Biocatalyst Design for Stability and Specificity - American Chemical

Chapter 25

Cross-Linking Techniques

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Applications to Enzyme and Protein Stabilization and Bioconjugate Preparation Munishwar N . Gupta Chemistry Department, Indian Institute of Technology, Delhi Hauz Khas, New Delhi-110 016, India

Chemical crosslinking technology was used for (a) enhancing the stability of trypsin, β-galactosidase, and concanavalin A and (b) forming protein-protein conjugates viz. trypsin-chymotrypsin, trypsin-β-galactosidase, and concanavalin Α-β-galactosidase. For stabilization of the three proteins, dimethyl adipimidate was found to give the best results. The intramolecularly crosslinked trypsin, with an average of 9.5 groups modified out of 14 free amino groups present, showed a much slower rate of autolysis at 40°C compared to native trypsin. Crosslinked β-galactosidase entrapped in polyacrylamide hydrolyzed 47% of milk lactose in 6 h and at 55°C. Entrapped native enzyme hydrolyzed only 31% substrate under the same conditions. Besides preparing an insoluble aggregate of trypsin-chymotrypsin and β-galactosidase, conjugates of trypsin-chymotrypsin, trypsin-alkaline phosphatase, and Concanavalin Α-β-galactosidase were also prepared and evaluated.

There is near unanimity about unfolding of the polypeptide chain as being a primary event in the denaturation of proteins (1). For example, in thermal denaturation, this is the initial reversible step (2). Thus, it follows that any approach which makes proteins rigid (reduces conformational flexibility) should impart stability. Three distinct approaches attempt to do this by creating additional linkages with parts of the polypeptide chain:

0097-6156/93/0516-0307$06.00/0 © 1993 American Chemical Society

Himmel and Georgiou; Biocatalyst Design for Stability and Specificity ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

BIOCATALYST DESIGN FOR STABILITY AND SPECIFICITY

308 (1) .

Immobilization (2-4)

(2) .

Introduction of disulfide bridges by protein engineering (5)

(3) .

Chemical crosslinking (6)

The present chapter would limit itself to the third approach. Apart from protein stabilization, another major application of crosslinking technology has been linking of different biological molecules by intermolecular crosslinking. Such bioconjugates have already found considerable applications in such diverse areas as bioconversion (7), medicine (S), and bioseparation (9). This chapter would also describe some work on heteroenzyme conjugates and other bioconjugate preparations using intermolecular crosslinking. Crosslinking Methodology When a crosslinking reagent reacts with a protein molecule, several kinds of reaction products are possible, including intramolecularly crosslinked proteins, oligomers formed due to intermolecular crosslinking, and insoluble protein aggregates. However, it is possible to design a crosslinking experiment so as to obtain the desirable product as the major reaction product. Some general guidelines mentioned by Wold (10) many years ago are quite useful for this purpose: (1) .

High protein concentration would favor intermolecular crosslinking over intramolecular crosslinking.

(2) .

Ph corresponding to minimum net charge on the protein would favor intermolecular crosslinking.

(3) .

High reagent to protein ratio and prolonged reaction time would favor extensive crosslinking and may result in insoluble protein aggregates.

(4) .

Successful formation of crosslinks depend upon the availability of suitable reactive groups within the effective range of the reagents. In the context of intramolecular crosslinking, this means that the "span" of the crosslinking reagent is a crucial parameter.

Enzyme Stabilization Chemical Crosslinking of Trypsin. The property of trypsin to undergo autolysis in solution has resulted in this enzyme being a favorite target of various approaches of enzyme stabilization (11-13). Attempts at chemical crosslinking of trypsin with glutaraldehyde and two bisimidoesters [i.e., dimethyl suberimidate (DMS) and dimethyl adipimidate (DMA)] indicated that bisimidoesters are a better choice for obtaining trypsin preparations with decreased autolysis (74). Best results were


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obtained with D M A (14.3 mg/ml) using trypsin concentration of (0.25 mg/ml) in Tris-HCl buffer (0.2 M , pH 8.3) containing 25 m M C a ~ (75). Chromatography on CM-Cellulose showed that the crosslinking preparation did not contain any unmodified trypsin. Free amino group analysis showed that an average of 9.5 residues out of 14 present in trypsin were modified. SDS-PAGE analysis showed that no intermolecular crosslinking has occurred. Some characteristics of the crosslinked trypsin are summarized in Table 1 (75). The crosslinked preparation showed much slower rate of autolysis at 40°C as compared to native trypsin (Figure 1). In this particular system, the formation of crosslinks was presumed and not verified. It was also not determined whether the decrease in autolysis was caused by decrease in number of bonds susceptible to tryptic cleavage or crosslinking per se.

Lactose Intolerance; Whey Disposal Through Chemical Crosslinking. Another enzyme chosen for chemical crosslinking was β-galactosidase. The choice of this enzyme was based upon its biotechnological usefulness (16). This usefulness arises because of lactose intolerance and whey as a biomass (16). Lactose Intolerance. It is a metabolic disorder which is associated with the lack of adequate β-galactosidase activity. There are significant differences in the incidence of lactose intolerance among different ethnic groups. The adult intolerance has so far been observed only in northern Europeans (90%) and in the members of two nomadic pastoral tribes of Africa (80%) (17). Hydrolysis of milk lactose yields low lactose milk. Such a preparation, apart from being low in lactose content, also retains most of the other nutrients present in the milk. Many commercial technologies for production of low lactose milk utilize immobilized β-galactosidase (lactase) (18). Whey as a Biomass. A large amount of milk is converted into whey during the manufacture of cheese. The use of lactose present in whey in food industries has certain associated problems. Whey, as a waste product, cannot be disposed of easily as such because of its high biological oxygen demand (BOD) value. The hydrolysis of whey lactose by β-galactosidase to glucose and galactose solves these problems. These sugars have greater fermentation potentials as compared to lactose. Also, the hydrolyzed whey can also be used in food industries as such (16). Crosslinking of β-Galactosidase. The above considerations prompted us to attempt crosslinking of E. coli β-galactosidase—a commercially available and well characterized enzyme. The crosslinking reagents employed in this work (79), i.e., glutaraldehyde, D M A and D M S , were specific for free amino groups in proteins. Therefore, they were a safe choice for E. coli β-galactosidase since it is reported that the lysine groups in the enzyme are not involved in the catalysis and the enzyme retains 80% of its activity even after all its lysine groups are modified (20). Exploratory experiments indicated that the crosslinked derivative obtained with D M A was most thermostable (79). The free amino group analysis showed that 38% free amino groups were modified. The SDS-PAGE analysis showed that neither


310


Table 1. Properties of Trypsin Crosslinked with Dimethyl Adipimidate A l l percentage values are relative to the observed value for native trypsin taken as 100

Property 1. Amidase activity towards benzoyl DL-arginine p-nitroanilide (ΒΑΡΝΑ)

113%

2. Esterase activity with p-tosyl L-arginine methyl ester (TAME) as substrate

65%

3. Proteolytic activity towards: (a) Casein (b) Haemoglobin

27% 22%

4. 1^ for ΒΑΡΝΑ at pH 8.2

1 m M , same as that for native trypsin

5. pH optimum with ΒΑΡΝΑ as substrate

Broad range of 7-9, similar to native trypsin

4

6

10

TIME (hrs)

Figure 1.

Autolysis of native and DMA-crosslinked trypsin at 40°C. The protein concentration was 125 μg/ml. (Reproduced with permission from ref. 15. Copyright 1988 ButterworthHeinemann.)


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intersubunit nor intermolecular crosslinks had been formed. When the enzyme was treated with ethyl acetimidate (a monofunctional analog of D M A ) , although extent of free amino group modification was comparable (26%), the o-nitrophenyl β-Dgalactopyranoside (ONGP) activity of the modified preparation, both before and after heat treatment (i.e., the enzyme preparations were heated at 55°C in 0.3 M sodium monophosphate buffer p H 8.0 containing 3 m M M g C y was quite different as compared to the crosslinked enzyme (Figure 2). While this data did not completely rule out the possibility of some simple chemical modification also having taken place in case of reaction with D M A , it did confirm that the desirable change in the enzyme was the result of formation of intramolecular crosslinks (79). Continuous hydrolysis of milk lactose at 50°C was monitored by using both native and the crosslinked enzyme (Figure 3). Whereas the native enzyme hydrolyzed 40% milk lactose, the crosslinked enzyme hydrolyzed 55% milk lactose in 12 h (75). This kind of conversion rate is considered adequate for obtaining low lactose milk based dairy products at the pilot plant level (27). In order to be able to reuse crosslinked enzyme, it was entrapped in polyacrylamide (22). The optimization of entrapment conditions was carried out with the native enzyme, and our results show that 50% of the enzyme activity on ONGP was entrapped and the enzyme lost 20% activity during entrapment. The activity of the entrapped enzyme was considerably enhanced when a protective mixture of bovine serum albumin, cysteine, and lactose was present during entrapment. The enzyme crosslinking with D M A and entrapped was also found to be more thermostable as compared to other entrapped enzyme preparations (Figure 4). The hydrolysis of milk lactose was carried out using native and D M A crosslinked enzymes (Figure 5). The crosslinked preparation entrapped in polyacrylamide hydrolyzed 47% of milk lactose as compared to 31% hydrolysis by entrapped native enzyme in 6 h at 55°C (22). The above system illustrates the usefulness of combining crosslinking with immobilization for obtaining a reusable product with enhanced thermostability. Insoluble β-Galactosidase Aggregate (23). Another way of obtaining a reusable enzyme preparation by crosslinking is to form chemical aggregates. E. coli β-galactosidase aggregates prepared by extensive crosslinking with glutaraldehyde retained 63% of the activity on ONGP provided bovine serum albumin and lactose were present during the crosslinking at 4°C. This activity increased to about 70% when the aggregate was homogenized for about 90 s in a mixing blender. A l l further work discussed below was carried out with the aggregate which had been subjected to this treatment. The 1^ value of the aggregate for ONGP was found to be 6 χ ΙΟ M at p H 7.5 and 25°C as compared to a value of 2.8 χ 10 M for the native enzyme. The p H optimum was found to remain unchanged at 7.5. The aggregate showed considerable improvement in thermal stability at 55°C (Figure 6) which was reflected in its improved performance during continuous hydrolysis of milk lactose (Figure 7). One reason why enzyme aggregates have not become popular is that they are difficult to handle and would give poor flow rates in columns. A solution.to this -4

-4


312


-

NATIVE β-GALACT OSIDASE

100-

DMA TREATED β - GALACT0SIDA5E

ETHYL ACETIMIDATE TREATED β - GALACTOSIDASE

0

\

- \ *

40|

NI

20H

1

1

t

2

4

6

TIME OF INCUBATION (hrs)

Figure 2.

Comparison of the stability of native β-galactosidase with β-galactosidase modified with ethyl acetimidate and D M A respectively. (Reproduced with permission from ref. 23. Copyright 1988.)

NATIVE β-GALACTOSIDASE D M A CROSSLINKED β-GALACTOSIDASE

3

6

TIME OF INCUBATION

Figure 3.

9

12

(hrs)

Continuous lactose hydrolysis of milk by D M A modified and native β-galactosidase at 50°C. The enzyme concentration for both native as well as modified preparation was 25 pg in 2.0 ml reaction volume. The initial lactose concentration in the milk was 5%. (Reproduced with permission from ref. 25. Copyright 1988.)


25. GUPTA


CD*

01 0

! 1

1 2

1 3

ι 1 4

TIME OF INCUBATION (hrs)

Figure 4.

Thermal stability of entrapped β-galactosidase preparations at 55°C in sodium phosphate buffer (20 m M , pH 7.5 containing 0.1 M NaCl and 3 m M MgCl ). O ; Native enzyme entrapped in absence of any protective agent Δ ; D M S crosslinked enzyme entrapped in absence of any protective agent • ; D M A crosslinked enzyme entrapped in absence of any protective agent · ; Native enzyme entrapped in presence of bovine serum albumin (BSA) + cysteine + lactose D M S crosslinked enzyme entrapped in presence of B S A + cysteine + lactose • ; D M A crosslinked enzyme entrapped in presence of B S A + cysteine + lactose (Reproduced with permission from ref. 32. Copyright 1988.) 2


314


INCUBATION TIME

Figure 5.

Time course of hydrolysis of milk lactose at 55°C. O ; Native enzyme entrapped in absence of any protective agent · ; Native enzyme entrapped in presence of B S A + cysteine + lactose • ; D M A crosslinked enzyme entrapped in absence of any protective agent • ; D M A crosslinked enzyme entrapped in presence of B S A + cysteine + lactose (Reproduced with permission from ref. 33. Copyright 1988.)

TIME OF INCUBATION

Figure 6.

(hrs)

(hrs)

Thermal stability of β-galactosidase preparation at 55°C. The enzyme activity was determined with ONGP as the substrate. (Reproduced with permission from ref. 35. Copyright 1988 Indian Academy of Sciences.)


25. GUPTA


315

problem may be to entrap proteins by aggregation within commercially available beads. Chemical aggregation of β-galactosidase by glutaraldehyde inside Sephadex G-200 beads showed that about 17% of the total enzyme activity added at the start was present inside beads (24). The 1^ towards ONGP (6.3 χ ΙΟ M) in this case was nearly the same as that of the simple aggregates (24). There is obviously need to improve upon these results but they are encouraging. -4

Thermostabilization of Concanavalin A (25). Concanavalin A (Con A), is a lectin of considerable biological interest. Our interest in thermostabilization of Con A stems from the usefulness of this lectin in affinity separations and bioaffinity immobilization. Crosslinking of Con A (1 mg/ml) was carried out at pH 7.5 using D M A (50 mg/ml) in the presence of α-methyl mannoside—a sugar specific for Con A . The crosslinked preparation (112% activity when measured in terms of its ability to precipitate glycogen) was fractionated on Mono S column of F P L C . It resolved into three fractions. The major fraction containing 52% of the total protein was further purified on Mono Q column of F P L C . About 80% of the protein did not bind to the column. This fraction (which did not bind to Mono Q column) showed a single band on SDS-PAGE corresponding to the position of Con A monomer. This, apart from establishing homogeneity, also ruled out inter subunit and intermolecular crosslinking. The crosslinked derivative had 98% activity as compared to native Con A and free amino group analysis showed that 17% of amino groups were modified in this derivative. This corresponded to about 2 amino residues per Con A subunit. The gel filtration of the derivative on a calibrated column of Fractogel HW-55F showed its molecular weight to be identical with native Con A . Thermal stability of this crosslinked derivative, native Con A and ethylacetimidate reacted Con A at 70°C is shown in Figure 8. Thus, mere modification of the amino groups in Con A with monofunctional analog did not lead to thermostabilization. These results indicate the formation of one crosslink per subunit in Con A molecule as the cause for thermostabilization. Based upon earlier structural data in the literature, the position of the crosslink can be tentatively assigned between Lys-135 and Lys-138. Becker et al. (26) have pointed out that residues 131-168 form part of disordered structure in the molecule. Perhaps, this crosslink introduces an element of order in this region, it reduces conformational flexibility and hence leads to considerable thermostabilization. Bioconjugate Preparation One class of bioconjugates are the protein-protein conjugates. Enzyme linked immunoassays (27), immunotoxins (2

Biocatalyst Design for Stability and Specificity - American Chemical

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