Protein Based surfactant‘s Production:

 Surfactants are based on renewable raw materials (i.e., proteins

and fatty acids)
 Solution to waste disposal for animal and vegetable protein
byproducts.
 Proteins are by nature amphipathic or amphiphilic molecules
(contain both a hydrophobic (non polar) and a hydrophilic (polar)
moiety) and are similar to synthetic surfactants.
Hence, affords them certain degree of surface activity.
 Natural proteins are not used as commercial surfactants. Rather,
proteins are modified by chemical or enzymatic means to
products with surface-active properties.
 Basic form of amino acids has two functional groups:
a) amine group and b) carboxyl group and a Side group, R
 Surfactant Type Surfactant Structure Obtained by:

N - Substituted Anionic by condensation of a fatty

surfactants acyl radical with the α-
Eg: Long chain N- amino group of a
acyl amino acid neutral or acidic amino
acid.

C - Substituted Cationic long chain alcohols or

surfactants amines react respectively
Eg: Amino acid with the α- carboxyl
long chain esters or group of a neutral or
amides basic amino acid

N- alkyalated Amphoteric reaction of primary,

surfactants secondary or tertiary
Eg: Long chain N- long chain alkyl
alkyl amino acids halides with amino
2 acids as starting material.
The main molecular properties of proteins responsible
for their surface activity are:
Influences adsorption Determines the
and orientation of surface activity of
proteins at interfaces proteins in a
Polar, non- particular system
and correlates with polar, and
surface activity. charged
amino acids

Lipophilicity
Structural
features
MOLECULAR
PROPERTIES
Allows the
protein to bind
with surfaces of
different chemical
nature Stability
Amphipathicity
 Acylation
 Acylation: Most important chemical means to modify protein functionality.

 Acylated protein hydrolysates are known to be very mild surfactants and

when added along with irritating bulk surfactants gives good compatibility
with the skin.

 For N-acylation - fatty chain can be introduced via the amine or carboxylic
function of amino acid.

 Acylating agents: acyl anhydrides, acyl chlorides, and N-hydroxy succinimide

ester.

 Hence, the hydrophobicity and surface activity of the protein

molecule is thus tailored by controlling the molar ratio of the
acyl-chlorides to proteins.
 Protein based synthesis of surfactants using meal.
 Study of surfactant properties - emulsifying, detergency, wetting,
solubilizing, foaming, or dispersing functions and compare it with standard.
 Try to produce surfactant by green and natural way.
 The procedure is illustrated as:
 Raw Material for
Castor meal as a protein base:

Castor Meal Contents ( % ) • Castor De-Oiled Cake is obtained from

castor cake after the multistage solvent
Moisture 12 (max) extraction.

Crude protein 32 (approx) •De-Oiled Castor Cakes are excellent product

used for organic fertilizer.
Nitrogen 4 (min)

Potassium 1 (min)

Phosphorus 1 (min)
Reference:
Oil Content 0.5 (max) Proximate Analysis Of Castor seeds and Cake.
ANNONGU, A. AZOR; JOSEPH, J. K. J.
Appl. Sci. Environ. Manage. March, 2008
Vol. 12(1) 39 - 41
 Amino acid composition of Castor protein (%)
Amino acid, g/16gN Undecorticated castor seed Decorticated castor seed cake
Arginine 8.00 9.36
Histidine 1.38 1.88
Lysine 4.11 4.48
Serine 2.13 3.02
Tryptophan - 1.30
Alanine 3.96 5.02
Cystine 0.53 0.86
Methionine 2.06 2.86
Threonine 3.00 3.88
Leucine 5.90 8.56
Isoleucine 3.09 1.61
Valine 5.46 4.93
Proline 2.76 3.08
Glycine 0.56 1.11
Glutamic acid 13.19 15.64
7
Aspartic acid 6.42 7.66
 Contd…

 Fatty acyl chloride: Acid chloride of C12 or C16.

 Preparation of Fatty Acid Chlorides:

 In a four necked round bottom flask equipped with stirrer and thermo
pocket charge 0.1mole of Fatty Acid at room temperature and heat to 40°
C.
 Add 0.15mole of thionyl chloride under Nitrogen atmosphere over a
period of 1 hour
 Heat the reaction mixture to reflux for 1.5 hr to obtain crude Acid
Chloride.
 Reactions
 Protein content in a castor meal:
Kjeldahl method: heat (Burner)
Sample + conc. H2SO4 mixture will turn green
(CuSO4 + K2SO4 )(1:10) ( end of digestion of the sample
due to release of nitrogen)
%N = (Blank – Sample) * N * 1.4/ wt. of sample
%Protein = %N * factor
Given: Factor for soybean protein = 5.71

 Isolation of protein from oilcake: Salting out process.

Castor meal  Grind  Powdered meal + 0.5% solution of NaOH (1:20 w/v cake: alkali)

50oC, 3 hrs Reflux

continuous stirring
Centrifuge the mixture

Discard the Residue Aqueous part

Adjust pH to isoelectric point of soybean protein,
i.e., 4.5 by addition of dil. HCl
Protein precipitates out and further wash with water and dry.
i) Acid Hydrolysis- Heat 107oC, 22hrs
Protein isolated from castor meal + 6N HCl continuous stirring, reflux

(1:3 Protein : HCl) Protein hydrolysate

(aqueous solution)

ii) N-acylation (Schotten–Baumann method) – To give surfactant mixtures

1) acid chloride + amine  amide + H+ + Cl-

2) Add aqueous solution of base slowly to the mixture to absorb this acidic proton,
or the reaction will not proceed. (During reaction, pH 11-12)

Acid chloride
(C12 and C16) Na acyl amino acid
pH =1
 Acylation of amino acids:
 Acylation of :L- Valine, L- Serine

 Solvent Extraction of surfactant:

Solvents used: Ethyl acetate, acetone, carbon tetrachloride, benzene,
toluene.
Proportions: 1:1 V/V
Extraction time: 1 hr
Seperation: after 1 hr, centrifuge @ 10000rpm, 30 min

Observations: Emulsion breaks fast in acetone with clear layer.

Carbon tetrachloride gives reverse layers.
Biosurfactants containing amide bonds are a particularly attractive
class of compounds !!!
- Potential substitutes for emulsifiers derived from petroleum

- Lower potential toxicological effects of amide than those of

emulsifiers derived from petroleum.

- Skin tolerance, good biologic degradability and low toxicity.

- Amide linkages are very stable chemically and are not easily
degraded in alkaline media.
 Potential alternative to chemical synthesis is based on the use of
biocatalysts.
 Lipoamino acid or lipopeptide compounds constitute an interesting
alternative to conventional synthetic surfactants in which the three
fundamental requirements for industrial development are present:
(a) multifunctionality
(b) low toxicity and
(c) renewable sources of raw materials.

 Enzymatic synthesis presents several advantages:

[1] Enzymes work under mild conditions,
[2] These are regio-, stereo- and chemoselective
[3] There is no need for protection/deprotection of the precursor reagent,
and
[4] No by-products.
 Lipoamino acid or lipopeptide compounds could be defined as
amphiphilic molecules that contain one amino acid residue as the
hydrophilic part and at least one long chain as the hydrophobic part.

 Many amino acid derivatives with different structures can be obtained by

the reaction of amino acids with long chain compounds such as fatty
acids, fatty esters, fatty amines and fatty alcohols.

 The fatty chain can be introduced into the amino acid structure through
(a) acyl, (b) ester, (c) alkyl or (d) amide linkages.
Amino acid based surfactants
The hydrophobic group is attached to the amino acids either at the amine moiety or at the
carboxylic moiety as follows :
Reflux
Conc.H2SO4 / HCl as a catalyst

Amphiphilic esteramine -H2O

Amidoacid
Fatty alcohol Fatty acid

Conc.H2SO4 as a catalyst

Alkyl amine Alkyl halogen

long-chain alkyl amino acid
Amphiphilic amidoamine
Reactions of -Carboxyl Group

In presence of
strong acid
 Formation of peptide bonds

 Amino acids contain two functional groups : amines and

carboxylic acids.
 Amino acids can be linked by a condensation reaction in which
an −OH is lost from the carboxyl group of one amino acid
along with a hydrogen from the amino group of a second,
forming a molecule of water and leaving the two amino acids
linked via an amide—called, in this case, a peptide bond.
 Reaction with Fatty alcohol:
 - OH group from amino acidis replaced with –OR of alcohol.
i.e the COOH group from the amino acid will form an ester bond with
the -OH group on the alcohol, with the elimination of a molecule of H20.

 Amino acids form esters when heated with alcohols using dry hydrogen
chloride as a catalyst . In these reactions the carboxylic acid group takes
part in a condensation reaction, while the amino group forms a
hydrochloride salt with the acid catalyst.

 The H+ ions can then be removed by treatment with a suitable alkali.

Plan of work:1. Formation of amide surfactants via
enzymatic amidification:
Enzymatic reactions:
 1:1 mole ratio of reactants, i.e. 0.1mmol of ethanolamine (6.1 mg) + 0.1mmol of fatty acid (17
mg of capric acid, 20 mg of lauric acid, 22.8 mg of myristic acid or 25.6 mg of palmitic acid).

 Mix with 0.5 ml of solvent (Acetonitrile, chloroform, and methanol all of HPLC quality) in
stoppered glass bottles.

 Place the solution in a thermo-constanter orbital and hold at the reaction temperature for five
minutes.

 Subsequently add 25 mg of Novozyme 435 and shake the at 200 r.p.m. for the indicated time in
the corresponding experiment.

40°C 20 min
The purification protocol for amides of different
fatty acids is as follows:
1. Carry the reaction using an equimolar ratio of fatty acid
and ethanolamine.
2. Upon completion of the reaction, remove the by filtration
through a sieve with a characteristic dimensions of
0.32mm.
3. Dissolve the product in a mixture of methanol and
chloroform [50/50 (v/v)].
4. Eliminate the solvent by evaporation in a rotovap.
5. The resulting solid is amide.
 In acetonitrile, the major product obtained from an equimolar
ratio of fatty acid and ethanolamine is the amide
(90%, together with 6% of the amide-ester in 4 h).

 When a 2–4 fold molar excess of the fatty acid is employed, one
obtains 70–90% of the amide-ester in 4–6 h.

 By contrast, in cases when an excess of ethanolamine is employed,

the intermediate amide product is the only product formed at
conversions of 95–99%.
 γ-alkyl Surfactant synthesis

Synthesis route of γ-alkyl aspartic acids

 Synthesis of γ-octyl L-aspartate:
1) Dissolve the mixture of L-aspartic acid (4g, 30mmol) and 1-octanol
(4.7g, 36.5mmol) in tert-butanol (60ml).
2) Add 95%-98% sulfuric acid 2.4 ml to the mixture dropwise by a
syringe.
3) Heat the suspension up to 65°C for 1 hour.
4) After that add ethanol (50ml) and triethylamine (6ml) to precipitate
the product.
5) Filter the solid and wash with methanol and water.

Similar procedure is applied for the synthesis of :

γ-dodecyl L-aspartate
γ-dodecyl L-glutamate
Synthesis of Di-alkyl amino acids

12 carbons
 Synthesis of aspartic acid di-tetradecylester
1) Dissolve L-aspartic acid (4g, 30mmol) and p-Toluene sulfonic acid (PTSA)
(6.5g, 32mmol) in toluene (100ml) and reflux for 1 hour at 110°C.
2) Add 1-Tetradecanol (13.7g, 64mmol) to the solution, followed by stirring
for 12 hours under reflux.
3) Evaporate the reaction mixture and dissolve in ethyl acetate (100ml).
4) Wash the ethyl acetate solution with 10% sodium carbonate solution
(100ml 2) and distilled water (100ml 1).
5) After that evaporate ethyl acetate and recrystallize the residue from
methanol to obtain a white powder.
6) Further dry such powder in vacuum.

Similar procedure is applied for the synthesis of :

glutamic acid di-tetradecyl ester
 Applications
 γ-alkyl amino acids :
- These are insoluble in water and in most common organic solvents
due to intermolecular electrostatic attraction between ammonium
and carboxyl group.
- Can be employed as the functional additives in the formulation of
powder cosmetics.

• Di-alkyl amino acids :

- These are very soluble in organic solvents but insoluble in water at
room temperature.
- They are able to form stable water in oil emulsions due to good
dispersing ability and double hydrophobic chains.
- Can be employed as emulsifier and emulsion stabilizer.
Application of enzymatically synthesized
surfactant:
 Amides/ esters can be employed in the manufacture of foods,
cosmetics, pharmaceutical products, and a wide variety of specialty
chemicals which exploit the surface active properties of these
materials.
 Several important pharmaceutical products are derivatives of
amino alcohols and long chain fatty acids.
 Both biodegradability and the relatively non-allergenic character of
mono- and diethanol amides of fatty acids depend on both the
chemical nature of the substituents on the nitrogen atom and their
chain lengths.