stabilization of restriction endonuclease bam hi by cross-linking reagents

Stabilization of Restriction Endonuclease Barn Hi by Cross-Linking Reagents

A. K. Dubey, V.S. Bisaria, S. N. Mukhopadhyay,* and T. K. Ghose Biochemical Engineering Research Centre, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi- 1100 16, India

Accepted for Publication July 18, 1988

Bacillus amyloliquefaciens H produces a restriction endonuclease enzyme BamHI which is heat labile even at low temperatures. Studies were conducted to enhance thermal stability of BamHI using cross-linking reagents, namely, g lu ta ra ldehyde, d ime thy l ad ip imida te (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3'- dithiobispropionimidate (DTBP). Reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. However, the DMA-, DMS-, and DTBP- cross-linked preparations of Bam HI exhibited significant improvement in thermal stability. Studies on thermal denaturation of the cross-linked enzyme preparations revealed that these do not follow a true first-order kinetics A possible deactivation scheme has been proposed in which the enzyme has been envisaged to go through a fully active but more susceptible transient state which, on prolonged heat exposure, exhibits a first-order decay kinetics. At 35°C' which is close to the optimum reaction temperature of 37°C for BamHI activity, the half-line of DMA-, DMS-, and DTBP-cross-linked preparations were 4.0, 5.25, and 5.5 h, respectively, whereas the native enzyme exhibited a half-line of 1.2 h only. The apparent values of deactivation rate constants for native, DMA-, DMS-, and DTBP-cross-linked Bam HI were 1.13, 0.39, 0.29, and 0.26 h-', respectively, at the same temperature, and the apparent values of activation energies for denaturation of native, DMA-, DMS-, and DTBP-cross- linked BamHI were 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. The DTBP-crosslinked Bam HI was, therefore, the best heat-stable preparation among those tested. The unusually low values of activation energies for denaturation of Bam HI represent their highly thermolabile nature compared to other commonly encountered enzymes such as trypsin, having activation energies of more than 40 kcai/mol for their denaturation.

INTRODUCTION

The physicochemical properties of restriction enzymes in relation to activity and stability have been discussed in a recent review.' The low stability of highly efficient and specific enzymes in native form has attracted much atten- tion on enzyme stabilization during recent years, as the de- velopment of stabilized enzymes permit their application in various technological processes and laboratory techniques. 2.3

The loss of activity of enzymes can be caused by metal ion

* To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 33, Pp. 131 1-1316 (1989) 0 1989 John Wiley 81 Sons, Inc.

inhibition, chemical modifications such as bond cleavage, or denaturation. Denaturation is probably the most common form of inactivation and is defined as a process, or a se- quence of processes, in which spatial arrangements of the polypeptide chains within the molecule is changed from that of the native protein to a more disordered arrange- ment.4 Significant advances have been made by immobi- lizing enzymes on water-insoluble matrices to enhance their ~tability.~ However, transformation of soluble enzyme to the insoluble state by immobilization sometimes restricts its practical applicability. Enzymes immobilized on water- insoluble polymers lose their capacity to react with water- insoluble substrates. When soluble substrates are used, the effectiveness of such catalysts is usually markedly reduced because of steric hinderances of the matrix.

Attempts have been made to prepare matrix-bound restriction EcoRI and BamHI coupled to CNBr- activated Sepharose 4B have been reported to show significant enhancement in thermal stability.6 Because of the highly specific nature of interaction between a restriction enzyme and its substrate (DNA), the matrix-bound enzyme is likely to exhibit poor efficiency. In order to circumvent this problem, the approach in the present investigation involved the use of bifunctional cross-linking reagents for stabilizing Bum HI in solution. This was achieved by using bifunctional cross- linking reagents, glutaraldehyde, and imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3'-&hobispropionimidate (DTBP). Glutaraldehyde is reported to react nonspecifically with amino acids involving the a-amino groups, N-terminal amino groups of some peptides, the sulfhydryl group of cysteine, the +amino group of lysine, and the phenolic and imidazole rings of tyrosine and histidine.'-" On the other hand, imidoesters react specifically with lysine. This article reports the studies carried out on stabilization of Bum HI using these cross-linking reagents.

MATERIALS AND METHODS

Barn Hi

Bacillus amyloliquefaciens strain H was the source of the restriction endonuclease BamHI. The enzyme was pro-

CCC 0006-3592/89/01001311-006$04.00

duced in a 6-L laboratory fermentor and purified by using phosphocellulose and hydroxyapatite column chromatogra- phy.I3 The yield of the enzyme in the purified fractions was 2500 units/mg protein.

Reagents

Glutaraldehyde was obtained from Riedel Dehaenag Seelza, Hannover (West Germany). Dimethyl adipimidate hydrochloride (DMA), dimethyl suberimidate hydrochloride (DMS), and dimethyl 3,3'-dithiobis propionimidate hydrochloride (DTBP) were procured from Sigma Chemi- cals Co. All other reagents used were of analytical grade.

Preparation of Cross-linked Barn HI

Reactions were conducted in a volume of 10 pL in lOmM potassium phosphate buffer (pH 8.0) containing 20 pg BamHI protein at a level of 5 units/pL and the cross-linking reagents at levels ranging from 0.01 to 0.2% (w/v). The temperature of the reaction was 25°C. The reaction with glutaraldehyde was stopped after 30 rnin by addition of ter- mination buffer which contained lysine in 100 mM Tris-HC1, pH 8.0 (mole-temole ratio of glutaraldehyde to lysine being 1 : 27, as optimized experimentally). The reactions with imidoesters were allowed to proceed for 1 h. No termina- tion step was considered necessary because bisimidoesters hydrolyze rapidly themselves. l4

Thermal Stability of Native and Cross-linked Barn HI

Temperature sensitivity of BamHI (native or cross- linked) was monitored by incubating it at temperatures ranging from 25 to 50°C in 10 mM potassium phosphate buffer, pH 7.0, for varying lengths of time. In each case, 10 pL enzyme preparation (5 units/pL) containing 20 pg protein was subjected to heat treatment. The residual activity aamfter heat treatment was determined by the serial dilution method. I 3 Adequate dilutions of the enzyme were prepared in the dilutian buffer (20 mh4 K2HP04-KH,P04, pH 7.0; 0.2M NaCI; 1mM EDTA; and 7mM 2-mercaptoethanol di- luted 1 : 1 with glycerol). In a typical assay, 0.5 pg A-DNA was digested with BamHI in a reaction mixture containing 20 mh4 Tri-HC1 (pH 8.5), 10 mM MgCl,, l00mM NaCl, and 7mM 2-mercaptoethanol in a total volume of 20 pL. The digestion was conducted at 37°C for 1 h and was ter- minated by heating at 70°C for 5 min after adding tracking dye. The DNA digestion was observed by electrophoresis using 0.8% agarose. One unit of BumHI was defined as the amount which completely digested 1 pg of X-DNA under the reaction conditions. Based on the residual activity, percentage loss of initial enzyme activity was computed.

RESULTS AND DISCUSSION

Studies on heat inactivation of native and cross-linked BamHI preparations revealed that the enzyme deactivation

did not follow a true first-order kinetics. There was an initial lag period, which varied with the temperature and the BamHI preparation, after which the enzyme denatured with first-order kinetics. A detailed discussion on thermal deactivation of the enzyme is provided in the following sections.

Thermal Stability of Native Barn HI

Native BamHI did not exhibit any inactivation when subjected to a heat treatment at 25°C for 2 h (Fig. 1). Fur- ther exposure beyond 2.0 h caused a decline in enzyme activity which increased with the length of exposure period. At 35"C, the enzyme was rendered completely inactive upon exposure for 2.66 h but did not show any inactivation for the initial 0.5 h. BamHI was found to undergo quick inactivation at higher temperatures. When compared with other enzyme from this organism (B. amyloliquefuciens), e.g., a-amylase, BamHI was found to be much more heat labile as the latter was completely inactivated within 0.66 h at 50°C while the former retained 40% activity at 70°C after 0.5 h." The results show that BamHI is very heat labile at temperatures higher than 40°C.

Effect of Cross-linking Reagents on Activity of Barn HI

With a view to select a suitable concentration of the cross-linking reagents which causes minimum inactivation of the enzyme, the effect of different concentrations of the cross-linking reagents, namely, glutaraldehyde, DMA, DMS, and DTBP, on the activity of BumHI was studied. At a concentration level of 0.01% (w/v), none of the reagents caused inactivation of the enzyme. The loss in the enzyme activity was observed with increasing concentra-

Time ( r n i n )

Figure 1. Thermal deactivation of native BarnHI. Ten microliters BamHI preparation (5 units/pL) containing 20 pg protein was exposed to various temperatures in range of 25-50°C in lOmM potassium phosphate buffer (pH 7.0): Er , activity of transient state of enzyme and equal to initial activity; E, , residual activity of the denatured enzyme after heat treatment (see text for details); (0) 25°C; (A) 30°C; (0) 35T; (0) 40°C; (A) 45°C; (.) 50°C.

1312 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 33, APRIL 1989

I \\ I I I I I b

0 0.OL 0.08 0.12 0.16 0.20 0

R e a g e n t concent ra t ion (wlv I

Figure 2. Effect of various cross-linking reagents on activity of BamHI. Reaction of BamHI with cross-linking reagents was conducted as described in Materials and Methods. After reaction was over, residual activity of BamHI in cross-linked preparations was measured and expressed as percentage of initial activity. (0) Glutaraldehyde; (0) di- methyladipimidate (DMA); (0) dimethyl suberimidate (DMS); (A) dimethyl 3,3’-dithiobispropionimidate (DTBP).

tion of the reagents, and at a concentration level of 0.2% (w/v), all the cross-linking reagents except DTBP led to 90% or more inactivation of BamHI (Fig. 2). The activity loss due to DTBP at this concentration was 80%. Glu- taraldehyde was found to have no effect on Bum HI activity up to a concentration of 0.04% (w/v), whereas DMA and DTBP reduced the activity 10% and DMS by 20% at the same concentration level (Fig. 2).

Olszewski and WassermanJ6 studied the effect of glutaraldehyde on BamHI activity. Our results are in confor- mity with those reported by these workers. The loss in activity on exposure of the enzyme to higher concentrations of the cross-linking reagent is possibly due to either in- volvement of amino acids at the active site or increased substitution which may lead to modification of less acces- sible groups, resulting in conformational changes in the enzyme molecule. No (or little) loss of BarnHI activity at lower concentrations of glutaraldehyde and imidoesters reflect that the amino acids are involved in such a way that there is no (or minimal) conformational change in the enzyme molecule.

Thermal Stability of Cross-linked Preparations of Barn HI

The cross-linked preparations of Bum HI obtained at various reagent concentrations were screened for their temperature sensitivity by subjecting them to a heat treatment at 40°C for 30 min in order to select the best-stable preparation. Since native BamHI retained 70% of its original activity after this treatment, the cross-linked preparations retaining more than 70% activity were selected for detailed kinetic studies on their thermal stability. The criteria for

selection of an optimum concentration of a cross-linking reagent was the minimum loss of enzyme activity due both to treatment with the reagent and to heat treatment at 40°C for 30 min. The results pertaining to the effect of heat at different concentration of cross-linking reagents are shown in Table I.

Glutaraldehyde, a very common reagent used for cross- linking, was found not to enhance the thermal stabiIity of BamHI. Glutaraldehyde was reported to enhance the thermal stability of a number of enzymes.” Boudrant et al.I8 reported an increase in thermal stability of subtilopeptidase upon reticulation using glutaraldehyde. But in the case of cata- lase, no change in thermal stability was noted upon cross- linking with glutaraldehyde.” Similar observations were made with BamHI in the present study. Location of the cross-links formed is very important in conferring stability to the enzyme.” Thus, it appears that the cross-links formed by glutaraldehyde in BarnHI are not positioned appropri- ately so as to provide either conformational rigidity to the enzyme or protection to the susceptible bonds which are involved in the denaturation process.

Contrary to the effect of glutaraldehyde, the imidoesters (DMA, DMS, and DTBP) resulted in the preparations of BarnHI which displayed enhanced heat stability. With increasing concentration, the thermal stability of DMA-, DMS,- and DTBP-crosslinked preparations increased compared to that of the native enzyme. The increase in the degree of cross-linking with increased concentration of imidoesters may account for enhanced thermal stability of cross-linked preparations of BarnHI. The best thermally stable preparations of BamHI were obtained at the DMA concentration level of 0.12% (w/v) and DMS and DTBP concentration level of 0.08% (w/v); the corresponding concentration of these reagents in terms of millimoles per liter are 4.88 for 0.12% DMA, 2.9 for 0.08% DMS, and 2.58 for 0.08% DTBP. DMS and DMA have been employed to cross-link several other enzymes and proteins. Rajput and Gupta14 reported that the reaction of trypsin with bisimidoesters (DMA and DMS) produced a product which retained significant amounts of its various biological activities. At the same time, it showed significantly less autolysis compared

Table I. heat stability of cross-linked BamHI.’

Effect of different concentrations of cross-linking reagents on

Percentage activity retained by cross-linked BamHI after heat treatment at 40°C for 30 minb

Reagent concentration (% w/v) Glutaraldehyde DMA DMS DTBP

0.01 70 70 70 70 0.04 70 80 90 85 0.08 70 90 100 100 0.12 70 100 100 100 0.16 60 100 100 100

“Loss of BamHI activity which occurred following chemical treatment

bActivity just before start of heat treatment is taken at 100%. but before heat treatment is given in Figure 2.

1313 DUBEY ET AL.: STABlLlZATlON OF RESTRICTION ENDONUCLEASE BamHl

to the native enzyme. Enhancement in thermal stability of ribonuclease A and a-chymotrypsin was achieved by retic- ulating them with DMA.

Since glutaraldehyde did not lead to any thermally stable preparation, it was not used in further studies. However, detailed studies on thermal inactivation of DMA-, DMS-, and DTBP-cross-linked preparations were conducted.

Deactivation Kinetics of Cross-linked Barn HI Preparations

The cross-linked preparations of BamHI obtained at the optimum concentrations of the reagents were subjected to a wide range of heat treatment to study their deactivation kinetics.

(a) DMA-cross-linked Bum H I : DMA-cross-linked preparation of BamHI when exposed to 25°C retained all its activity for 3.5 h; the exposure beyond 3.5 h resulted in a loss of activity whch went down to its 70% level after 5 h. At higher temperatures, considerable loss of enzyme activity was noticed with time (Fig. 3). The results when compared with that of the native enzyme showed an improvement in the stability of Bum HI against thermal inactivation.

(b) DMS-cross-linked Barn H I : DMS-cross-linked BamHI did not exhibit any inactivation at 25 and 30°C up to 3.5 h (Fig. 4). A comparison of thermal denaturation data of native and DMS-cross-linked Bum H I indicated that

Time( rnin)

-2.2 -'"I

T i m e [min)

0 90 120 150 180 2R) 240 270 300

-1.2 -I -1.L < Figure 4. Thermal deactivation of DMS-cross-linked BamHI. Cross- l i e d preparation of BumHI obtained at DMS concentration of 0.08% w/v was subjected to heat treatment at indicated temperatures for varying length of time. See Figure 1 and text for details: (0) 25°C; (A) 30°C; (0) 35°C; (0) 40°C; (A) 45°C; (0) 50°C.

cross-linking of Bum HI using DMS had conferred notable resistance against thermal denaturation.

(c) DTBP-cross-linked Barn HI: At 25"C, this preparation retained all its activity for 5 h. When heat treatment was conducted at 30°C, no deactivation was noticed for 3.5 h. DTBP-cross-linked BamHI retained 50% activity after 4.5 h at 45°C and 4 h at 50°C (Fig. 5). Thermal inactivation of DTBP-cross-linked BamHI with respect to that

lime (min)

0 90 120 150 150 210 240 270 300

-0.2

W

-1.0

Figure 3. Thermal deactivation of DMA-cross-linked BamHI. Cross- lied preparation of BumHI obtained at DMA concentration of 0.12% w/v was exposed to temperatures of 2550°C for various time intervals. See Figure 1 and text for details: (0) 25°C; (A) 30°C; (0) 35°C; (0) 40°C; (A) 4 5 0 ~ ; (m) 50°c.

Figure 5. Thermal deactivation of DTBP-cross-linked BamHI. Cross- linked preparation of BamHI obtained at DTBP level of 0.08% w/v was given heat treatment at indicated temperatures for various time intervals. See Figure 1 and text for details: (0) 25°C; (A) 30°C; (a) 35°C; (0) 40°C; (A) 4 5 ~ ; (m) ~ O T .


of the native enzyme showed a remarkable enhancement in thermal stability at both lower and higher temperatures.

Comparison of the deactivation rates of DTBP-cross- linked Bum HI with that of DMA- and DMS-cross-linked preparations revealed that cross-linking of Bum HI using DTBP provided better resistance against thermal inactivation, particularly at higher temperatures and with respect to DMA-cross-linked Bum HI.

The period leading to 50% decay of enzyme activity is considered the half-life of Bum HI at a particular temperature. The half-life for inactivation of both the preparations of BumHI (native and cross-linked) decreased with an increase in temperature (Table 11). At a particular temperature, the half-life for inactivation of reticulated enzyme preparation was significantly higher than that of the native; at 50°C, the DTBP-cross-linked Bum HI displayed the highest (20-fold) increase in half-life as compared to the native one. Thus, the higher half-life of cross-linked preparations of BamHI compared to the native one proved that stabilization against thermal denaturation was achieved as a result of cross-linking.

Possible Thermal Inactivation Mechanism

The mechanism of thermal inactivation of BumHI (native or cross-linked) appeared to involve a two-step process. In the first step, no apparent inactivation could be observed as measured by activity determination, but it resulted in a transient form of enzyme which was more susceptible to heat treatment and was inactivated upon prolonged exposure to heat. The deactivation process could be illustrated as follows:

(1)

where En is the initial enzyme, E, is the intermediate or transient form of the enzyme, and Ei is the denatured or inactivated enzyme. Since En and E, were equal as shown by their activity values, k, , and k- , could not be measured. Here, E, followed first-order inactivation kinetics and was converted to E, . The “apparent” deactivation rate constant k d for this step was determined by measuring the activity of the denatured enzyme.

k+1 En ET E; ‘k-I

Apparent Deactivation Rate Constants and Activation Energies for Deactivation of Native and Cross-linked Barn HI

Enhancement against thermal inactivation can be ascer- tained by comparing the deactivation rate constants and ac-

tivation energies for deactivation of native and cross-linked preparations of Bum HI. These parameters were therefore computed at different temperatures for both the native and the cross-linked enzyme. Since the intermediate form of Bum HI (E,) followed fist-order deactivation kinetics, the following step was used to compute the apparent deactivation rate constant k,:

E, A Ei (2)

The above change occurred at a rate r, proportional to the active enzyme concentration E , of the intermediate form:

rd = kdET (3)

Consequently, the time course of enzyme inactivation is described by

= -k,E, dE, dt (4)

so that

ln(E, / E T ) = - kdt ( 5 )

Thus, the slope of the plot between ln(E,/E,) and time gave the value of k,. The best-fit lines for these plots were obtained by linear regression techniques and are presented in Figures 1 and S 5 . The apparent deactivation rate constants for different preparations of BumHI are shown in Table III. It is clear that the apparent deactivation rate constants increased with an increase in temperature for the native and cross-linked BumHI. Also, the deactivation rate constants of all cross-linked enzyme preparations were lower than that of the native enzyme at all studied temperatures, im- plying an increase in their stability over the native enzyme. DTBP-cross-linked BamHI with the lowest value of k, at a particular temperature was therefore the most heat-stable enzyme. The apparent activation energy for deactivation was calculated by employing an Arrhenius relationship:

k, = AePEiRT (6)

(7)

where k, is the deactivation rate constant (h-’), A is a fre- quency factor (h-I), E is the activation energy for deactivation (cal/mol), R is a universal gas constant (cal/mol K), and T is absolute temperature (K).

An Arrhenius plot, In k, vs. 1/T, gave a straight line (Fig. 6 ) with a slope of -E /R in accordance with equa- tion (7). The activation energies for native and DMA-, DMS-,

or

In k, = 1nA - E/RT

Table 11. Half-life of native and cross-linked EamHI at different temperatures.

Half-life (h) BamHI

preparation 25°C 30°C 35°C 40°C 45°C 50°C

Native EarnHI 3.0 2.8 1.2 0.7 0.3 0.2 DMA-cross-linked Burn HI - 4.8 4.0 3.5 3.2 2.6 DMS-cross-linked Barn HI - - 5.3 4.3 3.8 3.5 DTBP-cross-linked Bum HI - - 5.5 5.3 4.5 4.0

DUBEY ET AL.: STABILIZATION OF RESTRICTION ENDONUCLEASE BamHl 1315

Table 111. Apparent values of deactivation rate constants k,, of native and cross-linked BamHI.

l . b t

1 2 - E 10- 0 - 2 0.8- 0 : 0 6 -

5 0 4 -

I e

c 0 2 - “ 0 al 0 0.2

kd 0- l )

Temperature Native DMA-cross-linked DMS-cross-linked DTBP-cross-linked (“C) BamHI Ram HI Bum HI Bum HI

-

-

25 0.90 0.25 30 1.05 0.35 35 1.13 0.39 40 1.22 0.42 45 3.12 0.52 50 5.10 0.68

0.23 0.25 0.29 0.34 0.39 0.43

- 0.18 0.26 0.29 0.36 0.39

h

I 1 I 3.2 3.3 3.1

0.1 L // ’ 3.0 3.1

ip x T O ~ ~ K - ’ )

Figure 6. Arrhenius plot for native and cross-linked BamHI. Activa- tion energies were calculated from slope of line and were as follows: (0) native BamHI, 2.63 kcal/mol; (A) DMA-cross-linked BamHI, 5 .24 kcal/mol; (0) DMS-cross-linked BamHI, 6.55 kcal/mol; (0 ) DTBP-cross-linked BamHI, 9.20 kcal/mol.

and DTBP-cross-linked enzyme were calculated to be 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. Thus an increase in the activation energies of cross-linked BamHI over that of the native Bam HI proved that stabilization against thermal deactivation was achieved upon cross-linking . The best heat-stable cross-linked enzyme preparation was that of DTBP, as it had the highest activation energy value (9.2 kcd/ mol) among all the cross-linked Bam H I preparations.

The lower value of activation energy for denaturation of an enzyme represents its lower heat stability. The unusually low value of activation energy for denaturation of BamHI observed in the present investigation, besides conf i ing the well-known fact of its heat-labile nature, gives quantitative information on its thermal denaturation. It is also to be noted that the activation energy for denaturation of the best (DTBP) cross-linked BamHI is still considerably lower than that of a number of enzymes such as trypsin, pepsin, ATPase, etc.,

having activation energies of more than 40 kcal/mol with concomitant high thermal stability.2o

The authors thank Dr. M. N. Gupta for his valuable suggestions. A research fellowship awarded to A. K. Dubey by the Council of Scientific and Industrial Research, New Delhi, and the fund sup- port received from the Department of Biotechnology, Government of India, are gratefully acknowledged.

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stabilization of restriction endonuclease bam hi by cross-linking reagents

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