Enzymes stability

Govt Holkar Science CollegeIndore (MP)

DEPT OF BIOTECHNOLOY

PAPER 1- ENZYMOLOGY

TOPIC ENZYME

STABILITY

Contents-

bull Short Introduction to Enzymes

bull Need for enzyme stability

Biological and industrial use of enzymes

bull Enzyme Stabilization

bull Techniques for enzyme Stabilization1 Immobilization

2 Enzyme Engineering

3 Solvent Engineering

4 Modification of chemical structure

5 Through Nanotechnology

6 Freeze drying

bull Conclusion

Introduction to ENZYMESbull Enzymes are proteins that catalyze chemical reaction The term

ldquoenzymerdquo was given by Kuhne in 1878 although the first observation of enzyme activity in a test tube was reported by Payen amp Persoz in 1833

bull Enzyme increases the rate of such reaction by 103-106 fold that occur at a very slow rate

bull Enzymes are usually proteins They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds

bull Enzymes can be denatured and precipitated with salts solvents and other reagents They have molecular weights ranging from 10000 to 2000000 daltons

bull Despite their obvious advantages as process catalysts biocatalysts as proteins they are labile molecules Therefore biocatalyst stability and stabilization is a central issue of biotechnology today In fact biocatalyst operational stability will determine to a large extent the viability of the process

Need for ENZYME STABILITYbull Enzymes are protein-based molecules whose function is to catalyze

chemical reactions in the body

bull Specific enzymes are produced for particular chemical reactions

Thus enzymes have highly specific reactions and an enzyme converts

a particular substrate into a particular product

bull Enzymes are very important to maintain metabolic processes in the

body A defect in the level of enzymes can cause abnormalities

bull Thus research is going on to develop methods to isolate enzymes

However the biggest challenge in this is the instability of enzymes

bull The specific action of enzymes depends on the sequence and the

overall 3-dimensional arrangement of the amino acids If not handled

carefully the protein structure of the enzymes gets altered and they

lose their function

Biological Functions of Enzymesbull Enzymes serve a wide variety of functions inside living organisms

bull They are indispensable for signal transduction and cell regulation

often via kinases and phosphatases

bull They also generate movement with myosin hydrolyzing ATP to

generate muscle contraction and also moving cargo around the cell

as part of the cytoskeleton

bull Other ATPases in the cell membrane are ion pumps involved

in active transport

bull Enzymes are also involved in more exotic functions such as

luciferase generating light in fireflies

Conthellipbull An important function of enzymes is in the digestive systems of

animals Enzymes such as amylases and proteases break down large

molecules (starch or proteins respectively) into smaller ones so they

can be absorbed by the intestines

bull Different enzymes digest different food substances In ruminants

which have herbivorous diets microorganisms in the gut produce

another enzyme cellulase to break down the cellulose cell walls of

plant fiber

bull Several enzymes can work together in a specific order

creating metabolic pathways In a metabolic pathway one enzyme

takes the product of another enzyme as a substrate After the catalytic

reaction the product is then passed on to another enzyme

bull Enzymes determine what steps occur in these pathways Without

enzymes metabolism would neither progress through the same steps

nor be fast enough to serve the needs of the cell

Application Enzymes used Uses

Food processing

Amylases catalyze the

release of simple sugars

from starch

Amylases from fungi and plants

Production of sugars from starch such as in

making high-fructose corn syrup In baking

catalyze breakdown of starch in the flour to

sugar Yeast fermentation of sugar produces

the carbon dioxide that raises the dough

ProteasesBiscuit manufacturers use them to lower the

protein level of flour

Baby foods Trypsin To predigest baby foods

Brewing industry

Germinating barley used

for malt

Enzymes from barley are released during the

mashing stage of beer production

They degrade starch and proteins to produce

simple sugar amino acids and peptides that are

used by yeast for fermentation

Industrially produced barley enzymes

Widely used in the brewing process to

substitute for the natural enzymes found in

barley

Amylase glucanases proteases Split polysaccharides and proteins in the malt

Betaglucanases and arabinoxylanasesImprove the wort and beer filtration

characteristics

Amyloglucosidase and pullulanasesLow-calorie beer and adjustment of

fermentability

ProteasesRemove cloudiness produced during storage of

beers

Acetolactatedecarboxylase (ALDC)Increases fermentation efficiency by

reducing diacetyl formation[88]

Indstrial Application

Enzyme Stabilization

bull Enzyme Stabilization is gaining importance due to the wide range of enzyme applications Other than medical research they can be used in food chemical and pharmaceutical industries

bull To use these biocatalysts called enzymes to their full potential it is very important to achieve progress in enzyme stabilization

bull Factors affecting enzyme stability are-

bull pH (optimum pH- 45 ndash 80 )

bull Temperature (optimum temp-25C -37C)

bull Ionic strength

bull Solvent

The techniques that have been attempted to achieve

enzyme stabilization can be divided into broad

categories Some of them are

1 Immobilization

2 Enzyme Engineering

3 Solvent Engineering

4 Modification of chemical structure of enzymes

5 Through Nanobiotechnology

6 Freeze drying

IMMOBILIZATION OF ENZYMES

bull Enzyme immobilization may be defined as confining the enzyme molecules to a distint phase from the one in which the substrates and products are present this may be achieved by fixing the enzyme molecules to or within some suitable material

bull Immobilization of enzymes molecules does not necessarily render them immobile For eg-

i Entrapment amp Membrane Confinement-enzyme molecules move freely within their phase

ii Adsorption amp Covalent Binding- enzymes are immobile

MATERIAL USED- Material used for immobilization of enzymes is called Carrier

Matrices (usually inert polymers or inorganic materials)

Some of the commonly used matrices are ion exchange matrices porous carbon clays hydrous metal oxides glasses and polymeric aromatic resins agarose cellulose polyacrylamide(ion exchange matrices are costly but they can be readily regenerated by a simple operation eg washing off the absorbed enzyme with a concentrated salt solution)

The ideal matrix should have the following properties- Low cost

Inertness

Physical strength

Stability

Regenerability

Enhancement of enzyme specificity

Reduction in product inhibition

Reduction in microbial contamination and non-specific adsorption

METHODS OF IMMOBILIZATION

bull There are four principal

methods available for

immobilising enzymes

a Adsorption

b Covalent binding

c Entrapment

d Membrane

Confinement

A ADSORPTION-bull The enzyme molecules adhere to the surface of carrier matrix due

to a combination of hydrophobic effects and the formation of

several salt links per enzyme molecule

bull The binding of enzyme molecules to the carrier matrix is usually

very strong but it may be weakened during use by many factors

eg addition of substrate pH or ionic strength etc

bull Adsorption of enzymes to the matrices is very easy and widely

used The enzymes is mixed with a suitable adsorbent under

appropriate conditions of pH and ionic strength

bull After incubation for a sufficient period of time the carrier is

washed to remove un-absorbed enzyme molecules and the

immobilized enzyme is ready for use

bull This method usually produce a high loading (1g enzymeg matrix)

B COVALENT BINDING -

bull In this the enzyme molecules are attached to the carrier matrix by

formation of covalent bonds

bull The strength of binding is very strong and there is no enzyme loss

during use

bull The covalent bond formation occurs with the side chains of amino

acids of the enzyme their degree of reactivity being dependent on

their charge (Lysine residues are the most useful in covalent binding)

bull The most commonly employed matrices are agarose celluloses

polyacrylamides

bull Sepharose an agarose is available commercially as beads is highly

hydrophilic and is generally inert to microbial attack (Sepharose is

activated by a treatment with cyanogen bromide which forms highly

reactive intermediates with the ndashOH groups of sapharose the enzyme

molecules bind to these activated groups)

C Entrapment -

bull In this approach enzyme molecules are held or entrapped within suitable gels or fibres and there may or may not be covalent bond formation between the enzyme molecules and the matrix

bull Covalent binding is also can be generated enzyme molecules are usually treated with a suitable reagent For eg acryloyl chloride is used to prepare lysine residues for binding by forming acryloyl amides these are then copolymerized amp crosslinkedwith acrylamide and bisacrylamide to form gel containing entrapped enzyme

bull Enzymes may be entrapped within cellulose acetate fibres

bull Enzyme loading in the entrapment

procedures is very high

(1gg gel or fiber)

Due to this diffusion of substrate

to the enzyme and of the product

away from the enzymes creates difficulties

D Membrane Confinement -bull Enzymes molecules usually in aqueous solution may be confined

within a semipermeable membrane which ideally allows a free movement in either direction to the substrate and the products but does not permit the enzyme molecules to escape

bull No of strategies are employed for this purpose-

1 The reaction vessel is partitioned into two chambers by a semipermeable membrane One chamber contains the enzyme while the other has the substrate and the product

2 Hollow fibre membrane units contain the enzyme in the fibre lumen or hollow space and the fibres themselves are sumbmerged in the substrate This strategy provide large surface area per unit volume (gt20m2l) but for only such substrates that are much smaller than the enzyme molecules

3 Enzymes may be packed in microcapsules formed by polymerization reaction (16-diaminphexane) They may be enclosed within liposomes(small spheres made up of concenteric lipid membranes)

CharacteristicsAdsorption Covalent

bindingEntrapment

Membrane

confinement

Preparation Simple Difficult Difficult Simple

Cost Low High Moderate High

Binding force Variable Strong Weak Strong

Enzyme leakage Yes No Yes No

Applicability Wide Selective Wide Very wide

Running Problems High Low High High

Matrix effects Yes Yes Yes No

Large diffusional barriers No No Yes Yes

Microbial protection No No Yes Yes

Generalised comparison of different enzyme

immobilisation techniques

Enzyme Engineering

bull Improvement in the activity and usefulness of an existing enzymes(stability) or creation of a new enzyme activity by making suitable changes in its amino acid sequence is called Enzyme Engineering

bull When this approach is used for any protein whether enzyme or non-enzyme it is termed as Protein Engineering

bull Enzyme engineering utilizes recombinant DNA technology to introduce the desired changes in the amino acid sequences of enzymes

bull The recombinant strain produce the enzyme in considerably higher quantity than did the originalparent strain which reduce production costs amp enhance enzyme purity

Objective of Enzyme Engineering

bull The chief objective is to produce an enzyme that is more useful for industrial or other applications

bull Through Enzyme Engineering various properties of enzymes that may be modified are-

i Improved kinetic properties

ii Enhance substrate and product specificity

iii Increased thermostability

iv Alteration in optimal pH

v Suitability for use in organic solvents

vi Increaseddecreased optimal temperature

vii Increased stability to oxidizing agents heavy metals etc

viiiResistance to proteolytic degradation

ix Fusion of two or more enzymes to create bi- and poly-functional enzymes

Principle and Steps in Enzyme Engineering

bull The structure and function of an enzyme molecule are chiefly

determined by its amino acid sequence ie its primary structure

bull Therefore any change in the amino acid sequence should alter the

properties of the enzyme

bull But it is not always the case because the enzymatic properties are

changed only when amino acid changes are introduced in certain critical

regions of the protein This is possible through SITE DIRECTED

MUTAGENESIS

bull Therefore it is of great importance to know the critical regions for the

various functions of an enzyme

bull The case of alkaline protease being a paradigm Already in the market

thermostable proteases capable to withstand harsh washing conditions

(high pH high concentration of strong oxidants) are products of protein

engineering produced by point aminoacid substitutions in the most labile

region of the molecule

Expression of the

modified gene in a

suitable HOST eg Ecoli

Isolstion and cloning of the

conserned gene desired change in

base sequence by SITE-

DIRECTED MUTAGENESIS

Analysis of Structure Function

Relationship Molecular Modelling

Prediction of the Desired Amino

Acid Seqence

Isolation and Purification of

Enzyme Determination of

Structure and Function

Database

Summary of various steps involved in Enzyme Engineering

Solvent Engineering

bull Solvent engineering seeks to usefully modify properties of enzymes simply by using appropriate organic solvents to carry out the enzyme-catalysed reactions

bull This is in contrast to enzyme engineering which aims to modify the properties of an enzyme by making appropriate changes in its amino acid sequence

bull A recent development is to use PEG modified enzymes those are soluble in organic solvents and form homogenous reaction systems which do not pose diffusion problems

bull The main purpose of medium engineering in biocatalysis is associated with the utilization of robust commercial hydrolytic enzymes in organic synthesis thermostability in organic media is an additional bonus of great significance in process economy

The Effect of Organic Solvents on the

Enzymesbull A hydrophobic solvent does not change the three dimensional

structures of enzymes while hydrophilic solvents extract the water associated with the enzyme which often leads to their inactivation

bull Organic solvents can modulate enzyme specificity and enzymes in organic media often show altered substrate specificity as compared to that in water For eg- chymotrypsin prefer hydrophobic nonpolar substrates in water while in organic media they show a preference for hydrophilic substrates

bull Enzymes suspended in organic solvents often show enhanced thermostability under optimal conditions they remain stable upto 150C

bull Enzymes may be engineered for a better performance in organic solvents It was found that a substitution of charge residues by uncharged residues on the protein surface and enhanced hydrogen bonding abilities had a +ve effect on enzyme stability

Stabilization by chemical modification

bull Stabilization by chemical modification of the protein molecule is attractive

but has not received much attention Increased stability has been obtained by

the introduction of hydrophilic groups in the surface of the enzyme molecule

that reduces the contact of hydrophobic regions with water thereby

preventing incorrect refolding after reversible denaturation

bull Extrinsically enzyme stability may be changed by the addition of suitable

stabilizing effectors (cations cross-links peptides etc)

bull Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability

bull Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts

bull PEGylation increases solubility in organic solvents

bull Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase

bull Enzymes can be made suitable by adding following substances-

bull Additives used includes metal ionssalt sugar surfactants organic solvents(eg polyols) and soluble inert molecules ie polyethylene glycol

bull Metal ion such as Ca++ have been used to stabilize trypsin chymotrpsin and amylase Ca++ is known to result in the formation of ionic bridging in enzyme molecules

bull Addition of Co++ and Mn++ has been shown to improve the thermostability of the enzyme cyclic phosphodiesterase

bull The effect of salt containing ions such as Li+ Na+ Cs+ K+ I- F- NO3

- SO4--etc on enzyme stability has also been studied For

eg- alcohol dehydrogenase is activated by low conc Of Na+ K+ and Li+ but inhibited by high conc Of the same cations

bull Increasing ionic strength of enzyme solutions by adding salts such as Na2SO4 NaCl KCl stabilizes chymotrypsin and sodium chloride stabilizes amylase during storage

bull The stabilizing sugar includes glucose fructose maltose sucrose trehalose mannitol sorbitol and glycerol Stabilizing action of these substances is associated with hydrogen bonding hydrophobic interactions ionic and Van der waalsforces between sugar and enzyme molecules

bull The effectiveness of a compound as a stabilizing agent depends upon the no of hydroxyl group it contains

bull Surfactants are other possible stabilizers For eg- Triton X-100

bull Organic solvents like Glycerol has been shown to stabilize both lactate and malate dehydrogenase during storage at 4C

bull Ethanol methanol propanol isopropanol butanol acetone etc have also been shown to stabilize these enzymes

bull Addition of polyvinyl alcohol at low conc Enhanced activity of Aspergillus niger glucomylase

bull The stabilizing effect of solvent is associated with their ability to displace water molecules from vicinity of enzymes

Nanotechnologybull Enzymes are proteins that function as biocatalysts in living systems One of the major

concerns in environmental biomedical and other applications of enzymes is their short lifetime

bull Enzymes lose their activity due to oxidation or other processes which results in less stability and a shorter lifetime thereby rendering them less efficient

bull An effective way to increase the stability longevity and reusability of the enzymes is to attach them to nanoparticles

bull With this aim two different catabolic enzymes trypsin and peroxidase were attached to uniform magnetic core-shell iron nanoparticles (MNPs)

bull By doing so the lifetime of enzymes increases dramatically from a few hours to weeks and that MNP-Enzyme conjugates are more stable efficient and economical

bull We predict that nanostructures shield the enzymes preventing them from getting oxidized or self digested

bull Significant enhancement of enzyme stabilization was realized as introducing cross-linked

enzyme aggregates (CLEAs) via glutaraldehyde mediated coupling with various

nanostructured materials such as nanofiber carbon nanotube or mesoporous silica

bull Nanostructure -enzyme conjugates could efficiently be reused which makes enzymes more productive We also found that the enzyme structure plays a major role in efficient attachment of nanostructures

bull In nanotechnology a particle is defined as a small object that behaves as a whole unit in terms of its transport and properties Particles are further classified according to size(in terms of diameter) nanoparticles are sized between 100 and 1 nanometers

bull Nanoparticles may include gold silica glass metal oxides etc

bull Properties of nanoparticles include biocompatibility superparamagnetism small size and low toxicity

bull Application of nanotechnology-bull Fluorescent biological labels bull - Drug and gene deliverybull - Bio detection of pathogensbull - Detection of proteinsbull - Probing of DNA structurebull - Tissue engineeringbull - Tumour destruction via heating (hyperthermia)bull - Separation and purification of biological molecules and cells

bull Examples for explaning use of nanoparticles in enzyme immobilization-

bull Lipases (glycerol ester hydrolases) are an important group of enzymes which have been widely used in the catalysis of different reactions

bull These enzymes have been applied in chemical and pharmaceutical industrial applications due to their catalytic activity in both hydrolytic and synthetic reactions However free lipases are easily inactivated and difficult to recover for reuse

bull Therefore especially in large-scale applications lipases are often immobilized on solid supports in order to facilitate recovery and improve operational stability under a wide variety of reaction conditions

bull Some lipase immobilization strategies involve the conjugation of lipases via covalent attachment cross-linking adsorption and entrapment onto hydrophobic or hydrophilic polymeric and inorganic matrixes

bull In recent years magnetic nanoparticles (MNPs) based on iron oxides have attracted much interest thanks to their multifunctional properties such as biocompatibility superparamagnetism small size and low toxicity They have been applied in magnetic resonance imaging (MRI) biosensors and as anti-cancer drugs carriers Due to their high specific surface area and easy separation from the reaction medium by the use of a magnet they have been employed in enzymatic catalysis applications

bull Typical strategies for immobilizing lipase onto MNPs rely on surface grafting via low molecular weight linkers or polymers containing amino or epoxy functional groups to which lipases are reacted via covalent conjugation methods Using such methods the maximum reported loading capacity of lipase on nanoparticles is approximately 130 mgg

bull Working-bull In this work we present approach to immobilize

lipases onto iron oxide MNP surfaces modified with polydopamine an in-situ formed coating inspired by the adhesive proteins secreted by marine mussels

bull The ortho-dihydroxyphenyl (catechol) functional group found in dopamine is also present in mussel adhesive proteins in the form of the amino acid DOPA where it is highly adhesive to oxide surfaces and under alkaline conditions oxidizes to form quinone a species that is reactive toward nucleophiles such as primary amines

bull Dopamine containing both catechol and primary amine was previously found to produce conformal coatings on surfaces by self-polymerization which are further capable of immobilizing biomolecules

bull In the method described here polydopamineserves as a conformal coating for the purposes of lipase immobilization onto MNPs Our results demonstrate that PD-MNPs exhibit high efficiency for lipase immobilization under aqueous conditions and the enzyme retains high activity after many cycles of magnetic separation and reuse

bull Effect of temperature and pH on free and immobilized lipase activity- The effect of

temperature on the activity of free and immobilized lipase at pH 70 is shown in Figure1 The

activity of both free and immobilized lipase is not adversely affected at temperatures below 40degC

The relative activity of free lipase dropped significantly above 40deg C and decreased to just 265

of the initial activity at 60degC In comparison lipase immobilized on PD-MNPs retained 593 of

activity at 60degC A possible explanation for this is enhanced thermal stability in the immobilized

state leading to less denaturation of protein

bull The effect of pH on the relative activity of free and immobilized lipase is shown in Figure2

According to our data the activity of lipase immobilized on PDMNPs was retained throughout a

wider pH range compared to free enzyme At pH 6-7 free lipase was stable for short incubation

periods but was adversely affected at longer incubation times retaining only 60-70 of the activity

after 96 h incubation At pH 5 and 8 only 30- 40 of the activity of the free enzyme was retained

after 96 h and complete loss in activity was observed for free lipase upon incubation at pH 4 and 9

In contrast PD-lipase immobilized on MNPs retained more than 90 of activity at pH 6 and 7 and

even retained more than 50 of activity after 96 hours incubation at pH 4 5 and 8 The increased

stability of immobilized lipase may result from multipoint covalent linking between lipase and the

PD-MNPs which prevents lipase denaturation in acid or alkaline environments

bull Recovery and reuse of immobilized lipase- The stability of immobilized lipase under conditions of repeated magnetic isolation and reuse was studied After each enzyme run the lipase containing PDMNPs were magnetically isolated and washed twice with hexane and ultrapure water to remove any remaining substrate and product species before the next experiment The residual activity of the immobilized lipase after each cycle was normalized to the initial value (the initial activity was defined as 100)

Figure- Magnetic isolation

of PD-MNP

Photographs of an aqueous

suspension of PD-MNPs

before (right) and after (left)

magnetic isolation

FREEZE DRYINGbull Removal of water from this mixture by lyophilization in the presence of stabilizing agents results in a

lyophilized enzyme that is stable at room temperature over extended periods of time

bull This invention also relates to an improved combination of stabilizing agents useful in a process for

lyophilizing an enzyme The combination is capable of providing enhanced stability of enzymes stored at

temperatures above freezing

bull Enzymes are sometimes unstable in aqueous systems at room temperature and so are typically stored

either in a frozen state or liquid at -20deg C (-70deg C in some cases) in the presence of stabilizers such as

glycerol that have low freezing points and low vapor pressures

METHOD-

bull Lyophilization is a drying process in which water molecules are removed from a frozen solution under a

vaccum

bull Lyophilization first requires that the aqueous solution be frozen and preferably quick-frozen One means

of quick-freezing a solution is immersing it into liquid nitrogen

bull A high vacuum is then applied to the frozen sample which results in sublimation or vaporization of ice

phase at subzero temperatures (primary drying)

bull Residual moisture can be subsequently removed by allowing the temperature to gradually rise (secondary

drying)

bull The resulting freeze-dried product is a dry crystalline substance or a powder Substances that are

lyophilized are often hygroscopic that is they will tend to absorb atmospheric moisture and lose their

stability In the presence of certain additives however it is possible to produce material that is not

hygroscopic

bull Stabilization and storage of materials using carbohydrates as cryoprotectants can be done

bull The formulation comprises a carrier protein one or more sugars one or more disaccharides

one or more disaccharide derivatives optionally one or more sugar polymers andor branched

sugar polymers The formulation may either be aqueous or substantially free of water (dried

formulation) The dried formulation may be reconstituted to the aqueous phase before use

bull Eg- A preferred formulation comprises the sugars trehalose and maltitol the sugar polymer

dextran along with a carrier protein preferably an albumin more preferably bovine serum

albumin (BSA) in a buffer solution Neither the BSA nor sugars alone confer the thermal

stability required for long-term storage

bull Enzymes that have been stabilized with sugars during lyophilization include

phosphofructokinase which was stabilized with glucose galactose maltose sucrose and

trehalose and alkaline phosphatase which was stabilized with mannitol lactose and

trehalose

bull Enzymes stabilized by the method of the present invention may be selected from the group

consisting of T7 RNA Polymerase AMV-Reverse Transcriptase RNase H etc

Conclusionbull Biocatalyst stability is a key issue for bioprocess economic viability

Industrial bioprocesses will keep increasing in the future as far as stable and robust biocatalysts are developed to withstand harsh conditions of operation

bull Research on intrinsically stable enzymes from extremophiles is very active and important outcomes for biocatalysis are expected in the near future

bull Gene cloning of such enzymes on mesophilic hosts and protein engineering of mesophilic enzymes are being exploited already by major enzyme companies to develop stable and robust biocatalysts It is foreseeable that in the near future most industrial biocatalysts will be products of genetic and protein engineering

bull Stabilization of biocatalysts by conventional means like immobilization and new methodologies like cross-linked enzyme crystals is broadening the scope of biocatalysis

bull Increased stability of enzymes in non-aqueous media is a relevant technological asset for the development of biocatalysis in organic synthesis

REFERENCES

bull Books-bull Biotechnology- Expanding horizons BDSingh

2nd Edition Pg no- 650-677bull Immobilization of enzymes Joseacute M Guisaacuten - 2006

- 449 pages

bull Websites-bull Wu Y Wang YJ Lou GS Dai YY In situ preparation of magnetic

Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution BioresourceTechnol 2009 1003459-3464