India n Journal of Biochemistry & Biophysics
Vol. 39. Augu st 2002. pp. 220-228
Applied Biocatalysis: An Overview
Munishwar N Gupta* and Ipsita Roy
Chemi stry Department. Indi an Institute of Technology. Delhi , Hauz Khas. New Delhi 110 0 16. India.
Received 12 Jllly 2002
" Rather I prize the doubt.
Low kinds exist with out,
Finished alld finite clods.
Untroubled by a spark "
Robert Brownillg
Introduction
The title of thi s overv iew/mini-sym pos ium may
rai se a few eyebrows. Wh y applied biocatalys is? Wh y
not si mply biocatalysis? ]s th ere a difference? The
foll owi ng anecdote may give a clue. Michael Faraday
prese nted hi s discovery of electricity to the Briti sh
government. The Lord of Exchequer asked him the
poss ible use of such a discovery. Faraday 's answer
showed his brilliance. He repli ed: " My Lord, one day
yo u will be ab le to tax it!" He was given the gree n
signal for further work . It is a thin line whi ch di vide:;
fund amenta l and applied sciences. If you put a
biocatalys t to use for a spec ifi c purpose and the
applicati on is economi call y viabl e (or at leas t shows
potential ), we can call it appli ed biocatal ys is. The
adj ec ti ve keeps Ollt co nv enti onal enzy mology, e.g.
purify ing an enzy me with 5-10 steps.
A few years bac k, Whites ides an d assoc iates wro te
a very good articl e on perceived advan tages an d
disadva nt ages of usi ng enzy mes l . The laller included
"expens ive, un stab le, restricted to predo minantl y
aqueo us enviro nments an d di fficult to manipul ate".
Thi s overview will show that these disad vantagcs
have been more or less overco me. (I n fact , the vari ous
papers in thi s mini-sy mpos ium ill ustrate thi s with
specific exampl es) . Much of the biotechno logy today
deals with either prod ucing enzy mes/pro tein s or using '
them! Appli ed Bi ocatalysis is an are:.! wh ich is trul y
multi-disc ipli nary in nat ure: mi crobiologists . tiss ue
;, Author to who m correspondence: Illay be add ressed.
rc I.: 91 - 11-659 1503
rax:9 1-tt-658 tOn
Ema il : mn_g upta@ho tmail.co lll
culture ex perts, chem ical engineers, biomedical
engineers and immunologists merely consti tute an
illustrativ e list.
The hi story of app lied biocatalysis is nearl y as old
as human civili zation. Earliest applications were
em pirical and were more of recipe than science.
Moreover, these app li cati ons were there eve n before
th e co ncep t of biocatalys t or enzy me was known to
us. Table I summarizes th ese earl y app licati ons and
discoveries and some correspond ing current
perspecti ves.
P,-oduction of Enzymes
T he production of th erapeuti c proteins, whic h has
bee n made possib le by new discoveries in
biotechno logy, has in 200 I, generated sales exceeding
$25 bi lli on 1o This, co mbin ed with the commercial
need of vaccines, ind ustrial biocatalysts, enzymes for
syn thesis of fine chemica ls by 'green' rou tes and
di ag nosti c kits, have necessitated a 'seco nd look' at
th e cost factor in downstream processing of proteins.
Thi s stage also faces challenges from the fact th at th e
target product form s only a minor part of a hi ghl y
comp lex broth.
Apart from co nventi onal methods of ex tracting
enzy mes from anima l, plant and mi crobial sources,
today one can produce enzy mes by cloning, ti ss ue
cu lture or transgen ic technique ". All stages fro m the
poi nt of producti on to obtaining the enzy me with
"adeq ua te purity le vel" co nstitu te dow nstream
proccss ing' c. In th e case of intrace llul ar enzymes,
dow nstrea m process ing starts with cell d is rupti onl.1 by
chem ical, mec hani cal or bi oc hemical means. Fo r all
cnzy mes . the next step is so li d/l iquid separatio n for
re mov ing the su pend ed impurities and cell debri .
Beyond thi s, there are no set rul es but the trend is to
use met hods of increas ingly hi gher resol ution. Thus,
vec ipitat io n ~ ch romatog raphy ~ affinity chro matogra phy have, ove r th e years, fo rmed th e basic outl ine
for the purification protocol.
221
GUPTA & ROY: APPLIED BIOCATALYSIS : AN OVERV IEW
Table 1•
•
Hi storica l Pe rspective and So me C urre nt Tre nds in Appli ed Bioc3 talys is
Produc ti o n o f soy de ri ved foods in Ch in a and Japa n. Mi crobi al a myl ases a nd proteases were in vo lved
2
2
Home r desc ribed " the producti o n of c heese by sti rrin g mil k w ith a twi g of fig tree". Fic in , obv io usly, was in vo lved
Milk kept in a bag made fro m th e sto mac h o f a rece ntl y slaug ht e red calf gave a dri e r substance wh ic h gain ed a fl avour
2
afte r so me time. Thus was bo rn the art of cheese makin g w ith the he lp of re nnin Our mo re recent know ledge te ll s us th at
I06
lo
ca lf re nnin catalyzes the proteo lyti c clea vage o f a sin g le pep tide bo nd (Phe '_ Me t ) in th e K-ca se in mo lecu le, ini ti atin g
pro tein prec ipita ti o n).
We now also know th at c han ges in tex ture a nd fl avo ur o f c heese a re associated with th e furth er hyd ro lys is of milk
.
pro te ins .
Proteins -7 Peptides -7 A mino ac ids -7 Vo latile fl avours and aro mas
Occasio nal sho rt ages of ca lf re nnin (c hy mos in ) have pro mpted the use of fun ga l proteases from Elldothill pa/'Gsitica.
Mu co r meihei a nd M. puci/lus as substitutes . G lobal demand fo r chymos in is estim ated to be wo rth abo ut $ 100 milli o n a
yea r. O n March 23, 1990, c hy mos in became the first gene ti ca lly e ng ineered protein to be used in food process ing'.
Rennet (a mi xture of re nnin a nd pepsi n) inc ide ntall y was also the first e nzy me prepara ti o n to be mar keted, by a
company called C hritian Hansen (Copen hagen, Denma rk ) in 1875 2.
~
•
Otto Ro hm obtained try psin-in-dete rgent patent in 19 13. Thi s led to the introduction o f the first enzymati c deterge nt
' Burnus' co nsistin g of pancreatin and sodium ca rbo nate 6 . However, it was q ui te so me time before enzy me-based
detergents became co mmo nl y acceptab le. Like many o the r app li catio ns of enzy mes, thi s story is linked wit h the iss ues of
stabi lity and stab ili zati on of e nzy mes. Th e enzy mes incorporated in the detergents sho uld be stable at alk alin e p H, at
fairl y hi gh tempe ra tures a nd in the prese nce of c hel ati ng agents and surfac tants. Around the earl y sixties, "everybody
wanted Biotex , the protease co ntai ning dete rge nt ,,2 However, th ere was the scare of all e rgic respo nses in the la te sixti es.
Thus, it is inte restin g to note that by the ea rl y seventi es, e nzy mes were w ithdraw n fro m detergents 3 . Granu lat io n
tec hni ques all owed the ir reintroduc ti o n in th e de te rgents as it was found th at it was the inhalation of d ust formed in the
e nzy me powde r which was responsib le fo r th e all e rg ic responses 3
Prote in e ng ineered subtili sin addressed the aspect of operationa l stab ili ti. Today, c nzy mc-based detergents constitute
a major applicatio n of biotechno logy. T he dete rge nt ind ustry acco unt s for - 30 % o f to tal sales of e nzymes. Apart from
pro teases and lipases, amy lases and cellul ases are also used.
Apa rt from detergents for tex tiles, e nzy mes are also used in clean in g agents for o th e r purposes, e.g. lipases in contact
lens cleaners and w indshi eld washing fluids, e tc. 2
•
Starch processing :
Today, po lysacc haride-deg rad in g enzy mes constitute a majo r co mpo nent o f th e market for industrial e nzymes . Wi thin
th is, starch-degradin g enzymes represent the biggest porti o n. a- Amylase a nd g lucoamylase y ie ld s uga rs. The latter ca n
e ithe r be fe rmented to alcohol (a n e nergy source) o r convert ed into prod uc ts like hi gh fructo se corn syrup (HFCS )
(industri al sweete ner) by g lucose isomerase. The e nzy mo logy o f starch deg rada ti o n is dep ic ted in Fig. I.
A maj o r cons tra int has been th at whil e indus trially use fu l (t hermo tab le en zy mes fo r sta rch liqu efac ti o n) a-amy lases
are uns tab le be low p H 6.0, g lucoamy lase req uires p H of aro und 4.2-4.5 for furth c r hydrolysi s to g ive g lucose . pH
adju sting mean s additio nal cost, mo re so if g lucoamylase ac ti o n has to be fo ll o wed by glu cose iso merase (w ith pH
o pti mum aro un d 7-8). Fe w years back, S haw et al. ( 1999) have rev iewed protei n e ng ineeri ng of a- amyla se for
fun ct io nin g at low p H7. Thi s perh aps g ives a good idea of w ha t app lied biocata lysi s is all abo ut!
•
PeClill hydrolysis:
It was in 1930 that Kertesz tiled a pate nt for "enzy matic clarifi cati o n of unfe rme nt ed ap~l e juice"S Today, en zy me s
are used in a variety of ways : in ex tracti on. clarifi cat ion and modifi cati o n of frui t jui ces . While pec tinase is a key
en zy me (see Fig. 2), man y othe r e nzy mes are a lso employed in specific cases . Fo r example, add ition of cellulases helps
in releasing desired colou red co mpound s in the case of black currants and red g rape. So me fruits like pear a re ri c h in
arabans an d arabanase is required for c larifi ca ti o n of pear juice. Naringi nase is req ui red fo r deb inering, espec ially in
cases lik e grape fru it ju ice. Another int e resti ng requi re me nt is that of g lucose ox idase for re moval of O 2 from
"headspace" in the case of wine. beer and fruit j ui ce. Thi s c uts dow n non-enzy matic browning of these beverages. Thi s is
o ne a rea w here know ledge of bioca taly sis has beco me a n acceptable practi ce at industrial level. For example. whil e
tec hnology fo r production of Fre nch cider ha s esse nt ia ll y rema ined un changed since 1909, in about 30 % cases, the
industry has switched ove r to the use o f Rapi J ase Po maliq (a mix ture of pcc tin ases, heillicellulases an d cel lulases by
Gi st-Brocades, Nc therlands ) fo r c uttin g down the process time by enzymati ca ll y hyd ro lyz in g pec ti c material in appl es s
•
Today, there is hardly any indu stry whcre en zy me s are not used. Ani mal feed. prod uc ti o n of low lactose milk and whey
hydrolysi s. cdib lc oil ex trac ti on. textiles, leathe r a nd paper indus tri es are so me oth er notab le exa mpl es.
INDI AN J. BIOCHEM . BIOPHYS., VOL. 39, AUGUST 2002
222
Starch
me mbran e
interacti o ns
with
prectpltati on,
separati ons, fluidized beds, two-phase a nd threeph ase partiti o nin g me thods.
1a -amylase
Glucoamylase
J
Oligosaccharid e
).
Pullulanase
Gl ucose
l
p-amylase
Maltose
11 Glucose isomerase
Fructos(
Fig. I- Schematic diag ram of enzy mati c deg radatio n of starch.
Ia-A mylase hydro lyzes internal a- l ,4 linkages (endoh ydrolase) .
p-A mylase removes maltose units from the non-reduc ing end
(exo hyd rolase). Glucoa mylase hydro lyzes a- l,4 glycos idi c bonds
and removes glucose units from the nonrcducin g end of the
oligosacc harid e. Pullulanase, on the other hand . hydro lyzes a-I,6
glycos idic bonds. Glucose isomerase converts glucose to fru ctose
and is used for producing industrial sweeteners as fru ctose is
much more sweet th an glucose].
00B-0-v>
1
E,Opolyg,Lumn",
COOCH)
cleaves links between
non-reducing
ends of chains Endopolyga!acturonase and
pectate lyase cleave bonds
Pectin lyase
between non-esterifi ed
galacturonic acid residues cleaves bonds
between
esterified
galacturonic
acid residues
Fig. 2- Schematic diagram of enzy matic degradati on of pectin
Modern downstream processin g is based upon the
rea lization that a limited number of unit processes is
an essential feature of a n economical protocol. Thi s
has resulted in two broad approaches :
(a) To deal with crude suspensions directly and skip
the solid/liquid separation step. This has been
possible by
using two-phase extractions,
ex panded bed chromatography a nd in recent
years, three phase partitioning.
(b) To bring up affinity-based processes ri ght in the
beginning and not at the polishing stage. Thi s has
been made poss ible by inte rfacing affinity
Both broad approaches coin cid e in shapin g ma ny
modern bioseparati on techniques. Thi s is o ne area of
bi ocata lys is where much has been happe ning in the
las t decade or so. He nce thi s wi ll be dea lt with in
so mewhat grea te r detail. A rev iew of so me of th e
important deve lo pme nts fol lows .
Expanded Bed Chromatography
In thi s approach, the use of tai lor-made media
all ows one to o pe rate c hro matography in a fluidi zed
bed mode. Particulate adso rbents, if packed in a
co lumn, can not dea l w ith crude suspe nsio ns as
clogg ing of adsorbent beds will take place. Batch
adsorption in stirred tank is an opti on in such cases,
but sho ws poor reso luti on and capacit y. A fluidized
bed in whi ch inte r-parti cle di stance in adso rbe nt is
increased , creating voids th rough whi ch suspe nded
impuriti es pass th rough, is best suited to capture th e
protein from the crude extract. The bes t way of
fluidizing th e adso rbe nt particl es in practice is by a
liquid fl ow directed upwards. Such flui dized beds can
co mbin e cla rifi cati on, co nce ntrati on and (varying
ex tent of) fractionation in a simple un it process 14 .
More sophi sticated designs and more deta i led
di sc uss io n can be found in so me of the revi ews I5.1(i.
Operation all y, an ex panded bed chromatography
protocol fo llows the standard sequen ce (of packed
beds): equilibration , sampl e applicatio n, wash, elution
(CIP) .
Expanded
bed
and
c1eaning-in-place
chromatography has already been succes full y applied
in many cases. Some illustrative examp les fro m our
laboratory are g ive n in Tabl e 2.
Affinity Precipitation
Affinity prec ipitation involves preC Ipitati on of th e
target protein in free solution, mostly using a
I7 IS
reversibly so lubl e-insoluble macroaffinity li gand . .
Thi s macroaffinity li gand consists of a polymer which
is so designed that its solubility is contro lled by
altering some so lvent parameter such as pH,
te mperature, addition of metal ions, etc. In so me
fortuitous cases, the polymer itself has an affinity for
th e protein. In other cases, a ligand is coupled to the
polymer. Since the target protein has affini ty for the
macroaffinity li gand, it is se lectively precipitated
along with the macroaffinity " li gand upon the
necessary change in the controlling parameter. It can
be recovered us in g specific or non-spec ific elue nts
GUfYfA & ROY : APPLIED BIOCATALYSIS : AN OVERVIEW
223
Table 2- Some examples of the use of the technique of expanded bed chromatography (Adapted fro m Ref. 12)
Enzyme/
Protein
Source
Yield
Matrix
(%)
Alginate
92.5
99.7
89.8
80.2
Aspergillus niger
Cellulose beads
Chitosan
91
80
Amy lase inhi bitor
Wheat ge rm
Cu2+- linked agarose
83
Phospholipase D
Peanut
Cross linked alginate
78
Chicken intestine
Dye-linked cellulose
beads
70
Bacillus acidopullulyticlis
Alginate
97
Duranta plulllieri
Streamline DEAE
77
a-A mylase
Bacillus amylo/iqll efac iens
Porcine pancreas
Wheat germ
Scyralidiull1 th ennophihllll
Cellulase
Alkaline phosphatase
Pullulanase
Polyphenol ox idase
and the macroaffinity li gand is ava ilabl e for reuse.
The process thu s results in co nce ntrati o n and
purification of the target protein and is amenable to
sca le-up. It can also be used direc tl y with crude
c ulture broth, unlike chromatographi c techniques
where fouling of the column (especially in the packed
bed mode) is a recurring problem when using ex tracts
containIng particulate matter. Some illu strative
examples are tabul ated in Table 3.
Table 3-- Some examples of proteins purified by affin ity
precipitation
Polymer
Enzy me/Protein
puri fied
Eudragit S- IOO
Xylanase
IgG-type monocl onal
anti body
20
Alginate
Pectinase
a-Amylase
Phospholipase D
Lipase
21
22
23
24
Chitosan
Wheat germ lectin
Lysozy me
25
26
Copolymer of
I-viny limi dazole
and N-viny lcaprolacltim
Soy bean tryps in
inhibitor
27
Poly (N-i so propyiacrylamide)
Alkaline protease
28
Copolymer of
I-vin ylimid azo le
and N- isopw pylacryl ami de
Am ylase inhibitor
29
Gal actomannan
Human IgG
30
Poly (N-isupropy lacry lamide)
Avi din
31
Three Phase Partitioning
Precipitatio n wi th salts and org::lIlic so lvents are
well-establi shed techniques for co ncentration of crude
protein extracts . Three phase part iti o ni ng (TPP) was
first devel o ped as a bridge between upstream and
32
downstream processes . The basic protocol involved
is outlined in Fig. 3. The mechanism of ho w
partitioning o perates is not very c learl y understood. It
is believed to be the result of a co ll ective operation o f
pri ncipl es in vo lved in numerou s techniques such as
convention al
salt ing
o ut.
Morton 's Il-butan o l
extraction method , isoi onic precIpItati o n, co ld
coso lvent precipitati ~ n and osmo lyte and ko", motropi c
precip itation of proteins 33 . In an effort to understand
the mechan is m involved , proteinase K was s ubj ected
to three phase partitioning. Th e X-ray d iffracti o n
patterns of crystal of pure nati ve proteinase K a nd
l
TPP-treated prote inase K (TPK) were com pared.l . As
a resul t of this treatment, the sPecific ac ti vity of TPK
had gone li p by 2. 1 tim es. The atte nti o n was thus
foc ussed o n the binding reg Io n o f th e e nzy me. The
Reference
19
224
INDI A J. BIOC HEM. BIOPHYS., VOL. 39, AUGUST 2002
Crude extract
Table 4-- Li st of enzymes/proteins purifi ed by three phase
partitioning
Add ammonium sulphate
and t-butanol
Lower
Aqueous phase
Interfacial
protein precipitate
Upper
organic laye r
~
Source
EnLy melProtei n
Chicken intestine
A Ika line phosphatase
35
Dacus carota
Phospholipase D
36
Wheat ge rm
a-Amy laselProteinase K
inh ibitor
37
Tomato
Pect inase
38
Soybean
Trypsin inhibitor
39
Aspergillus lIiger
Xylanasc
40
Reference
Recover and dissolve in aqueous buffcr
Fig. 3-- Flowsheet for three phase partit ioning
most striking change in TPK was found in the
conformation of the side chains of a number of
residues. The residues indicated more than one
conformati onal state for their side chains. This
structure (TPK) corresponds to the highest observed
B-factor for proteinase K. Thus, the increased
flexibi lity of the mo lecul e is responsible for hi gher
activity of the enzyme.
Some exa mpl es from our laboratory , listing th e
success of thi s technique, are shown in Table 4. It has
been show n that interfacing a metal affinity based
step with TPP protocol makes th e latter a hi gh ly
selecti ve technique. Metal affinity based separati ons
(of which
immobilized
metal
ion affin ity
chro matography (IMAC) is th e most frequently used
version) exploit th e affinity of surface hi stidine,
cysteine and tryptophan residues in the protein
mol ecule for metal ions like Cu 2+, Zn 2+ and Ni 2+.
Soybean tryps in inhibitor (ST!) has surface hi stidin e
residues and has been purifi ed via metal affinity. The
selectivity and usefulness of meta l ion-ass isted TPP
has been evaluated by purifying STI from soybean
meal39 .
A modification in this process has been introduced
by macroaffinity ligand facilitated three phase
partitioning (MLFTPP). As discussed above, th ere are
many polymers which show affinity for th e enzy mes.
Using these polymers to capture th e target enzymes in
the TPP mode, enhances th e selectivity of this simpl e
process. Thi s has been used to purify xy lanase usi ng
Eudragi t S-IOO and the polymer is ava ilable for reuse
, after the end of one purification cycle4o .
Other Emerging Techniques
Simulated Moving Bed Technology
Continuous chromatographic separat ion processes
based on the si mul ated mov ing bed (SMB) tec hno-
logy have been gatntng increasing importance since
the beginning of the last decade for app lications in the
fine chemical and pharmaceutical industries, in
pa rti cular for the resolution of enantiomers~'. The
foc us has shi fted from sugar and petrochemical
industries for very large scale fractionation when the
technology was introd uced in the late 1950s. The
technology has evo lved out of true moving bed
(TMB ) concept. The so lid adsorben t particles flow
alo ng th e column co unterc urrent to the fluid stream at
a ve locity th at pu shes mixture co mponents (wh ich are
to be separated) in oppos ite directions. The mi xture is
fed in th e middl e and co mponen ts A and B can be
co llec ted from the top and bottom portions. Apart
from the most criti cal choice of flow rates, desig n of a
TMB (or 5MB) also has to cons ider feed
concentrati ons, number of co lumn s per zo ne, column
length, column diameter and particle size.
Nicoud has described the use of moving bed for
removal of ammo nium sulph ate from a protein~2 .
Purification of trypsin from porcine pancreatic
ext racts, purificat ion of human serum alb umin,
separation of myoglobin and lysozyme have shown
th e potential of thi s approach42 . Gottschli ch and
Kasc he have purified a monoclonal antibody from a
cell culture supern atant with yie ld exceedi ng 90%43.
SDS-PAGE of th e feed and th e pooled stream
confirm ed the removal of more than 99% of th e
co ntaminating proteins. 5MB technology offers
several advantages over conventional preparative
chromatographic techniques: a continuous process, it
ca n be allowed to run unattended and ensures stable
qu ality of the product. Prod uctivity per unit mass of
the stationary phase is hi gh (the column works in the
non-linear region of the adsorption equilibria of the
GU PTA & ROY : APPLI ED BIOCATALY SIS: AN OV ERVI EW
components to be separated, i.e. under overload
conditi ons, thus ensuring optimal use of the stati onary
phase) and the recycling of the fluid ph ase implies
th at th e solvent requirement is low. Al so, th e
technique is more robust since hi gh purity of th e
produ ct can be attained with a smaller number of
theoreti cal pl ates. At prese nt, however, thi s
technology is limited to effi cient and complete
separati on of a mi xture of two proteins.
Perfusion Chromatography
rn co ntrast to co nventional chromatog raph y medi a,
perfusion chromatography medi a particl es have two
di sc reet classes of pores. Large "throu ghpores " all ow
convecti on fl ow to occur through the parti cles
th emselves, quickly carrying sample molecul es to
short' "diffu sive" pores in side. By reducing th e
distance over whi ch diffusion needs to occur, the time
required for sample mol ec ules to interact with interi or
binding sites is reduced. Diffusion is no longer
limiting and fl ow rates can be dramaticall y
increased - without any loss of resolution or
capacity. Separati ons can be achi eved at I ,000 to
5,000 cm h( l compared to 50 to 360 cm h( l for
conventional medi a. The technique is associ ated with
hi gh throughput and hi gh dynami c capacity . Fahrner
Blan k have shown
th at an on-line
and
chromatographic assay can reli abl y co ntrol antibody
in
real-time
protein
A
affinity
loading
chro matographi c puri ficati on of a recombinant
antibody from clarified Chi nese hamster ovary cell
cultu re f1 uid 44 . Hizel et al. have described a new
procedure fo r the iso lation, puri fica ti on and
quantification of th e product of the oncosuppressor
gene brcal in tumour cells 45 . It invo lves esS]
methi onine labelling of intrace liular proteins followed
by two perfusio n chromatographies. DNA-binding
affi nity
proteins were isolated by heparin
chromatography on Poros 20 HE. BRCA I, which
inc ludes a zinc fin ger, could be eluted along with all
the nucleic acid-binding proteins using a salt grad ient.
Monoliths
Monoliths are co ntinuous stati onary phases th at are
cast as a homogeneous colum n in a single pi ece and
prepared in vari ous dimensions with agg lomerati ontype of fi brous mi crostructu res. They exhibit hi gh
effi ciencies even at hi gh flow rates du e to their fast
co nvective mass transfer and can th erefore be used at
very high mobile phase velocities (up to 400 mlmin- 1),
leading to hi gh producti viti es 46 .47.
225
Monoliths have been applied fo r the fas t separat ion
and purificati on of proteins, DNA, smaller molecul es
like
organi c
acids48,
hydroxybenzoates,
·
Ieotl'des and peptl'des 4950
..
o IIgonuc
' . Quantltatlve
analy sis of affinity interactions betwee n antibodi es
and immobilized group specifi c li gands (protein A, G
and L) has been attempted using hi gh perform ance
monolith affinity chromatograph y (HPMAC)51.
Monoliths have been used' for in situ solid phase
and
immobili zati on
of
peptide
synthesis52
polynucleotide phosphoryl ase 53 . The benefits of using
monolithic columns include mini aturi zati on leadi ng to
extremel y fast separation (within seconds) and easy
sca le-up. Further applicati ons of thi s upcom ing
include the areas of capillary
techn ology
elec trochromatography , chip technol ogy and scale-up
in purificati ons4 .
Tailoring Enzyme Properties or Biocatalyst
Engineering
Early efforts in tailorin g enzymes to suit spec ific
needs utilized chemical modifications and chemical
crosslinking55. The key iss ue has been of enh ancing
stability
(especially
thermal
stabili ty)56.
Immobilizati on techniques brought in reusability and
possible stabili zati on57 . Both noncovalent and
covalent methods of immobili zation have proved
use ful. Two relatively recent developments in this
area have been:
(i) Introduction of cross li nked enzy me cry. ta ls,
CLEC™, by Altus Bi ologicals. These microcrysta lli ne preparati ons are reported to be stable
in both aq ueo us and nonaq ueous media58 .
(ii) Design of smart biocatalysts. Thi s has mostly
consisted of creating bioconjugates of enzy mes
with smart polymers59 . Thi s enab les the
biocatalys t to be in free so luti on and one can
transfo rm
macromolecular
and
insol ubl e
substrates. After use, the biocatalyst can be
recovered by app lying suitable stimulus which is
chosen depe nd ing upon the smart pol ymer.
Te mperature, p H and add ition of chemicals are
so me of the stimuli whi ch have been used. An
attrac ti ve des ign is to build in a smart domain
with in the enzy me stru ctu re 60 .
Protein engineerin g has bee n a more powerful tool
for alterin g specific properti es of enzymes 61 . While
earl y appli cati ons again focussed on enh anci ng
stability, more innovative approaches like changi ng
pH optima (for effi cient bi odegradati on of starch)
are beginning to emerge 7. Directed evolution is the
226
I ND IAN J. BIOCHEM. BIOPHYS .. VOL. 39, AUGUST 2002
lates t tool whi ch attempts at creatin g enzymes with
suitab le reg io- an d stereo-selecti vity and suitab le
kineti c properti es 62 . Thi s co ntinues to use
reco mbinant methods bu t takes advantage of hi gh
throughput screen ing methods to reach a pre-selected
property.
Bioconversions, Organic
Aq ueous Enzymology
Synthesis
and
Non-
Convertin g biomass to va lue-added products has
been a majo r class of bioconvers ions. Some of th ese
have already been menti oned in Tab le I. Hydro lys is
of cellulose and ligni n to suga rs and finall y to
bioethanol has been an act ive area which has recentl y
been reviewed by Himme l et al. 63 . Starch degradati on
has already been mentio ned. Starting with corn starch,
prod uction of hi gh fructose corn syrup (HFCS) is one
of the top few applications of enzy mes in th e
industryl. Enzymes are increasingly bei ng employed
to develop 'green technol ogy' for replac ing processes
based upon aggressive chemi cals64 . A less- known
exam ple in thi s category is the ex tracti on of edible
oil s by aq ueous enzy mati c oil ex tracti on process 65 .66.
The process essentially degrades macro molec ul ar
struc tures entrapping oil bodies and thus fac ilitates the
oil recovery.
Development of methodology for use of enzymes
under low water conditi ons constitutes a milestone for
applied biocatalysis. Both "nearl y anhydrous
systems" pioneered by the groups of Klibanov 67 ,
Mattiasson68 and Halling69 and "reverse micell ar
systems" developed by th e gro up led by Levashov
and Martinek 7o , Luisi 71 and more recentl y by Cabral 72 ,
have made it possible to use hydrolases for sy nthesis.
Lipases, undoubted ly, have been used more often,
fo llowed by proteases and carbohydrases. Several
interesting phenomenon like pH memory, unusuall y
high thermal stability and molec ul ar imprinting have
been observed with the use of enzymes in "neat
organic solvents,,73. Again the major iss ue here has
been that of stabili zing th e enzymes against
inactivation by organi c solvents: Polar so lvents are
know n to be more damaging as com pared to nonpolar solvents74 . Again, a vari ety of techniques like
chem ical modification , immobili zation and protein
engineering (used for enzyme stabilization in aqueous
med ia against denaturin g co nditions) have bee n used
with varyi ng success 75. Recent work with directed
evo lution is especially releva nt to the use of enzymes
for obtaining drug intermediates 76 . This is an area
where much is go ing on and it is likely to prove
immensely useful for th e overall purpose of synthesis
of fine chemicals and pharmaceutical s.
Ex pos ure to organic solvents as such can have
so me unusual and interes ting co n eq uences . It has
bee n shown th at in the presence of about 5- 10%
organic solven t, vari ous enzy mes sho w hi gher
. 77
acti. vity
. I t has also been observed that heating
enzy mes in the presence of organic so lvents at
moderately hi gh temperatures res ults in obtaining
enzymes with hi gher turnover num bers78 .
Biosensors and Diagnostics
Biosensors are basicall y created by interfac ing a
biocatal ys t with a transducer. The fo rmer may be an
enzy me, ti ss ue sli ce, or whol e organi sm. The purpose
of the biocatalys t component is to detect the presence
of a chemica l substance and amplify th e 's ignal' via
its turn over number. The transducer converts it into an
observab le parameter. Depending upon the latter, we
have enzy me electrodes 79 , fibre optic se nsors
(o ptodes)8o, field effect transi stors (FETs)81 and
th ermi stors82. The applications of biosensors ex tend to
diverse areas like clinical chemistry and health care,
veterinary,
agriculture
and
food
sciences,
fermentation and pharmaceutical production and
environmental co ntrol and pollution monitoring. With
the advent of smart materi als, the last few years have
seen des ign of ' molecular gates ' and 'valves ' which
sense and respond to the presence of certain
metabol ites 83 .
ELISA, based upon anti gen-enzy me or antibodyenzy me bioco njugates is the best examp le of use of
biocatalyst diagnostics84. On-line anal yzers using flow
injection (FIA) in combination with expanded beds
and perfu sion chromatography are relatively recent
examples 85 .86.
Proteomics and Beyond
Proteo mics represents the most recen t and still
fashionable face of applied biocatalysis. Genomics
has given us the inform ation. Proteomics has to tell us
the "action part". Which enzyme/protein occurs
where, how much , along with what other proteins and
doe what, are the questi ons which define th e
co ntours of this emerging science. It may be
interes ting for the reader to refer to a recent debate on
what proteo mi cs should mean 87! Ri ght now, for most
of th e people, it means mass spectrometry (electro
spray and occasionally MALDI-TOF) or 2-D electrophoresi s. There are so me more sensible views
ex pressed, which foresee that more sophisticated
GUPTA & ROY : APPLIED BIOCATALYS IS: A
bioseparation strategies at th e semi-preparati ve leve l
will have to be deve loped 88 . Thus, once again, we
may come back full circle. The old protei n chemi stry
. uddenly became fashionable and was reborn as
protein fo ldi ng. May be, we will see renaissance of
down stream processing as a part of proteo mics.
Thus, th e area of appli ed biocatalys is, which
perhaps started by stirring milk wi th th e twig of a fi g
tree, has come a long way . Today it in vo lves more
sophi sti cated too ls and strategies .
15
" Where is th e knowledge we have lost in
ill/ormation ? "
T. S. Eliot
20
Acknowledgement
The publi cati on of thi s arti cle and the work qu oted
herein fro m the auth ors' laboratory were supported by
fund s from Coun cil of Scientifi c and Industri al
Research (Extramural Di vision and Tec hnology
Mi ssion on Oil seeds, Pulses and Maize), Department
of Science and Technology, Department of
Biotechnology and Nati onal Agri cultural Tec hnology
Project (lndi an Council fo r Agri cultural Research).
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