[acs symposium series] emerging technologies for materials and chemicals from biomass volume 476 ||...

Chapter 17

Emerging Polymeric Materials Based on Soy Protein

Thomas L. Krinski

Protein Technologies International, Checkerboard Square, St. Louis, MO 63164

This chapter will describe the chemical modification of native soy protein, a globular protein primarily used as a food source. A review of the past technology will describe the early industrial uses of soy protein and how the protein was modified to alter its function as an adhesive. Current and future directions will show how chemical modification and alteration of protein chain association can further enhance soy protein polymers function. The past unfavorable attribute of biodegradation of the soy polymer chain may now guide the future of soy polymer derivatives as the need increases for biodegradable polymers from renewable resources.

Industrial Soy Protein of the Past

Industrial soy protein used are primarily in the coating of paper and paperboard, with minor areas in water based inks and water based adhesives. The water based adhesives include bottle label adhesives, foil laminating adhesives, and cone/tube winding adhesives.

Protein Composition and Isolation. The protein present in the soy bean is not a simple singular globulin but a mixed assortment of different types of protein fractions. These fractions consist of:

Bowman-Birk Trypsin Inhibitor. A globular protein of 2 4M molecular weight that can form dimers and trimers due to high cystine content.

Kunitz Trypsin Inhibitor. This is another low molecular weight protein (molecular weight of 21M) which possesses disulfide linkages.

Hemaglutinin. A glycoprotein with a molecular weight of 100-110M without any disulfide crosslinks.

0097-6156/92/0476-0299$06.00A) © 1992 American Chemical Society

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In Emerging Technologies for Materials and Chemicals from Biomass; Rowell, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

300 MATERIALS AND CHEMICALS FROM BIOMASS

Lipoxygenase. An enzyme with a molecular weight of 102M which can be disassociated into two near equal fragments using guanidine hydrochloride.

7S Globulin. A large associated polymer of 180-210M molecular weight consisting of nine polypeptide chains. These peptide chains can be disassociated into 2S and 5S subunits at pH 2.0. At neutral and slightly alkaline pH (pH of 7.6), the 7S can dimerize into the 9S fraction. Fully disassociated, the polypeptide chains that make up the 7S have molecular weights in the 20-25M range.

11S Globulin. A large 330-350M molecular weight associated polymer which easily disassociates into 7S size fragments. These fragments can likewise further disassociate into various smaller subunits capable of associating with one another to form dimers and trimers.

Urease. An enzyme having a sedimentation fraction equivalent to an 18S form (1).

Soy proteins are isolated by aqueous alkaline extraction of defatted soybeans. The native protein can be isolated prior to any additional treatment by precipitation at their average isoelectric point of pH 4.5 and separated from the soy sugars and salts. The molecular weight distribution by HPLC in 6 Molar guanidine hydrochloride i s represented in Figure 1. The native protein has an average molecular weight (Mw) of 192,000 daltons.

Native soy protein i s d i f f i c u l t to work with as an adhesive because the many different globulins disassociate and reassociate as the protein i s solubilized on the alkaline side. Even though the molecular weight of the native protein i s quite high and i t has good adhesive qualities, the problem of solution rheology control usually discourages i t s use (2).

Physico-chemical Treatment of Soy Protein (Caustic Treatment). Native soy protein has been modified using a controlled alkaline heat treatment of the protein while in solution. This processing u t i l i z e s three factors to control the protein reorganization: pH, temperature and time (3).

Table 1 shows the primary events which occur during physico-chemical treatment. Protein globulins slowly unfold with a minimum of backbone chain cleavage. As the chains unfold, they reorganize or reassociate, this time by hydrophobic/hydrophilic regions, this exposes more potential hydrophilic groups to aqueous contact.

A portion of the asparagine and glutamine residues are hydrolyzed to the free acids. This increases the anionic character of the protein as seen in the ti t r a t i o n curve of Figure 2.

Finally, we also achieve some internal chemical crosslinking through the formation of lysinoalanine (LAL). This occurs when the cysteine or serine residues undergo a β elimination reaction (Figure 3) in the presence of elevated ph and temperature, forming a dehydroalanyl residue. This residue can then react with lysine amine through a base catalysed reaction achieving potential crosslinking of two independent protein chains.

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17. KRINSKI Emerging Polymeric Materials Based on Soy Protein 301

6M GUANIDINE HYDROCHLORIDE pH 10 %/Min. TSK-G5000PWxl

1000 10,000 100,000 1,000,000 10,000,000

Molecular Weight

Figure 1. Native protein Mw distribution.

Table I. Caustic Treatment (pH>10.5, 40°C<Temp<70°C. Time!

1. Encourages unfolding of native globular structures.

2. Encourages Reorganization of unraveled globules by hydrophilic/ hydrophobic regions.

3. Partial hydrolysis of asparagine and glutamine primary amides.

4. Protein chain crosslinking by formation of lysinoalanine.

A C I D - B A S E B A C K TITRATION O F S O Y PROTEINS

l g PRODUCT IN 50g SOLUTION pH +5 mis. 0.5N NaOH 141

0 1 2 3 4 5 6 7 8 9 10 Mis. 0.5N HCI

Figure 2. Protein titration curve.

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Figure 4 shows the molecular weight distribution of a caustic treated protein compared with the native. With an average molecular weight of 150,000 daltons, there i s a marked shift to lower molecular weights as compared with the native protein.

The physico-chemical treatment step achieves reorganization of the protein globules/chains. Hydrophilic residues are exposed to more aqueous contact, while hydrophobics are buried. Combined with increased anionic character, the treatment greatly reduces the protein globules tendancy to self associate, which changes their rheological function and how they interact when used in coating paper (4).

Paper Coating Applications The major use of soy protein polymers is in the coating of paper and paperboard. Paper i s coated (Table 2) to improve i t s surface appearance and to make i t whiter and more uniform in color. The coatings impart a smoother surface which accepts ink uniformly without feathering of the ink.

A paper coating consists of three classes of components (Table 3) . pigment makes up most of the coating weight. The combined use of different levels of protein, latex, and starch provides the adhesive which holds the pigment on the paper surface.

Coating rheology influences how well the coating can be applied to the paper surface. The hercules rheogram (52% solids, 4400 RPM) of the native protein (Figure 5) has a much higher viscosity than the caustic treated protein rheogram (Figure 6). Physico-chemical treatment reduces not only the internal reactivity of the protein chains but also the reactivity of the protein with pigments such as clay. This leads to more uniform application of the coating to the paper surface with improved overall surface properties.

Chemical Modification of Soy Proteins

Recent technology indicates that chemical modification of some of the amino acid residues can provide the a b i l i t y to design new protein polymers with more specific functionality changes than simple caustic treatment can achieve. Both of these protein modification tools can be combined to further enhance the protein modification and extend the application.

In chemical modification of the proteins, we must keep in mind that the hydrophilic amino acids (Table 4) due to their reactivity provide the best targets considering the aqueous conditions under which these proteins are processed (5).

Specific amino acid residues can be targeted (figure 7), by choosing the proper reaction pH (6).

Anhydride Reactions. Acid anhydrides reacting with pendant free amine groups can be used to modify the protein. A primary anhydride such as acetic (Figure 8), can be used to react with the amine groups negating their cationic charge. This tends to decrease solution and paper coating viscosities of the soy protein (7).

The use of di and tricarboxylic anhydrides enables the modification not only to negate the cationic amines, but also to

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Ρ Η Η R - N ,

τ / \ Ε — C CH 2 _SH I II Ν Ο

Ρ Η u R —Ν ;

τ / \ Ε - C C H 2 - O H I II

Ν Ο

OH"

° — - CH-CH2-NH(CH2)4-CH — Τ / \

^ Lysinoalanine

ρ Η R — Ν ° C • CH2+H2S or Η 2 0

E - C /

I II Ν Ο

OH" ρ Η 9 . 0

Ρ / R

H2N(CH2)4-CH 0

Ν Τ Ε I Ν

Figure 3. Protein crosslinking by lysinoalanine formation.

0 1000 10,000 100,000 1,000,000 10,000,000 Mo lecu la r Weight

Figure 4. Protein Mw distribution.

Table II. Primary Use Paper Coatings General Coating Applied to Paper

ο Improved appearance ο Improved color ο Improved printing surface

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Table III. Primary Use Paper Coatings Composition (Aqueous)

o Pigment Clay Calcium Carbonate Titanium Dioxide

ο Adhesive Soy Protein Latex Starch

ο Minor Additives

COATING RHEOLOGY OF NATIVE SOY PROTEIN

S h e a r Ra te

Shear Stress Figure 5. Native protein rheogram.

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COATING RHEOLOGY OF HYDROLYZED SOY POLYMER

S h e a r Ra te

Shear Stress

Figure 6. Soy polymer rheogram.

TABLE IV. AMINO ACID COMPOSITION OF SOY PROTEINS

H Y D

R Ο Ρ Η

L I C

AMINO ACID CYSTEINE PROLINE

THREONINE

TYROSINE

ASPARTIC ACID

ASPARAGINE

GLUTAMIC ACID

GLUTAMINE

HISTIDINE

LYSINE

ARGININE

SERINE

}

}

MOLE% 0.68

6.33

4.01

2.64

11.33

18.11

SIDE OROVP -CH2SH

2.19

5.50

5.56

6.34

Ν CO-

- CH (CH^OH

- C H 2 (5> OH - C H 2 C 0 2 Η

Ο Κ

- C H 2 C ^

NH 2

- C H 2 C H 2 C02H

•° - C H 2 C H 2 C^

NH 2

-CH 2 ^

ΗΝ Ν

-CH 2 C H 2 C H 2 C H 2 NH 2

Η NH

- C H 2 C H 2 C H 2 N C * '

• C H 2 OH NH 2

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306 M A T E R I A L S A N D C H E M I C A L S F R O M B I O M A S S

replace them with additional acid groups (Figure 9 & 10). The increased anionic character is evident in the t i t r a t i o n curves of Figure 11 where di and tricarboxylic anhydrides were used to modify the soy protein. This type of modification in coatings tends to decrease the viscosity of soy protein even further (Hercules Rheogram in Figure 12). Increased anionic charge on the protein further reduces the associative nature of the protein and increases i t s dispersant nature. A shift to lower average molecular weight (133,000 daltons - Figure 13) occurs due to increased processing time and increased dissociation of the soy globulins.

Crosslinking Reactions. Soy proteins can be mildly crosslinked through the use of various simple or complex reagents. Using epichlorohydrin (Figure 14), the soy protein molecular weight and solution viscosity can be increased by varying the amount of reagent used.

A more complex monomer such as methyl acrylamidoglycolate methyl ester (MAGME) which has the a b i l i t y to crosslink both during i n i t i a l reaction and later during application (Figure 15) can be used (8). Under basic conditions, the primary reaction with the protein can occur either at the double bond or at the activated ester. At elevated temperatures, the proton on the amide group i s acidic enough to encourage further crosslinking at the amide nitrogen. This second reaction could occur during the soy polymers use in the coating-drying-calendering of paper.

Base Catalysed Reactions. Due to the alkaline processing conditions, nucleophilic addition reactions are excellent f i t s for modification of soy protein and can be tailored even to selective amino acid residues, water soluble monomers such as acrylamide (Figure 16) and acrylonitrile provide excellent reactivity with lysine amine groups and sulfhydryl of cysteine.

Hydroxyacrylates can also be used to modify any free amine groups and add additional hydroxyl residues to the protein (9). In paper coating applications, this allows the protein to hydrate more thus adding water holding to the paper coating as i t i s applied. The runnability of the coating as i t ' s applied to the paper is improved.

Exotic monomers, such as glycidyl oxypropyl trimethoxy silane, can add a silane ester to the protein amines (10). This modification greatly increases the viscosity of the protein with clay pigments or causes total destabilization of the clay suspension.

Interpolymer Technology. When soy polymers are used in the coating of paper, a styrene butadiene latex is also used. The increased dispersant action and lower viscosity of the anionic modified soy protein has been used in the preparation of various latices (Figure 17). Monomers can be emulsified by using the soy polymer as the only surfactant. These miscelles can then be polymerized in the presence of the soy polymer resulting in a soy latex interpolymer whose core is stabilized by a "surfactant shell" of soy protein. Figures 18 and 19 show the particle size analysis of such a styrene butadiene latex with an average size of 0.2 micron. This is

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THEORETICAL IONIC STATES OF AMINO ACID DI^OCIATION S I D E CHAINS AS A FUNCTION OF THE pH.

1.0 ι

e x .

0.8

0.6

0.4

0.2

° S BH*

-COOH

Figure 7. Ionization of side residues.

OH-Prot-NH2 + (CH3CO)20 — Prot - NH - C - CH 3 + CH 3 COO "

Ο

Figure 8. Anhydride reaction with amine groups: monocarboxylic anhydride.

Prot-NH2 + C H 2 - C ^

ι y Qu-CH 2 - C ' • Prot - NH - C - CH 2 - CH 2 - C '

o H x o -o

Figure 9. Anhydride reaction with amine groups: dicarboxylic anhydride.

Figure 10. Anhydride reaction with amine groups: tricarboxylic anhydride.

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A C I D - B A S E B A C K TITRATION O F S O Y PROTEINS

lg PRODUCT IN 50g SOLUTION pH +5 mis. 0.5N NaOH 14,

ol 0 1 2 3 4 5 6 7 8 9 10

Mis. 0.5N HCI Figure 11. Protein titration curve.

COATING RHEOLOGY OF ANIONIC S h e a r Ra t e S O Y POLYMER

Shear Stress

Figure 12. Soy polymer rheogram.

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%/Min . 2

6 M GUANIDINE HYDROCHLORIDE pH 10 TSK-G5000PWxl

-An ion i c -Hydro lyzed * Nat ive

1000 10,000 100,000 1,000,000 10,000,000 Mo lecu lar Weight

Figure 13. Protein Mw distribution.

NaOH C H 2 - C H - C H 2 C I + Prot - N H 2 H 2 C-CH-CH 2 -HN-Pro t + NaCI

^ O V 2.

P ro t -NH-CH 2 -CH-CH 2 + Prot-NH 2 Prot-NH-CH 2 -CH-CH 2 -X-Prot \ / O H - 1

Ο + Prot-SH OH

+ Prot-OH

Figure 14. Crosslinking reactions using epichlorohydrin.

METHYL ACRYLAMIDOGLYCOLATE METHYL ESTER

C H 3 O C H 3

H 2 C = c' I C-NHCH-C-OCH3 n 11 • ο O I

A Β

Figure 15. Crosslinking reactions using MAGME.

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UNSATURATED CARBONYLS C A N REACT WITH UNPROTONATED AMINES

N H 2 pH 9.0 N H 2

C H 2 = C H - C = 0 +Prot- N H 2 — • Prot - NH - C H 2 - C H 2 - C = Ο

ARCYLAMIDE

Figure 16. Base catalyzed additions (nucleophilic).

· · · · · · · I taê M t f ^ f v N ^S!t\

· · · · · · · i · · « · • - {λ···\ • £ 3 K M

· J O V ' ^BSr Protein Soin Monomers Emulsified Polymerized

Monomer Interpolymer The Interpolymer is prepared by the free radical polymerization of styrene and butadiene in the presence of modified soy protein. The monomers are first emulsified by the protein. These emulsified particles are spherical in shape with the hydrophillic soy protein covering the surface and the hydrophobic monomers buried in the interior. The soy protein is polymerization.

Figure 17. Interpolymer preparation.

SOY INTER POLYMER PARTICLE SIZE ANALYSIS

Volume %

Particle Diameter (um)

Figure 18. Interpolymer particle size.

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SOY INTER POLYMER PARTICLE SIZE ANALYSIS

Number %

1 5 10 50 100 500 1000 Particle Diameter (um)

Figure 19. Interpolymer particle size.

comparable to commercial latices yet, when used in the coating of paper and paperboard, possesses the advantages of both soy polymer and S/B latex (11). Through the use of chemical modification, we have brought the old caustic treatment technology of soy protein out of the dark ages. Combining the caustic treatment step with chemical modification has enabled us to change substantially the functionality of the protein polymer by modifying a small number of amino acid residues. Using these "tools", we can alter the reactivity/associativity of the globular protein of the native soy with i t s e l f , alter i t s molecular weight and generally provide a more uniform protein.

Future Soy Polymers

Previously, most industries had considered soy proteins s t r i c t l y as adhesives (12). However, this attitude has dramatically changed as the chemistry of the soy protein has been modified. Modern soy polymers are now being used as paper pigment structuring agents and flow modifiers. Their amphoteric nature has shown they can be used as protective colloids and even surfactants in the stabilization of latices.

The use of chemical modification to change soy protein function is s t i l l in i t s infancy. As we learn how to use more reagents to modify the protein in conjunction with other treatments which can control protein reorganization, we can further specialize the functionality of the finished soy polymer. In doing so, we can expand the use of soy polymers into other industries.

Use of soy polymers in aqueous based inks has increased due to the environmental concerns of hazardous fumes from solvent based inks. As environmental concerns of biodegradability, waste

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disposal, and sewer effluent treatment become issues, new soy-polymers could be offered as solutions.

Creation of new soy interpolymers, solution viscosity modifiers, flocculating agents and ingredients for biodegradable plastics, are potential avenues for future soy polymer technology.

References

1. Smith, Α. Κ., and Circle, S. J . , Soybeans - Chemistry and Technology; 1978, Vol. 1; Proteins, pp. 114-128.

2. Coco, C. E. , Krinski, T. L. , Tappi Coating Binders Seminar, 1986.

3. Ishino, K, Okamoto, S., "Molecular Interaction in Alkali Denatured Soybean Proteins"; Cereal Chemistry 52, 9, 1975, p 19.

4. Coco, C. E. , Preprints Tappi Coating Conference, 1987. 5. IBID. 6. Means, G. E. , Feeney, R.E., Chemical Modifications of

Proteins, 1971, p 14. 7. Coco, C. E. , Krinski, T. L. , Graham, P. Μ., U.S. Patent

#4474694. 8. Steinmetz, A. L. , Krinski, T. L. , U.S. Patent #4554337,

11/19/85. 9. Krinski, T. L. , Steinmetz, A. L. , U.S. Patent #4687826,

8/18/887. 10. Krinski, T. L . , Steinmetz, A. L. , U.S. Patent #4713116,

12/15/87. 11. Coco, C. E. , Preprints Tappi Coating Conference, 1987, pp.

133-140. 12. Strauss, R. W., Protein Binders in Paper and Paperboard

Coating; Tappi; Monograph No. 36. RECEIVED July 9, 1991

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[acs symposium series] emerging technologies for materials and chemicals from biomass volume 476 ||...

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