-
~F-4a
PROTEIN
UV
CO NeENTRATIONS
ABSORPTION (280)
CRUDE
20 ug 1m'
DIALYSATE
18
ug/ml
.46
·13
Figure 1.
Chi tinasf~ acti vi ty of crude enzyme fraction of :;;r'-'-J.a
fol1oHinC exposure to zinc (Trial 1).
mixture containing
.5
The reaction
ml enzyme preparation, 20 ul
chitin, and ,25 ml ,~n ,)0'-1- (0. 0008 - 0.32 mg7,n/ml) was
incubatf~d for t hour at 37°'';'
Filterable nroducts
')
of ..JH-chi tin were determined after termina,tion of
the reaction with 1O's TeA.
COUNTS
UI
.
o
... 0
Oe
Ci)
n
o
Z
n
o·
... 0
o
-
N
....
Z
n
- ....
:I .
-0
.....
~L
o
PER
....
o
MINUTE
Figure 2.
Chitinase activity of crude enzyme fraction of
folloHing eXDosure to zinc (Trial 2).
mixture containing
1~F-4a
The reaction
.5 ml enzyme preparation, 20 ul
chitin, and .25 ml Zn30L~ (0.0008 - 0.32 mg Zn/ml) Has
incubated for 1 hour at 37°S.
Filterable products
of 3H-chitin Here determined after termination of
the reaction Hith
10;~ T~A.
COUN TS
o
"'0
o~
Ci)
n
o
Z
n
•
o·
",,0
o
N
Z
n
-
-~
.
-0
.
-
-o
PI.
-o
MINUTE (x 10
3
)
Figure 3.
Ghitinase activity of purified (lialysate of
folloHing exposure to zinc (Trial 1).
mixture containing
;~;;'-4a
The reaction
.5 ml enzyme preparation, 20 u1
chitin, and .25 ml ,~n30L~ (.0008 - 0.32 mg 7,n/ml) was
o
incubated for 1. hour at 37 G.
of
Filterable products
3H- chi tin were determinecl after termination of
the reaction with 10;1, TC_\.
.-
COUNTS
PE It
UI
.
0
,.0
0
...
0
Q
n
0
Z
n
•
...0 .
0
- ...
N
Z
n
3
--
II
3
- ...
0
....
...
o----------------
...
o
3
MINUTE (x 10 )
?iglrre 4.
Chitinase acti vi ty of purified. d:i.alysate of =~F-4a
followinG exposure to zinc (Trial 2).
mixture containing
.5
ml enzyme preparation, 20 ul
chi tin, and .25 ml Zn30
incubated for
1
The reaction
hour at
4 (0. 0008 - 0.32 me;
37°C.
:3n/ml)
Filterable C)roducts
of JH-chitin Hl;re determined. after termination of
the reaction with 1 Q'~ TCA.
was
'\
COUNTS
UI
0
~o
00
G)-
n
0
Z
n
0
"'0
....
-nZ
0
-
-a
--a
CD
-0
-.
-.o
PER
-o
MINUTE (x IO~)
-
UI
1.
Austin,? H., C:. J. Brine, J. i"I:. Castle, anel J. P.
1981 . Chitin:
new facets of research.
~3ikakis.
Science.
212:
7l~9-
753.
2.
Barnhart, Carole L. H. and J. Robi(~ 'festal.
1.983.
Sffects
of environmental toxicants on metabolic activity of natural
microbial communities.
Apnl. ;<;nviron. llicroblol.
46:
970-
977.
3.
Bewley, R.J."'.
1.980.
~~ffects
Oele leaf microorganisms.
of heavy f1J.etal pollution on
Appl.'~nviron.
)!licrobiol.
40:
1053-1059.
4.
Braclforo., H.
1976.
1\ rapid and sensitive method for the
quantitation of microcram quantities of protein utilizing the
principle of protein-dye binding.
5.
Handbook of Analytical Toxicology.
Anal. Biochem.
1969.
7 ') .
~.
1081 pp. (Irvinr,;
3un~,hine, ed.), Chemical Rubber COllmany, Cleveland.
6.
Houba, C. and J. Remacle.
1980.
Composition of the sapro-
phytic bacterial comffinnities in freshHater systems contaminatecl by heavy metals.
7.
11icrob. :Scol.
6:
Laube, V. S. Ramamoorthy, and D.J. Kushner.
55-69.
1979.
filobili-
zation and accumulation of sediment bound heavy metals by
algae.
Bull. Snviron. Contam. Toxicol.
21:
7ri3-770.
8.
A. Duran, and :<:. Cablb.
Holano, ,J"
1.977.
A rapid and
sensitive assay for chitinase using tritiated chitin.
Biochem.
9.
§J.:
Anal.
fh-8-656.
1977.
Huzzarelli, Ii.A.A.(ed.).
Shitin. Pergamon Press,
Oxford, 309 pp.
to.
1980.
;"I1lzzarelli, B.. A. A.
~nzyme !1!icrob. T(~chnol.
chitin and chitosan.
11.
Immobilization of emwmes on
2:
:t 77-184.
1)~'"'­
l'1uzzarelli, ].A.A., F. Tanfani., and r;. Scarpini.
Chelating, film-forming, and coagulating ability of the
chitosan-glucan comples from Aspergillus niger industrial
wastes.
12.
22:
Biotechnol. Bioeng.
885-896.
Ohtakara, A., 11. I-1itsutomi, and Y. Uchida.
1979.
Purifi-
cation and some properties of chitinases from Vibrio snp.
21:
J. Ferm. Tech.
13.
169-177.
Osborne, L.L., :::t.':!. uavies, p.rt,. Ventullo, T.I.ladd, and
1982.
J. '::. Costerton.
The effects of chlorina.ted municipal
sewage and temperature on the abundance of bacteria in
Sheep River, Alberta.
1L~.
Roberts,
n.L.,
chitinase:
and :~. Cabib.
3kerman, V.B.D.
Anal. Biochem.
1967.
Serratia marcesans
3mucke:::-, R. A.
1982.
1Z1:
402-412.
Chitin media. p. 255. In A guide
to the genera of bacteria.
16..
1982.
261-270.
one step purification and use for the determin-
ation of chitin.
15.
Z,2.:
'jan. J. Nicrobiol.
~-lilliams
and
~·;ilkins,
Baltimore.
;)etermination of chitinase hydrol:y·tic
potential in an estuary.
pp. 135-139.
In Proc. Second Int.
Conf. Chitin and Ghitosan. (3. Hirano and 3. Tokura, eds.),
Jap. Soc. of Chitin and Chitosan, Tottori.
17.
Young, II.ie:. and P.A. ';orroad.
1981.
Deuendence of extra-
cellular chitinase activity of Serratia marcesans:;E 1666
on continuous culture dilution rate.
11-1-2-144.
Gan. J. iUcrob.
n: