Κατευθυνόμενη εξέλιξη (directed evolution) ΠΡΩΤΕΪΝΙΚΗ ΜΗΧΑΝΙΚΗ

nuthookransomBiotechnology

Dec 10, 2012 (4 years and 6 months ago)

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Interdisciplinary

field

recognized

by

the

Organization

for

Economic

Cooperation

and

Development

(OECD)

as

an

important

component

of

sustainable

industrial

development
.



The

application

of

enzymes

and

whole

cell

biocatalysts

in

the

production

of

goods

and

services
.



Use

of

enzymes

in

competitive

large
-
scale

bioprocesses
.

1.
Detailed

analysis

of

protein

structure

and

function

at

the

basic

research

level

(Aim
:

understand

proteins

as

molecular

machines)


2.
The

emergence

of

proteomics

(Effort

to

understand

the

structure,

function,

interaction

partners,

processing

variants

and

regulation

of

all

the

proteins

encoded

by

an

entire

genome)


3.
Manipulation

and

improvement

of

enzymes
.

Optimization

of

their

efficient

use

as

commercial

biocatalysts



Χρησιμοποίηση του όρου «ένζυμο»







1878




Κινητική περιγραφή ενζυμικής δραστικότητας



1913




Πρώτη παρασκευή ενζύμου σε κρυσταλλική μορφή (ουρεάση)

1926




Περιγραφή ενζυμικών δοκιμών (
assays
)
1930




Πρώτη ακινητοποίηση ενζύμου


1953




Εφαρμογές ακινητοποιημένων ενζύμων & κυττάρων



1960’s






1970’s




Κλωνοποίηση, έκφραση γονιδίων ενζύμων σε μικροοργανισμούς

1980
’s


Κατευθυνόμενη μεταλλαξογένεση




Κατευθυνόμενη εξέλιξη (
Directed evolution)

1990’s


Food,

feed,

agriculture,

paper,

leather,

textile

industries



Products

as

well

as

raw

materials

consist

of

biomolecules

which

can

be

produced
,

degraded

or

modified

by

enzymatic

processes

(2)

Agrobiotechnological

processes



enzyme

assisted

silage

fermentation


bioprocessing

of

crops


production

of

feed

supplements

(phytases
:

improve

efficiency

of

nutrient

utilization)


Production

of

enzymes

in

plants

(trypsin,

laccase)


Production

of

chemicals

and

fuels

from

agricultural

waste

biomass

(cellulase
:

converts

cellulosic

biomass

to

fermentable

sugars)


Alcohol

industry
:

the

use

of

enzymes

for

the

production

of

fermentable

sugars

from

starch

is

well

established



Increasing

interest

in

fuel

alcohol

(increased

environmental

concern,

higher

crude

oil

prices)



Efforts

to

develop

improved

enzymes

to

enable

utilization

of

cheaper

substrates

(lignocellulose

to

make

bioethanol)




US

Department

of

Energy

awarded

$
32

million

to

Genencor

Novozymes

to

reduce

the

price

of

cellulase
.


Only

a

few

commodity

chemicals

are

produced

by

enzyme

technology



acrylamide
:

annual

production

40
.
000

tons


polylactic

acid


biobased

1
,
3



propanediol



Chiral

compounds


nicotinamide

from

3

-

cyanopyridine



(
3000

tons/

year)



Chemical

market

$
1
.
6

trillion


(
50

billion

accessible

for

biological

approaches)


1,3 propanediol based polymers have been known to have
unique properties



Lack of an economically viable chemical process



Construction of a recombinant E. Coli containing two
metabolic pathways



a)

Conversion of glucose to glycerol



b)

Conversion of glycerol to 1,3 propanediol



Economically attractive process

Two important features distinguish enzymes from other
types of drugs

a) Enzymes often bind and act on their targets with
great affinity and specificity


b) Enzymes are catalytic and convert multiple target
molecules to the desired products

STRUCTURAL GENOMICS INITIATIVE

AIMS:



1)
Organize known protein sequences into families

2)
Select family representatives as targets

3)
Solve 3D structure of targets by X
-
ray crystallography or


NMR spectroscopy

4) Build models for other proteins by homology to solved


3D
-
structures






Predict enzyme properties e.g.

substrate range, thermostability from (modeled) structure
.

High
-
throughput “ thinking”


-

High
-
throughput screening: Robotically screen huge libraries of


potential enzyme or cell growth inhibitors for pharmaceutical


development



-

High
-
throughput

protein expression, purification, crystallization


trials, structure determination


(In search of new structures and drug targets for pharmaceutical


discovery)



-

Simultaneous protein profiling of thousands of human proteins


from normal and diseased biological samples


(Diagnosis, tailored treatment, treatment monitoring of patients)

NEW TECHNOLOGIES FOR ENZYME

DISCOVERY


Natural microorganisms



Development of bioinformatics


Availability of sequence data



Enzyme engineering


-
Rational enzyme engineering


-
Directed evolution



High
-

throughput screening

COMBINATORIAL BIOCATALYSIS

-
An emerging technology in the field of


drug discovery


-

Use of enzymes and microbial catalysts


to generate libraries through


biotransformations of lead compounds



-

Enzymatic transformations: oxidation,


reduction, deamination, glycosylation,


acylation

Σταθερότητα



Αύξηση θερμοσταθερότητας


Αύξηση σταθερότητας σε χαμηλές/υψηλές τιμές
pH


Αύξηση σταθερότητας/ ενεργότητας σε οργανικούς διαλύτες


Ανθεκτικότητα σε οξειδωτική απενεργοποίηση


Ανθεκτικότητα σε πρωτεόλυση


Κινητική


Αύξησης μέγιστης ταχύτητας


Αύξηση συγγένειας για το υπόστρωμα


Τροποποίηση εξειδίκευσης σε υποστρώματα


Ανθεκτικότητα σε αναστολή από υπόστρωμα/προιόν


Αλλοστερική ρύθμιση ενζύμων


(απαλοιφή ανάδρομης αναστολής,
feedback inhibition
)


Απαλοιφή απαίτησης ενζύμου για συνένζυμο

1.
ΑΝΤΙΚΑΤΑΣΤΑΣΗ ΤΟΥ ΔΕΣΜΕΥΜΕΝΟΥ
ΜΕΤΑΛΛΟΥ ΣΤΟ ΕΝΖΥΜΟ


2.
ΧΗΜΙΚΗ ΤΡΟΠΟΠΟΙΗΣΗ


3.
ΕΝΖΥΜΙΚΗ ΤΡΟΠΟΠΟΙΗΣΗ


4.
ΚΑΤΑΛΥΤΙΚΑ ΟΜΟΙΟΠΟΛΙΚΑ
ΣΥΜΠΛΟΚΑ

ΕΝΖΥΜΟΥ
-
ΣΥΝΕΝΖΥΜΟΥ


5.
ΚΑΤΕΥΘΥΝΟΜΕΝΗ ΜΕΤΑΛΛΑΞΟΓΕΝΕΣΗ


6.
ΚΑΤΕΥΘΥΝΟΜΕΝΗ ΕΞΕΛΙΞΗ


7.
ΜΙΜΟΙ ΕΝΖΥΜΩΝ


Many

organic

chemical

substrates

and

products

are

sparingly

soluble

or

insoluble

in

aqueous

solvents



Novel

reactions

can

be

performed




Possible

to

regulate

certain

catalytic

properties

of

enzymes

(solvent

engineering)



Avoid

microbial

contamination


High skills in
:


1. Screening for new and improved enzymes

2. Fermentation for enzyme production

3. Large
-
scale enzyme production

4. Formulation of enzymes for sale

5. Customer liaison

6. Dealing with regulatory authorities


The

targets

of

pharmaceutical

compounds

are

chiral

molecules



Only

one

enantiomer

of

a

chiral

drug

has

the

desired

biological

activity



Most

manufacturers

will

increase

the

use

of

chiral

molecules

Regulatory

hurdles

Need

to

extend

patents

Requirements

to

improve

safety

&

effectiveness

of

their

drugs



Worldwide

sales

volume

for

single

enantiomer

drugs


>
$
100

billion



>

50
%

of

all

new

drugs

for

the

next

five

years

are

projected

to

contain

optically

pure

active

ingredients


Κατευθυνόμενη μεταλλαξογένεση


(
site directed mutagenesis)



Κατευθυνόμενη εξέλιξη


(
directed evolution)

(2)