ASSIGNMENT ON:

SUBJECT : Biotechnology of Fermentation and Biotransformation-II

TOPIC : Adhesive Protein Biopolymer, (Byssal Adhesive, Rubber Polymerase)

SUBMITTED BY:

NAVREET BDAENG

M.S.c. Industrial

Biotechnology

IInd Semester

CONTENTS :

INTRODUCTION: BIOADHESIVE

AND BIOPOLYMER. TYPES OF ADHESIVES. BYSSAL ADHESIVES. NATURAL RUBBER AND RUBBER

POLYMERASE: PRODUCTION PROCESS

FEEDSTOCKS FOR BIOPLASTIC. COMMERCIAL APPLICATIONS. CONCLUSION. REFERENCES.

IntroductionBioadhesiveS:

Bioadhesives are natural polymeric materials that act as adhesives. The term is sometimes used more loosely to describe a glue formed synthetically from biological monomers such as sugars, or to mean a synthetic material designed to adhere to biological tissue.

Bioadhesives may consist of a variety of substances, but proteins and carbohydrates feature prominently. Proteins such as gelatin and carbohydrates such as starch have been used as general-purpose glues by man for many years, but typically their performance shortcomings have seen them replaced by synthetic alternatives. Highly effective adhesives found in the natural world are currently under investigation but not yet in widespread commercial use. For example, bioadhesives secreted by microbes and by marine molluscs and crustaceans are being researched with a view to biomimicry.

Bioadhesives are of commercial interest because they tend to be biocompatible, i.e. useful for biomedical applications involving skin or other body tissue. Some work in wet environments and under water, while others can stick to low surface energy – non-polar surfaces like plastics. In recent years, the synthetic adhesives industry has been impacted by environmental concerns and health and safety issues relating to hazardous ingredients, volatile organic compound emissions, and difficulties in recycling or remediating adhesives derived from petrochemical feedstocks. Rising oil prices may also stimulate commercial interest in biological alternatives to synthetic adhesives.

BIOPOLYMERS

Biopolymers are polymers that occur in nature. Carbohydrates and proteins, for

example, are biopolymers. Many biopolymers are already being produced

commercially on large scales, although they usually are not used for the production

of plastics. Even if only a small percentage of the biopolymers already being

produced were used in the production of plastics, it would significantly decrease

our dependence on manufactured, non-renewable resources.

cellulose is the most plentiful carbohydrate in the world; 40 percent of all

organic matter is cellulose!

starch is found in corn (maize), potatoes, wheat, tapioca (cassava), and some

other plants. Annual world production of starch is well over 70 billion

pounds, with much of it being used for non-food purposes, like making

paper, cardboard, textile sizing, and adhesives.

collagen is the most abundant protein found in mammals. Gelatin is

denatured collagen, and is used in sausage casings, capsules for drugs and

vitamin preparations, and other miscellaneous industrial applications

including photography.

casein, commercially produced mainly from cow's skimmed milk, is used in

adhesives, binders, protective coatings, and other products.

soy protein and zein (from corn) are abundant plant proteins. They are used

for making adhesives and coatings for paper and cardboard.

polyesters are produced by bacteria, and can be made commercially on large

scales through fermentation processes. They are now being used in

biomedical applications.

A number of other natural materials can be made into polymers that are

biodegradable. For example:

lactic acid is now commercially produced on large scales through the

fermentation of sugar feedstocks obtained from sugar beets or sugar cane, or

from the conversion of starch from corn, potato peels, or other starch source.

It can be polymerized to produce poly(lactic acid), which is already finding

commercial applications in drug encapsulation and biodegradable medical

devices.

triglycerides can also be polymerized. Triglycerides make up a large part of

the storage lipids in animal and plant cells. Over sixteen billion pounds of

vegetable oils are produced in the United States each year, mainly from

soybean, flax, and rapeseed. Triglycerides are another promising raw

material for producing plastics.

These natural raw materials are abundant, renewable, and biodegradable, making

them attractive feedstocks for bioplastics, a new generation of environmentally

friendly plastics.

Starch-based bioplastics are important not only because starch is the least

expensive biopolymer but because it can be processed by all of the methods

used for synthetic polymers, like film extrusion and injection moulding.

Eating utensils, plates, cups and other products have been made with starch-

based plastics.

Interest in soybeans has been revived, recalling Ford's early efforts. In

research laboratories it has been shown that soy protein, with and without

cellulose extenders, can be processed with modern extrusion and injection

moulding methods.

Many water soluble biopolymers such as starch, gelatin, soy protein, and

casein form flexible films when properly plasticized. Although such films

are regarded mainly as food coatings, it is recognized that they have

potential use as nonsupported stand-alone sheeting for food packaging and

other purposes.

Starch-protein compositions have the interesting characteristic of meeting

nutritional requirements for farm animals. Hog feed, for example, is

recommended to contain 13-24% protein, complemented with starch. If

starch-protein plastics were commercialized, used food containers and

serviceware collected from fast food restaurants could be pasteurized and

turned into animal feed.

Polyesters are now produced from natural resources-like starch and sugars-

through large-scale fermentation processes, and used to manufacture water-

resistant bottles, eating utensils, and other products.

Poly(lactic acid) has become a significant commercial polymer. Its clarity

makes it useful for recyclable and biodegradable packaging, such as bottles,

yogurt cups, and candy wrappers. It has also been used for food service

ware, lawn and food waste bags, coatings for paper and cardboard, and

fibers-for clothing, carpets, sheets and towels, and wall coverings. In

biomedical applications, it is used for sutures, prosthetic materials, and

materials for drug delivery.

Triglycerides have recently become the basis for a new family of sturdy

composites. With glass fiber reinforcement they can be made into long-

lasting durable materials with applications in the manufacture of agricultural

equipment, the automotive industry, construction, and other areas. Fibers

other than glass can also be used in the process, like fibers from jute, hemp,

flax, wood, and even straw or hay. If straw could replace wood in

composites now used in the construction industry, it would provide a new

use for an abundant, rapidly renewable agricultural commodity and at the

same time conserve less rapidly renewable wood fiber.

The widespread use of these new plastics will depend on developing technologies

that can be successful in the marketplace. That in turn will partly depend on how

strongly society is committed to the concepts of resource conservation,

environmental preservation, and sustainable technologies. There are growing signs

that people indeed want to live in greater harmony with nature and leave future

generations a healthy planet. If so, bioplastics will find a place in the current Age

of Plastics.

Examples of bioadhesives in natureOrganisms may secrete bioadhesives for use in attachment, construction and

obstruction, as well as in predation and defense. Examples include their use for:

colonization of surfaces (e.g. bacteria, algae, fungi, mussels, barnacles).

tube building by polychaete worms, which live in underwater mounds.

insect egg, larval or pupal attachment to surfaces (vegetation, rocks), and

insect mating plugs

host attachment by blood-feeding ticks

nest-building by some insects, and also by some fish (e.g. the three-spined

stickleback)

defense by Notaden frogs and by sea cucumbers

prey capture in spider webs and by velvet worms

Some bioadhesives are very strong. For example, adult barnacles achieve pull-off

forces as high as 2 MPa (2 N/mm2). Silk dope can also be used as a glue by

arachnids and insects.

TYPES of adhesions:

Temporary adhesion

Organisms such as limpets and sea stars use suction and mucus-like slimes to

create Stefan Adhesion, which makes pull-off much harder than lateral drag; this

allows both attachment and mobility. Spores, embryos and juvenile forms may use

temporary adhesives (often glycoproteins) to secure their initial attachment to

surfaces favorable for colonization. Tacky and elastic secretions that act as

pressure sensitive adhesives, forming immediate attachments on contact, are

preferable in the context of self-defense and predation. Molecular mechanisms

include non-covalent interactions and polymer chain entanglement. Many

biopolymers - proteins, carbohydrates, glycoproteins, and mucopolysaccharides -

may be used to form hydrogels that contribute to temporary adhesion.

Permanent adhesion

Many permanent bioadhesives (e.g., the oothecal foam of the mantis) are generated

by a "mix to activate" process that involves hardening via covalent cross-linking.

On non-polar surfaces the adhesive mechanisms may include van der Waals forces,

whereas on polar surfaces mechanisms such as hydrogen bonding and binding to

(or forming bridges via) metal cations may allow higher sticking forces to be

achieved.

Microorganisms use acidic polysaccharides (molecular mass around 100 000

Da).

Marine bacteria use carbohydrate exopolymers to achieve bond strengths to

glass of up to 500 000 N/m2 .

Marine inverterbrates commonly employ protein-based glues for irreversible

attachment. Some mussels achieve 800 000 N/m2 on polar surfaces and 30

000 N/m2 on non-polar surfaces .

Some algae and marine invertebrates use polyphenolic proteins containing

L-DOPA

Proteins in the oothecal foam of the mantis are cross-linked covalently by

small molecules related to L-DOPA via a tanning reaction that is catalysed

by catechol oxidase or polyphenol oxidase enzymes.

L-DOPA is a tyrosine residue that bears an additional hydroxyl group. The twin

hydroxyl groups in each side-chain compete well with water for binding to

surfaces, form polar attachments via hydrogen bonds, and chelate the metals in

mineral surfaces. The Fe(L-DOPA3) complex can itself account for much cross-

linking and cohesion in mussel plaque, but in addition the iron catalyses oxidation

of the L-DOPA to reactive quinone free radicals, which go on to form covalent

bonds. An example of permanent adhesion is byssal adhesive.

Mucoadhesion

A more specific term than bioadhesion is mucoadhesion. Most mucosal surfaces

such as in the gut or nose are covered by a layer of mucus. Adhesion of a matter to

this layer is hence called mucoadhesion. Mucoadhesive agents are usually

polymers containing hydrogen bonding groups that can be used in wet

formulations or in dry powders for drug delivery purposes. The mechanisms

behind mucoadhesion have not yet been fully elucidated, but a generally accepted

theory is that close contact must first be established between the mucoadhesive

agent and the mucus, followed by interpenetration of the mucoadhesive polymer

and the mucin and finishing with the formation of entanglements and chemical

bonds between the macromolecules.In the case of a dry polymer powder, the initial

adhesion is most likely achieved by water movement from the mucosa into the

formulation, which has also been shown to lead to dehydration and strengthening

of the mucus layer. The subsequent formation of van der Waals, hydrogen and, in

the case of a positively charged polymer, electrostatic bonds between the mucins

and the hydrated polymer promotes prolonged adhesion.

Byssal adhesiveByssus generally refers to a filament created by certain kinds of marine and

freshwater bivalve molluscs, which use it to attach themselves to rocks, substrates,

or sea beds. In edible mussels the inedible byssus is commonly known as the

"beard", and is removed before cooking.

Byssus specifically refers to the long, fine, silky threads secreted by the large

Mediterranean pen shell, Pinna nobilis. The byssus threads from this Pinna species

can be up to 6 cm in length and have historically been made into cloth. The secret

to the mussels' staying power is tiny threads, called byssus. These tentacles, which

can reach more than two inches in length, are made of a protein with a high level

of stuff called phenolic hydroxyls.

Many species of mussels secrete byssus threads to anchor themselves on hard

surfaces, with Families including the Arcidae, Mytilidae, Anomiidae, Pinnidae,

Pectinidae, Dreissenidae, and Unionidae .

When a mussel's foot encounters a crevice, it creates a vacuum chamber by forcing

out the air and arching up, similar to a plumber's plunger unclogging a drain. The

byssus, which is made of keratin, quinone-tanned proteins (polyphenolic proteins),

and other proteins, is spewed into this chamber in liquid form, and bubbles into a

sticky foam. By curling its foot into a tube and pumping the foam, the mussel

produces sticky threads about the size of a human hair. The mussel then varnishes

the threads with another protein, resulting in an adhesive.

Byssus is a remarkable adhesive, one that is neither degraded nor deformed by

water, as are synthetic adhesives. This property has spurred genetic engineers to

insert mussel DNA into yeast cells for translating the genes into the appropriate

proteins.

California mussels Mytilus californianus owe their tenacity to a holdfast known as

the byssus, a fibrous extracellular structure that ends distally in flattened adhesive

plaques. The fabrication of strong and durable adhesive bonds between

macromolecules and metal or mineral surfaces in a wet environment is one of the

most persistent challenges for modern adhesive technology. Mussels (Mytilus)

thrive despite persistent surf and tides thanks in part to a robust holdfast structure

called the byssus, which consists of a bundle of threads each of which is tipped

distally by an adhesive plaque that bonds to mineral and metal surfaces. The

adhesive strategies of mussels and other sessile marine invertebrates are

increasingly envisioned as paradigms for designing tough water-resistant

adhesives.

The biochemistry of mussel byssus has been investigated in primarily two species,

Mytilus edulis and Mytilus galloprovincialis, both relatively sheltered species . Not

surprisingly, all of the proteins characterized so far in the two species show a very

high degree of sequence homology.

At least six different proteins have been characterized from freshly secreted

adhesive plaques of M. edulis. These are mefp1, 2, 3, 4, and 5 and various preCols.

An experiment was conducted as follows by Hua Zhao,Nicholas B.

Robertson,Scott A. Jewhurst, J. Herbert Waite  which is as follows:

MATERIALS AND METHODS

Protein Purification from Mussels-Mussels were collected locally from rocks and

jetties around Campus Point and Goleta Pier in Santa Barbara, CA and

immediately transferred to shallow holding tanks with circulating raw seawater at

15 °C. About 60 mussels were tethered to each 18 x 25-cm plate made of acrylic or

glass (thickness, 1 cm), from which byssal plaques were harvested daily using a

clean single-edge razor blade, briefly rinsed with 200 volumes of MilliQ water,

and stored at -80 °C. About 1000 accumulated plaques were thawed and extracted

in a small volume (5 ml/200 plaques) of 5% acetic acid (v/v) containing 8 M urea

and 0.1 mM tri(carboxyethyl)-phosphine by homogenization on ice using a small

hand-held tissue grinder (Kontes, Vineland, NJ). The homogenate was centrifuged

for 30 min at 20,000 x g and 4 °C. The supernatant was collected (10 ml), and half

was dialyzed against 4 liters of MilliQ water using membrane tubing with a 1,000-

dalton cut-off (Spectrum Industries). Dialysis resulted in a turbidity, which was

clarified by centrifugation (30 min at 20,000 x g and 4 °C), and sedimented

material was redissolved in 5% acetic acid with 8 M urea, whereas the supernatant

was freeze-dried at -80 °C.

The acetic acid/urea plaque extracts were subjected to reverse phase HPLC2 using

a 260 x 7-mm RP-300 Aquapore (Applied Biosciences Inc., Foster City, CA)

column eluted with a linear gradient of aqueous acetonitrile . Eluant was monitored

continuously at 280 nm, and collected 1-ml fractions were assayed by amino acid

analysis and electrophoresis following freeze-drying.

Electrophoresis-Routine electrophoresis was done on polyacrylamide gels (7%

acrylamide and 0.2% N,N'-methylenebisacrylamide) containing 5% acetic acid

with 8 M urea (10). This system is ideal for basic Dopa-containing proteins

because it can be processed for protein or Dopa staining with equal facility. The

proteins were stained with Serva Blue R (Serva Fine Chemicals, Westbury, NY),

whereas Dopa was stained with either the Arnow reagent or nitro blue tetrazolium

redox cycling.

Amino Acid Analysis and Sequencing of Peptides-The peptides and proteins were

hydrolyzed in 6 N HCl with 5% phenol in vacuo at 110 °C for 12-48 h to correct

for the losses of certain amino acids. Recovery of tryptophan required hydrolysis in

4 N methanesulfonic acid or in 6 N HCl with 30% phenol in vacuo for 20 and 40

min at 165 °C . Routine amino acid analysis was done by ion exchange and a

ninhydrin-based detection system (Beckman System 6300 Auto Analyzer) using a

previously described step elution program . Hydroxyarginine eluted at 80 min and

was quantified using the molar color yield of arginine. Because of its coelution

with ammonia, tryptophan was separately quantitated on System 6300 using a 40-

min program of NaD (5% sodium chloride and 1.9% sodium citrate at pH 6 at a

column temperature of 70 °C. The amino acid sequence of protein and peptides

was derived by automated Edman degradation using a Porton Instruments 2090

Microsequencer.

Adhesive Plaque Footprints-To obtain adhesive plaque footprints, the mussels

were tethered over glass coverslips, and when one or more plaques had been

deposited onto the substrates, the connecting threads were severed, and the

coverslips were removed. The coverslips were thoroughly rinsed with two changes

of MilliQ water. The underside of the glass coverslip was dried so that the

locations of the adhering plaques could be marked with a black marker pen. The

plaques were then removed with a clean single-edge razor. The footprints were

imaged by stereo light and scanning electron microscopy.

MALDI-TOF Mass Spectrometry-MALDI-TOF experiments were performed

using a Voyager DE (linear delayed extraction) mass spectrometer (Applied

Biosystems, Foster City, CA). The MALDI matrix was prepared by dissolving

sinapinic acid (10 mg/ml) in 70% acetonitrile. The Mcfp3 protein or peptides

derived thereof were dissolved in this matrix solution to give a final concentration

between 1 and 10 pmol/µl. About 1µl of this solution was applied to the target

plate and allowed to evaporate. The sample spots were irradiated using an N2 laser

with a wavelength of 337 nm and a pulse width of 8 ns and operated at a repetition

rate of 5 Hz. MALDI ionization generates protonated singly and doubly charged

ions and dimers for the Mcfp3 protein (mostly singly charged ions for peptides)

that were accelerated using either 20- or 25-kV accelerating voltage. The

resolution was about 1:500, which was sufficient to allow mass assignment of the

major peaks caused by the different hydroxylation states of the Mcfp3 protein.

Footprints of byssal plaques were screened for proteins by MALDI-TOF mass

spectrometry. The technique as currently practiced involves the following: glass

coverslips were collected shortly after plaque deposition. The coverslips were

cleaned of slime and debris by washing thoroughly with MilliQ water, after which

the plaques were scraped off using a clean single-edge razor. The glass surface

with the footprint residue was dried and mounted onto a MALDI sample plate with

double-sided tape. Two microliters of matrix mixture (30% sinapinic acid) were

applied to the footprint and air-dried before subjecting to pulsed laser irradiation.

With respect to matrix type, -cyano-4-hydroxycinnamic acid can be substituted for

sinapinate in fp3 analysis although with a lower signal-to-noise ratio. Singly and

doubly charged peaks for bovine insulin and thioredoxin (5734.59 and 5837.74,

respectively, for average masses) were used as internal calibrants. To compensate

for possible surface effects on MALDI by different materials such as glass and the

gold sample plate, internal standards were always recalibrated on each test surface.

RESULTSPlaque Footprint Proteins-MALDI-TOF mass spectrometry was used to interrogate

plaque footprints after plaque removal from glass coverslips so that little to no

residue could be observed by light microscopy or scanning electron microscopy

(Fig. 1, A and B). MALDI mass spectroscopy reproducibly detected small proteins

ranging from 5 to 7 kDa in the footprints (Fig. 1C), which are 1-2 orders of

magnitude more abundant than any other detectable proteins in the range of 1,000-

50,000 Da (supplemental material). All of the footprint proteins exhibit laddering. 

Mcfp3-1 and 1 differ only by a Gly to Arg substitution at position 13. Likewise

mcfp3-3 and mcfp3-3 differ only in an Ser to Asn substitution at position 10.

Mcfp3-5 has three polymorphs based on substitutions at position 10: has Asn, has

Thr, and has Ala. Mcfp3-1 and Mcfp3-2 have 4 or 5 Arg and 10 Tyr each, whereas

mcfp-3 to mcfp-6 have a single Arg and 14-15 Tyr each. This is expected to

increase the mass in steps of 16 Da to a maximum of at least 224 Da for 14

hydroxylation sites. In actuality, completely unhydroxylated and hydroxylated

forms were rare. For the two most abundant fp3 variants in footprints, for example,

a mass range of 5190.8 to 5446.8 and 6270.6 to 6510.6 is expected for mcfp3-1

and mcfp3-5, respectively.

Dopa in free or peptide-bound form is very prone to oxidation in seawater. Given

this, we speculated that Dopa detected in footprint fp3s might remain intact for one

of two reasons: 1) it is stabilized by the interface or 2) it is stabilized by factors

present in the plaque itself.

Adhesives made of 5 proteins Mefp-1,Mefp-2,Mefp-3 , Mefp-4, Mefp-5, with high

level of 3,4-dihydroxyphenyl-L-alanine, amino acid which allow of proteins to

cross link each other .

BIOENGINEERING

o Agrobacterium mediated transformation is used to clone genes involved in

synthesis of biopolymer into tobacco plant.

o Used for dental filling, tough elastic ropes ,surgical adhesives etc.

RUBBER:Rubber is an elastomer—that is, a polymer that has the ability to regain its original

shape after being deformed. Rubber is also tough and resistant to weathering and

chemical attack. Elastomers can be naturally occurring polymers, such as natural

rubber, or they can be synthetically produced substances, such as butyl rubber,

Thiokol, or neoprene. For a substance to be a useful elastomer it must possess a

high molecular weight and a flexible polymer chain.

Natural Rubber

Natural rubber is one of nature's unique materials. The Native Americans of

tropical South America's Amazon basin knew of rubber and its uses long before

Christopher Columbus's explorations brought it to the attention of Europeans. The

Indians made balls of rubber by smoking the milky, white latex of trees of the

genus Hevea that had been placed on a wooden paddle, to promote water

evaporation and to cure the substance.

Spanish navigator and historian Gonzalo Fernández de Oviedo y Valdes (1478–

1557) was the first European to describe these balls to a European audience. In

1615 a Spanish writer enumerated the practical uses of rubber. He reported that the

Indians waterproofed their cloaks by brushing them with this latex and made

waterproof shoes by coating earthen molds with it and allowing these coatings to

dry.

In 1735 interest was revived in this unusual substance when French mathematical

geographer and explorer Charles-Marie de La Condamine (1701–1774) sent

several rolls of crude rubber to France with an accompanying description of

products made from it by the South American natives. Although it met with some

use in waterproofing boots, shoes, and garments, it largely remained a museum

curiosity. Crude rubber possessed the valuable properties of elasticity, plasticity,

strength, durability, electrical nonconductivity, and resistance to water; however,

products made from it hardened in winter, softened and became sticky in summer,

were attacked by solvents, and smelled bad.

Rubber, sometimes called "gum-elastic," was known to the Indians by the name

of caoutchouc (from caa, "wood," and o-chu, "to flow or to weep"). In 1770

English chemist and Unitarian clergyman Joseph Priestley (1733–1804), the

discoverer of oxygen, proposed the name "rubber" for the substance because it

could be used to erase pencil marks by its rubbing on paper in lieu of previously

used bread crumbs.

RUBBER POLYMERASE

Rubber ,an elastomer type polymer has ability to regain its original shape.

Elastomers can be-

Natural

Synthetic(butyl rubber,thiokol,neoprene)

Rubber was first commercialized in 1791 when english manufacturer

Samuel Deal patented a method for water proofing cloth by treating

solution of rubber in turpentine.

In enzymology, a rubber cis-polyprenylcistransferase is an enzyme that

catalyzes the chemical reaction

poly--polyprenyl diphosphate + isopentenyl diphosphate diphosphate + a poly-

cis-polyprenyl diphosphate longer by one C5 unit

Thus, the two substrates of this enzyme are poly-cis-polyprenyl diphosphate and

isopentenyl diphosphate, whereas its two products are diphosphate and poly-cis-

polyprenyl diphosphate longer by one C5 unit.

This enzyme belongs to the family of transferases, specifically those transferring

aryl or alkyl groups other than methyl groups. The systematic name of this enzyme

class is poly-cis-polyprenyl-diphosphate:isopentenyl-diphosphate

polyprenylcistransferase. Other names in common use include rubber

allyltransferase, rubber transferase, isopentenyl pyrophosphate cis-1,4-

polyisoprenyl transferase, cis-prenyl transferase, rubber polymerase, and

rubber prenyltransferase. This enzyme participates in biosynthesis of steroids.

Production process:

Isolation of DNA from plant and other organisms:

Leaves from Hevea brasiliensis and guayule plants were harvested.

Surface sterilization.

Plant material was frozen quickly in liquid nitrogen.

Fine powder obtained was transferred to tubes containing Tris, EDTA, NaCl,

mercaptoethanol and SDS and centrifuged.

Precipitate obtained was dried and resuspended in solution containing Tris,

EDTA, and sodium acetate.

Isopropanol was added to reprecipitate the dissolved DNA.

Resuspended DNA was banded in a cesium chloride gradient.

DNA was recovered by centrifugation.

Isolation of mRNA from Hevea plant:

2-4 months old Hevea was grown. The plant obtained was cut into small

pieces and frozen and powdered.

Material was transferred to chilled blender to which added ice cold buffer.

Pellet was extracted and again centrifuged with the same buffer.

RNA precipitate from supernatant at 20∙C by adding sodium acetate and

ethanol.

Reprecipitation and resuspension process is repeated twice.

Pellet was washed with ethanol, RNA isolated was chromatographed.

The mRNA eluted from oligodT cellulose three times.

mRNA was then precipitated and extracted.

Synthesis and cloning of cDNA:

cDNA of Hevea was synthesized in-vitro from m RNA with the use of

reverse transcriptase .

cDNA library was constructed by using mRNA.

Formulation of nucleic acid probe for the identification of Hevea rubber:

Polymerase Gene

The probe is written in the conventional 5'➝3' direction and consisted of a mixture

of thirty-two nucleotide sequences, each being 17 bp in length, and one of which

would exactly complement the Hevea rubber polymerase gene.

The 5' end of the probe contained a 17 bp homology interrupted with three dI

residues. The 3' end of the probe contained a 15 bp homology interrupted with two

dI residues. At the wobble positions within the probe, A or T residues were chosen

when applicable because the gene as represented by the amino acid sequence was

predominantly A-T rich.

A17 base Hevea rubber polymerase gene specific oligonucleatide produced was

labelled with 32p-phosphate and utilized as probe.

Subcloning of the Hevea Rubber Polymerase DNA Sequence :

FEED STOCKS FOR BIOPLASTICS:

Some natural material is converted to biodegradable polymers.

LACTIC ACID- polymerizes to poly lactic acid, used in drug encapsulation.

TRIGLYCERIDES- to make long lasting durable materials used in

agriculture equipment ,automotive industries.

STARCH- based bioplastics, for utensils,plates and cups.

WATER SOLUBLE BIOPOLYMERS form flexible film for food packaging.

POLYSTER - from starch and sugar, used to make water resistant bottles.

COMMERCIAL APPLICATIONS:

Commodity wood adhesive based on a bacterial exopolysaccharide .

USB PRF/Soy 2000, a commodity wood adhesive that is

50% soy hydrolysate and excels at finger-jointing green lumber. .

Mussel adhesive proteins can assist in attaching cells to plastic surfaces in

laboratory cell and tissue culture experiments .

The Notaden frog glue is under development for biomedical uses, e.g. as

a surgical glue for orthopedic applications .

Mucosal drug delivery applications. For example, films of mussel adhesive

protein give comparable mucoadhesion to polycarbophil, a synthetic

hydrogel used to achieve effective drug delivery at low drug doses.

Several commercial methods of production are being researched:

direct chemical synthesis, e.g. incorporation of L-DOPA groups in

synthetic polymers

fermentation of transgenic bacteria or yeasts that express bioadhesive

protein genes

farming of natural organisms (small and large) that secrete bioadhesive

materials .

CONCLUSION:

DoPA- based adhesive strategies are fairly rare. It is observed only in

Mussel,tunicates and termatodes.

Deep studies on the mechanism of Byssal adhesion would lead to

fundamental researches.

Rubber has been used for centuries to make several products.

Time to time greater efficiency have been achieved. Petroleum is now much

favoured for production but is non-renewable so for future natural methods

shoulb be employed.

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3.Dalsin and Messersmith (1990) (2005) Materials Today 8, 53-57.

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Chem. Biol. 3 (1), 100-105.

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6. 8.Hua Zhao,Nicholas B. Robertson,Scott A. Jewhurst, J. Herbert Waite    作者单位：Department of Molecular, Cellular and Developmental Biology and

Department of Chemistry and Biochemistry, Marine Science Institute, University

of California, Santa Barbara, California 93106.

7.Graham, L.D. (2008) Biological adhesives from nature. In: Encyclopedia of

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16. Structural Effects of Crosslinking a Biopolymer

Hydrogel Derived from Marine Mussel

Adhesive Protein

Elena Loizou,1 Jaime T. Weisser,2 Avinash Dundigalla,1 Lionel Porcar,4 Gudrun

Schmidt,*1,3 Jonathan J. Wilker*2

1) Department of Chemistry, Louisiana State University, Baton Rouge, LA

70803, USA.

2) Department of Chemistry, Purdue University, West Lafayette, IN 47907,

USA.

Fax: þ1 765 494 0239; E-mail: wilker@purdue.edu.

3) Department of Biomedical Engineering, Purdue University, West Lafayette,

IN 47907,

USA E-mail: gudrun@purdue.edu.

4 )National Institute of Standards and Technology, Gaithersburg, MD 20899,

USA.

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