Subtilopeptidase a produced by Bacillus subtilis PR-70

Zbl. Bakt. II. Abt. 134 (1979), 275-281

[Microbial Enzyme Unit, Microbiology Research Dept., Agrio, Research Centre of Egypt]

Subtilopeptidase A Produced by Bacillus subtilis PR-70 I. Kinetic Behaviour of Solubilized Enzyme RASMY

M.

ATTIA

and

RAWIA

F.

GAMAL

With 6 Figures

Summary The kinetic behaviour of subtilopeptidase A was investigated. The enzyme was obtained from a local isolate of B. subtilis PR-70. The rate of enzyme catalyzed conversion of substrate to product is directly proportional to the enzyme concentration, v = K(E). Michaelis constant was determined using different methods. The average of K m value is equal to 0.01615. The V max and E t were determined being 0.71 and 1.467, respectively, using KUNITZ'S casein digestion method. The enzymeconcentration involved in the reaction system is equal to 97.8 %. A trial to calculate the molecular weight and the number of active groups were discussed.

Zusammenfassung Es wurde das kinetische Verhalten von Subtilopeptidase A untersucht. Das Enzym wurde aus einem einheimischen Bacillus subtilis-Stamm (B. subtilis PR-70) isoliert. Die Rate der von diesem Enzym katalysierten Umwandlung des Substrates zum Produkt ist der Konzentration des Enzyms direkt proportional: v = K(E). Die Michaelis-Konstente wurde mit verschiedenen Methoden bestimmt, der durchschnittliche Km·Wert betragf 0,01615. Die Werte Vmax und E t wurden an Hand der Kasein-Aufsehlufl-Methode nach KUNITZ mit 0,71 bzw. 1,467 gefunden. Das Enzym war im Reaktionssystem mit 97,8 % beteiligt. Eine Moglichkeit, das Molekulargewicht sowie die Zahl der aktiven Gruppen zu bestimmen, wird orortorb.

Enzymes are the most versatile and specific catalysts we know. They catalyze reactions at neutral pH values and low temperatures at very high rates. Thus we are presented with the challenge of finding out how these reactions are catalyzed. Quantitative kinetic studies have not a monopoly in this field, but they play an important role which is discussed in this paper. In recent years, proteases have assumed considerable industrial importance because of their ability to hydrolyze proteins nearly quantitatively to amino acins with little formation of reversion products. This paper deals with the kinetic properties of the purified enzyme isolated from the local strain of B. subtilis PR-70 which is used for the commercial production of protease in Egypt.

Materials and Methods 1. Source of enzyme Subtilopeptidase was prepared according to the following method, from B. subtilis PR-70, isolated by ATTIA. and SA.MIA. (1974). After removal of baeoacia by centrifugation, using Prima Floc at a concentration of 0.3 %, the enzyme was precipitated by addition of 400 g ammonium 18 Zbl. Bakt. II. Abt•• Bd. 134

276

R . M. ATTIA and R. F . GAlIIAL

sulfa te per litre plus so me H yflo Supercel to fa cilitate fil t erin g . T he fi ltercake contain ing salt was dried in a desi ccator. The enzyme was iso lated fr om t he crude po wde r after disso lving in water a nd a ddition of calcium clori de t o mak e a solution of 0.1 M in Ca 2 +. Addition of one volume of a cet on e at r oom tempera ture precip itated a dark, mucinous impurity which was r em ov ed by cen trifuga tion . Addition of a cet on e t o 75 % by v olume pr ecipit at ed t he enzyme . The p r ecip it at e wa s collected b y centr ifugation a n d m ost of t he a cetone was r em ov ed by a stre am of air. T o purify the enzy me, t he precipitate was d issolve d in water , filt er ed and dialyz ed ag ai ns t 0.05 M sod ium phosphate buffer, p H 6.5 (HAGlHARA ot al. 1958). The di al yz ed solut ion was adsorbed on a column of DuoIit e C-I O, and after washi ng wi th water a n d ac etone, t h e enzyme was eluted with a phospha t e b uffer con taini ng 0.5 M sodium clori de. The en zyme-r ich fra ct ion s of the eluate wer e pooled , and am mo nium sulfa te added to 40 g/ IOO mI. The enzy me containing p recipitate was collected b y fil t ering after addit ion of H yflo Supe rce I. Most of t he am monium sul fate was wa sh ed out from t he precipitate with 75 % aceto n e at 40 °C, and the r esidue wa s then sus pe nde d in 0.0 2 M sodi um aceta te. After r emoval of the Supercel by filtering, and after was hi ng wi t h sodium a cetat e solution, the en zy m e wa s precipitated fro m the combin ed f iltrate an d wa shings by addition of 2.5 v olum e of acetone at 5 -10 °C. The precipitate wa s collected by cen t ri fuga t ion , and ac eton e was removed with a stream of a ir until t he precipitate had alm ost d ried .

2. Assay of protease activity K UNITZ'S (1947) method a s m odified by HAGIHARA et al. (1958) wa s us ed. A I % solution of case in (H ammeratein quality) was disso lved in 0.02 M phosphate buffer (pH 7.3). The r ea cti on m ix ture contained 5 ml of t he I % casein solut ion and I ml of the en zy me prep aration. Digest ion was a llowed to proceed for 10 minu tes at 30°C. After the addit ion of 5 ml of T CA solution (TCA 0.1 M, NaO Ae 0. 22 M, HOAc 0.33 M) a n d v igo ro us shak ing, t he m ix ture was allowed t o stand a t room tempera t ure for 30 m inu t es a nd then fil t er ed. On e m l of 3-t imes d iluted F olin-Ciocal teau reagen t and 2.5 ml of 0.551\1 sod ium carb on a t e we re add ed t o I ml of th e fil trat e. Af te r the solution wa s k ep t at 30° for 20 minut es , t he absorbance at 660 nm was m easured.

3. Kineti c st ud ies Mich aeli s constan ts a nd V max values were d et ermin ed u sin g sev eral different m ethod s. The substrate wa s dilut ed to su ita ble lev els , and m ixed with t h e en zyme and incubat ed a t t he temper . atur e of 30°C for 10 minut es, a ft er whic h solutions were use d for determining t he rate of casein hy drolysi s.

Results and Discussion Figure 1 shows that the rate of enzyme catalyzed conversion of su bstrat e to pr odu ct is dir ectly proportional t o the enzyme concentration, i.e. v = K(E), at constant assay conditions of substrate concent rat ion , pH and t emp erature. This linear relationship between initial velocity and enzyme concentration will in general be observed when the method being employed to follow the r eaction rate truly reflects t he velocity of substrate-to-product conversion at all enzyme concentrations used. Thi s indicat es that there is no inhibitor or toxic impurity pr esent in the reaction mixture that would inactivate some of the enzyme pr esent. Also the concentration of subst rate and any required cofacto r ar e gr eatly in excess of the enzyme concentration (DIXON and WEBB 1964, PLOWMAN 1973, and ZEF}'REN and HALL 1973). In the mechanism of M ICHAELIS and MENTEN (1913), as generalized by BRIGGS and HALDANE (1925): k,

k,

E +S~ES-E +P k,

three st ages may be distinguished in a reaction following this mechanism. First, immediately after mixing solutions of enzyme and substrate, ther e is a rapid disappearance of subst rate and fr ee enzymat ic sit es as they react with each other to

277

Subtilopeptidase A Produced by B acillus subtili« PR-70. I

100..---

~

80

su 60

... ~

a.

O~

....L_

20

.....L.

.......I...

40

60 E

t

.....,jl___ _

80

___l

100

(V)

F ig. 1. Varia t ion of ra t e of enzyme ac tivity with (E l ) .

form the enzyme-subst rate complex. In the second stage a ste ady state is attain ed in which t he rate of form ation of compl ex is balanced by its rate of decomp ositi on to r egenerate fr ee enzyme and subs t ra te or to yield pr odu ct. In this ste ady state d(ES)/dt = 0, and t he rate of appear an ce of pr oduc t is constant . As t he substrat e is used up th e concent ration of (ES ) decreases and t he rate of the overall reacti on correspondingly decreases. In the third st age of the r eact ion d(ES)/dt is not equal to zero, but it is very small compared with d Sldt and so t he reaction is still referred to as being in a steady state. Studies of all three phases of the r eaction yield t he most complete nfor mation about the mechani sm and ar e referred to as transient-state st udies. CHANCE who in 1940 first st udied t he transient -stat e kineti cs of peroxidase, has pioneered this field of enzyme kinetics. Th e same rat e equation is obtained wheth er it is assum ed that the equilibrium is adjusted rapidly (MICHAELIS and l\'!ENTEN 1913) or not (BRIGGS and HALDANE 1925). The velocit y in the ste ady state is given by : v = V1(1 + Km)jS. Th e Michaelis constant (Km ) is a convenient quantity for summarizing experimental results becau se it is the concentration of substrate at which half the maximum velocity is reached. Various methods for plotting kin etic data for th e calculation of Vmax and K m have been used. Th e method of LINEWEAVER and BURK (1934) in which Ilv is plotted versus l is ha s the advantage that th e variables v and s are separated . DIXON (1953) has pointed out that the negative r eciprocal of the intercept of such a plot on the lis axis is equal to the K m . Michaelis const ant and maximum initial velociti es may also be calculated (EADIE 1942 and HOFSTEE 1952) from plots of v versus v js and sjv versus s. Thi s st udy is concerne d wit h different methods used for K m det erminat ion. Th e values of the slopes and intercepts in th ese various plot s ar e summarized in Tabl e 1. 1S*

278

R. M.

ATTIA

and R. F.

GAMAL

Table 1. Methods for ca lcula t ing K m and V max Plot

Intercepts

Slope

Ord inate

Ab scissa

-K m

I jv v

V jK m

I jV

sjv

-K m

KmjV

I jv v er sus I js v ve rsus v js sjv ver su s s

- l jK m

There are several different methods for the determination of K m. When l/v is plotted against l is, as in Fig. 2, corresponding to th e double reciprocal from of LINEWEAVER and BURK , l/v

=

Km/V · l is

+ l/V. 1/V

Vm;rx.=

0.71

Km

001639

3

2 y ..._ - - l/Vmax

o

_120

120

40 l/ S

Fi g. 2. Rate of I /V a s functi on of l iS. wh en S bound by enzyme is an a pp reci a b le fracti on of St.

0.9 Vmax

V.

Vmax :: 0.71

0.6

Km

-- 0.01605

0,4 Vmax / Krn

0.2

0

1-.._ _"--_ _"--_ _.1..-......._ " - -_ _" ' -....

o

10

20 .

30

40

50

60

VIS Fig. 3. R ate of V as function of V jS. whe n S bound by en zyme is an a p prec ia ble fraction of St.

Subtilopeptidase A Produced by Bacillus subtilis PR·70. I

279

In this figure, the graph cuts the vertical axis at a point which gives IjV and has a slope of Km/V. The K m is calculated as 0.01639 and Vis equal to 0.71. Fig. 3 shows that V is plotted against vis corresponding to the single reciprocal form of EADIE and HOFSTEE,

vis

=

-vlK m + V/K m

or v = V - K m • v Is.

Michaelis' constant can be calculated mathematically from Fig. 3, showing the value of 0.01605 and V is equal to 0.71. In this figure, the graph cuts the vertical axis at a point giving V and has a slope of - K m . Another method of plotting is shown in Fig. 4 where slv against s, corresponding to the equation given by HANES:

slv

=

Km/V

+ I/V' St.

In this figure, the graph cuts the vertical axis at a point which gives KmjV and has a slope of I/V. The K m is obtained by prolonging the graph to cut the base line, giving K m equal to 0.016. From the above metioned results, Michaelis' equation: v

= VII

+ Km/S

is plotted in several different ways for the determination of K m and Vmax values of the enzyme obtained from these several techniques. From the above equations, it. is convenient to introduce the REINER equation, St

=

r E,

+ Kr/(l-r)

in which relative rate (r) is defined by v/V. Plotting s against r, the first term of course represents a straight line on the graph. The line goes through the origin and has a slope E t . The second term starts out as almost a straight line, when r is very small compared to one, it is practically equal to Kr. As r increases the denominator l-r becomes smaller, the numerator r becomes larger, and so the whole term increases. As r approaches one, the denominator approaches zero, so that the whole term becomes

_----.uJOr------------------, S I V

Km

0.08

= 0.016

Vmax = 0.69S7

0.06

0.04

......- - - - Km / V max

a

0.01

0.02

0.03

O. 04

0.05

S Fig. 4. Rate of S/V as function of S when substrate bound by enzyme in appreciable fraction of St.

280

R. M.

ATTIA

and R. F.

GAMAL

O.DSr------------r----------,

s. 0.04

0.03

0.02

0.01

0.2

0.4

0.6

0.8

Fig. 5. Relation of S to relative rate (r).

infinitely large and the curve rises almost vertically as shown in Fig. 5. Plotting St against the combination of rf(l-r) rather than against r itself as in figure 6, we can solve REINER equation as: St = rEt + KrfO-r) for r in terms of u: r

= ufO

+ u)

0.0'"

0.02

Et = 1.4-67 mglml

l~

Slope= E,-+-I{

o

2

rl(l-r)

Fig. 6. Relation of S to rj(l-r) when rj(l-r) more than 1 (Note intercept E t ) .

Subtilopoptidase A Produced by B acillu s subtilis PR-70. I

281

the above mentioned equat ion can be rewritten as follows: St = E t . u/(1

+ u} + Ku

Wh en u becomes very mu ch larger than 1, the combined curve is therefore: St = E t + Ku and a stra ight line with slope K and intercept E t is obt ained as in Fig. 6. Th e fact t hat the intercept for large u is Et is indicated by the dotted line, which prolongs the limiting st raight line backwards t ill it intersects the St ax is. Th e init ial and final parts of the curve ar e part icularl y stra ight, so their slopes can be obtained graphically, giving E t K and K. The value of E t obtained by difference can be checked with the v alue of E t given by the inte rcept of the asymptote (th e dotted line in Fig. 6). Th e value of this graph is that the concentrat ion of E t is easy to get, being 1.467 mg/ml (146.7 LV/ml). As the t ot al enzyme used in the experiment was 1.5 mgjml, so 97.8 per cent of the solubilized subt ilopeptidase was envolved in the reaction system. If we are using a purified enzyme so that most of the protein added is enzyme , we can then get the molecular weight using M = W/E t and the quantity which we call here Et will actually be the number of active group s multiplied by the true molar concentrat ion of enzyme or in other words the equivalent concentration of active groups (REINER 1959).

+

References ATTIA, R. ~I. , and SAMIA, A. ALI: Isolat ion of Bacillu s subtilis produci ng sub tilope pt idase A. Agr ic. R es. R evi ew 52 (1974),111. B RIGGS, G. E ., an d HALDANE, J. B. S. : A n ote on t he kinet ics of enzyme actio n . B ioeh em. J . 19 (1925), 338 . DI XON, :\1., an d ' VEBB , E. C.: " E n zy mes " , 2nd ed ., London 1964. E ADIE, G. S.: Th e inhibiti on of cholines te rase by physostigmin e a nd p rosti gmine. J . BioI. Chern. 146 (19-t2 \, 85. H AGIHARA, B ., MATSUB ARA, R ., NAKAI, 1\1., and OKUNUKI, K .: Cr ystalli ne ba ct er ial prot einase. I. Prep ara tion of cryst alline prot eina se of B acillus subtilis. J. Bi och em . (Tok yo) 45 (1958), 185. H OFSTEE, B . R . J .: On t h e eva lua t ion of t he cons ta n ts V m a nd K m in enzy me reacti on s. Scienc e 116 (1952),329. K UNITZ, M.: Crystalline soybe an tryp sin in hib itor. II. Genera l prop erties . J. Gen . Phy siol. 30 (19 47), 291. LI NEWEAVER, R., and BURK, D .: T he d eterminat ion of enzyme d issocia t ion con st an ts . J. Am er . Chern . Soc . 56 (1934),658. MICHAELIS, L., and MENTEN, M. L.: In: " Out lin es of Enzyme Ch emistry" (NEILANDS, J. B ., and STUMPF, P. K., ods.) . New Y ork 1962; Biochem. Z. 49 (1913), 333. P LOWMAN, K. M.: "Enzyme Kin eti cs". N ew York 1972. R EINER, J . M.: "Behavior of Enzyme Sy st ems " . New York 1959. ZE F~'RE N, E ., and HALL, P . L.: "The Study of Enzyme Mech anisms". New York 1973. Authors' address: Dr. R ASMY M. ATTIA a n d Dr. R AWIA F . GAMAJ" Micr obi ology R es. D ep t ., Agr icul t ure R esearch Cen t re, Ga mma St., Giz a (E gy pt) .