Biotechnological Application of Psychrophiles and...
Transcript of Biotechnological Application of Psychrophiles and...
Journal o r Sc icnti ric & Industri ,iI RcscOi rch vol. St). Fchruary :W()() . pp X7- IOI
Biotechnological Application of Psychrophiles and Their Habitat to Low-Temperature
K V Ramana*, Lokendra Singh and R K Dhaked
Bi otcchnology Di vision . Defcncc Rcscarch ,lIld Dcvclopmcnt
EstOlhli shmcnl. Jhansi Road. Gwalior 474 ()()2. lndia
Rcccived : 16 M ;lrch 1l)l)'J: accepted: 19 November I 99()
MiCl"oorg; lIli~m s li ving in ex trcme environments ;lre di vided into ri ve categorics: thcrmophilcs. psychrophiles. alb li phil es.
ilcitiophi Ics ;lIld halophil es. Envi ronments that are considcrcd to he ex tremc are colonized by th e ~e spec ial microorga nisms which
OIre ;Idapted to these ecologi c; iI niches. Thc app lic,lIion or psychrophilic microorgani sms in industria l processes opens up a ne\V
cra ill hiotechnology. Due to its unique hiochem ieal reatures. it can be ex ploited ror use in biotechnologic,iI indust ri es bu sy in
m<l nur;lct uring rood. enzy mes . and va lue added pharmaceu tica ls products. Recent devclopments show that psychrophil es arc
good source or novel ca talysts or industr ial interes t. Some or the enzymes have been isol<l ted and the genes arc sueeessrull y
cloned and ex pressed in target hosts. Biopolymer degrad ing enzy mes like amy lase. pullulanases. xy lanases. ;lIld proteases play
;In import ,lnt role in rood. detergent s. and pulp industri es. Ce ll membranes or psyehrophi les contain surractan ls hearing unique
s(; lhil it y at low temperatures ;lIld th;1I Gin he used in pharmaceuti ca l rorm ulation. The cold adaptati on process or psychrophil es
encompasses w ide Illod iri cati ons in structun.: ami molecular architecture. physio logy. and hiochemist ry or these organisms.
These arc desnihed. in det;l i l. here.
Introduction At low temperature. growth and deve lopment of
microorganisms is dras ticall y red uced due to the low so lu bility of orga ni c and inorga nic nutri ents , decreased ion/so lute transport , reduced diffusi on, and os motic effec ts on pl as ma membrane and cd I wa ll I . Psyc hrophi I ic bacteria are most wide ly distributed than other types of cx tremoph i les for the si III pic reason that water covers approx. 70 per cent o f t he cart h' s sur face and a large part of it constitutes the marine environment. About 90 per
cent of the mari ne habita t ex ist at < SoC beyo nd the the rmoc line zo ne of ocea ns2
. Apart fromthe marine habitat , polar reg ions meas ure nearl y 14 per cent or the eart h' s surface , representin g an ex t reme low-te mperature ha bi tat s in the world. The growth charac teri sti cs of psyc hruphil es and psyc hrotTophs make them suitab le to grow in varioll s low-t emperature habitats i.e., in lake sediments, ; tS gut nora o r aquat ic animals , and in the form or perip hyti c Illi cro nora, and on rocks at low ll1 uisture co nditi ons. Psyc hrophilic bacteri a arc hi ghl y thermnsen .~ iti ve and ex p()sure to moderate room temperat ure could he detriment a l to the ir acti vit y. Most or the psychroph i I ic hacteria thus far isolated are gramnegati\'e and report s on the occ urrence of gram positi ve
':' Aut hor ror cmrespondencc
bacte ria are re lati ve ly few. To exe mplify the ir diversi ty. various bacteria be longin g to diffe rent genera have been iso lated in the recent past.
Extre r1'l ophil es and the ir enzy mes have att racted much attention because of their wide range of bi otec hnolog ica l applicati ons and also to understand the ir bioc he mi cal mec hani sms o f adaptat io n to ex tremes temperature, pH and salinity. Further, they are be ing utili zed in va rious bi oprocesses to be carried out at lowtemperature, in additi on to their role in natura l decolllposition of organic matte r and nutri ent recyc ling at low temperature habitats. Particularl y, studi es on psychrophili c and psyc hrotrophic microorganisms are re lati ve ly sparse in co mpari son to thennophiles and hypenherl11ophiles. According to the definiti on of Morita~ , psyc hro
philes show an optimum growth temperature of < ISoC
with upper growth temperature of 20°C, whereas psy
chrotroph s can grow at (j°C although the ir optimulTl
temperature is around 20-2S°C. Psyc hrot rophs wen: furth er c lassified as stenopsychrotrop hs and eurypsy
chrotroph s based on the i I' abi I ity to grow at 4()OC (re r.4) Stenopsychrotrophs arc abl e to grow sub-optimall y at
4()OC in contrast to eurypsyc hrotrophs . Conspicuolls ly the characteri sti c growth rate of these microorgani sms is not much affected at low temperature.
xx J SCIIND RES VOL S9 FEBRUARY 2000
The ability of psyc hrop hili c and psyc hrotrophic bacteria to grow at low-temperatures is due to the stabi lity of ce ll constituents such as enzy mes, fatty ac id compositi on of lipids in ce ll membranes, and mod ifi cations in nucl eic acid constituents and contro l over the gene expression through the mediation of transcription and translation with the production of cold-shock protein s. In the biosphere, both prokaryotes and eukaryotes are known to ex ist as psychrophiles. In addition to polar regions fresh water lakes. rivers , marine sediments and deep sea water are the best source of these bacteri a. In addition to the iso lat ion of bacteri a, several types of yeasts . fung i and algae ha ve also been reported. These microorganisms were found to be res ponsible for min
erali zati on and nutrient recyc ling at as low as _l aC in maritime Antarctica.
The presence of a wide va ri ety of hydro lases namely
amyla se, protease, ce llu lase, nuclease, lipase, ~- ga lactos idase, and phosphatase have been reported from Antarcti c bacteri a,,·7 This rev iew discusses the role and imporlance of psychrophili c mi croorgani sms in the bi odegradati on of organic pollutant s. In add iti on, var ious basic aspects of co ld adaptati on and bi otec hn ologica l potential of metabolites and hydrolyti c enzymes have also bee n rev iewed.
Phylogeny of Extremophiles The phylogeny of ex tremophi les is based on their 16S
rR A sequence and the uni versal tree has been divided into bacteria, archae and eukarya. The archae consist of two major kingdo ms, the crenarchaeota-thermoproteales branch and the euryarchaeota . The latter kingdom consists of ex tremophil es and methan ogensN The start in g lineage of bacteria is represented by the orders thermotogal es and aquifi ciales which again represent hyperthermophi les'} Signi fi cant I y, most of the psyc hrophilic bacteria have been assigned to bacteria. Excepti onall y, a few representat i ve spec ies of crenarchaeota and euryarchaeota ha ve been reported with the help of polymerase chain react ion and genecloning experi ments 10. Some of the recent Iy iso lated antarctic bacte ri al iso lates belongs to Cylop/lOga-F/a vobaclerilll11- Bacleroide.\ and they have been classified as Polaribacler fral7~I1/(/l/ii strain 3() I and Po/arihacler.fi/amenllls strai n 2 15 based on their 16S rRN A sequence analys is. Mosl of the bacteri al strains iso lated from Antarctica ha ve been c lass ified phylogenetica ll y based on the sequence homology of the ir 16S ri boso mal DNA or RNA indicating the presence of many new and prev iously unreported o rga ni s ms that in c lud e P/anoc()CCUS III cmeekinii,
Arthrobacler, Brachybacteriul1l and PsychroflexlIs IOr-. 11 . 12 qUiS gen, nov ., sp.nov .
Habitat in Relation to Structure and Molecu lar Architecture
Ecological Sign!/icance and Distribution It has wide ly been noticed that in cold habitats,
metabolic role of these microorganisms is essenti al in the biodegradation of organic matter and for recyc ling va ri ous carbonaceous and nitrogenous nutri ents in large quantiti esl ~ . I~. Most of the organi c ni trogen released in natural habitats under ex treme low-temperature is present in the form of hi gh mol ecular we ight proteins, nucleic acids along with uri c acid and urea which cannot be utili zed directly by the nati ve bacteri al populations. Proteins can only be utilized by bacteria after ex trace llul ar hydrol ys is by proteases.Several p. ychmphiles that produce substantial amounts of extrace llular enzy mes such as proteases, lipases, amylase, nucleases, phos
phatase, cellul ase, and ~-galactos idases have heen impi icated in biodegradat ion. 1 n low-temperature habitats. comparati ve ly large amount or enzy me is sy nthesized and secreted to compel. ;ate the ciecrea<; ing enzy me acti vity. The phenomenon was noti ced in the case of hydrolyti c enzy mes such as phosphatase, nuc lease, amylase, and lipase l
'i . 17 . Nutrient ri ch media with suitab le inducer substrate molecul es are effecti ve to stimulate the sy nthes is of hi gher quantiti es of enzy mes . Various enzymes produced by these bacteri a are li sted in Table I along with their optimum temperature and pH. Also the quantity if enzy me is known to affect the rate of bi odegradati on. Usua ll y, hydro lys is by extrace llul ar enzy mes is often a rate limiting step fo r bacteri al utilizati on of organic nitrogenous and carbon substrates lx.I'} .
Some Unique Features 0/ Bacteria (lnd thei r EII~vll1es
Psyc hrotrophic bacterial isolate of Vihrio sp strai n
5709 has an optimum growth at 20°C. The bacteria was isolated from a deep sea sedi ment and the protease from
this strain was optimally acti ve at 40°C. Retenti on of hi gh enzyme act ivity at low temperatu res is a characteri st ic feature of psyc hrophiles . For example. 16, 3"2, and 45 per cent of optimal activity was observed at ()O,
100, and 20°C res pecti vely. Alkaline phosphatase (EC 3. 1.3. 1) is one of the hy
drolytic enzy mes which is required for phosphorylat ion . I I b' I 10 E In mo ecu ar 10 ogy- . nzymes such as nitrate red uc-tase and arginino succ inate I ya~e from psychrophil ic green algae be longin g to the genus Ch/oml1IOIWS have
RAMA l A l'11I1. : BIOTECH OLOGICAL APPLI CATIO OF PSYCHROPHILES X9
Tahle I - Temreratu re and pH ort ima or r sychrurhili c enzyme~ Tah le I - Temrerature and pH ort ima or r sychrophili c enzyme~
... conld Microorganism
PS(' lldO/llfII WS
{/{TlIg ill{).1"lI
Sl'rmlill
/iqlll'fi u 'il' ll s
i!lIelllllls sp
Enzyme
Alkaline rrot ea~e
Lipa~c
Protease
Prot ea~e
Lipa~c
IJSl' lIdOIlWIWS sr Protea~e
Lipa~e
VI'I' lIdOIlI{) lIl1S ~r Prot ea~c
X{IIIIIUJlIIOIIOS
1I1l1110l,hilu
Prot ea~c
COlldidu hlll llico/u Protea~c
/Juc illlls co"g ll- Proteasc IIIIIS
I 'S{,IIII{)II/OIIIIS ~p Li p : \ ~C
PSI'//{"J/IIIJ/WS
lolllsii
Ar{)III{)II(/S
hwlrol",illl
Alle' lmlio/l lIS
hll"'I'/IIII('l is
MieT()C{)CCIIS sp
IVlic r {)('(J('CIIS sp
Micr {)('()(n IS ~ p
t l rthrohllcll'r ~ p
Li PiN:
Amy l : \~e
Protea~e
a- Amyla~c
Amylase
o.-Amy la ~c
Amylase :lI1d pullulanasc
Alka linc phosphata~c
Alk alinc ph(l~pha t asc
Optimum temr/r H
SSOC pH X.O
]SOC
pH 70
4S0C
pH 7.0- 1 1.0 pH X.O-9.0
2S0C pH Y.O- I 0.0
Rer~
2X
X4
X4
X4
107
lOX
109
11 0
III
11 2
II ]
11 4
li S
117
20
Microorgani sm
Vibri o II/OrillllS
P~ychrorh ile
F/ 0\ '( )/){Iel l' ri 11111
Vi /Jri o sp
Psychrophil e
RllOdococcliS sp
Enzyme
Triose- pho~ phate
i somera~e
~- I aclama~e
~- mannana~c
Asparta te transcarhamoyl tran s rera~e (ATCa~e)
ATCasc
Aliphatic aldehyde dehydrogenase
PSl'lIdOIl IOIWS sp 9-hexadccenoic acid ci~- t rans i ~omcra~e
Ct' lwu'hllt'11I11
.IT lll bioSIIIII
N i lrOSOI 'ibrio ~p
Vibrio sr
PSl'lldOIIlOllllS
.f/1/{)r{,S( ·('IIS
Vihrio ~ p
D A pol Y lll era~c
Ru BisCo Hydroxylamine oxida~e
ATPase FOFI-tyre
Rihonuc l ca~c
]- isopropyl malate dehydrogc n:\Sc
Optimum tem r /p H
SO°C ph S .] and X.O
A II;mIlWlIlIS sl'
K 14-2 Tryptoph:\Il ~y ntha~e 10°C
Arlhrohllc/{'r ~p ~-galact(ls i d ; \~e
Refs
II X
IIY
27
94
42
120
121
10
122
22
7
123
been in ves ti gated and their properties compared with enzymes obtained from a mesophili c Chlamfdn /l lOn{fs reinhardtii . The enzyme.~ were found to be co ld acti ve and thermolabile.The enzy mc arginino succinate lyase
(ASL) retained 2S per cent of it s ori ginal activit y at 4°C whereas mesophilic enzy me was completely inact ivated
" 1 at the same temperature- . The study also revea led th at Ch/nr()fl1()fws nitrate reductase and ASL, displayed different thermal properties . The former enzy me was found to be more thermolabile with 50 per cent of its acti vi ty loss after incubati on at 4()OC for jO min H
. More often, it was found that enzy mes from an organism ca n di splay
cOl1td diffcrent temperature and pH optima along with varying stability.
l)() J SCIIND RES VOL 59 FEBRUARY 20()()
The chitinase product ion is mainl y attributed to the biodegradati on of large quantities of chitin produced by in ve rtebrates that inhabit the water co lumn and sed iments of oceans and estuari es. The exoce llu lar chiti nases hydrol yze chitin into mi croparti cles. oli gomers and other small mol ecular weight sugars which can be further metabo li zed by bacteria as carbon and energy source2,.2.J. Apart from chitin the biomass resulting from sea weeds and animal activity is continu ously decomposed by microorgani sms mainl y by psyc hrophilic bacteri a.
In the marine ecosystem several o f chitin oc lastic bacteria have been reported in water and sediments of the antarctic maritime penin sula . In our ea rli er in vesti ga ti ons. we ha ve categori zed antarcti c psychrotrophi c bacteria iso lated from deC<lyin g blue green al gal mass as acidotolerants, alkalitoierants. and thermotolerant s. The bacterial isolates were able to grow at hi gher salt concentrati ons (up to 10.0 per cent) in the growth medium si gnifies that these isola tes are hal otolerant in their nature' 7
Antarctica psyc hrophili c bacteria display remarkable
act ivit y at O°e. Bacteria belonging to the genus Psychro/JoCl('/' sp are predominantl y found in antarcti c ornith oge ni c so il s" .
Man y enzy mes fro m psyc hrophil es di splay substantial ac ti viry at very low-temperatures . For example an ac id phosphatase with maximal activity at pH 6.0 and
~O°C has displayed 27 and 28 per cent of its maxima l
acti vit y at 0 and 5°C, res pec ti ve ly25 Several other enzy mes have al so been noticed to show hi gh catalytic activit y at low and intermediate temperatures with rapid
inact iva ti on at or above 40°e. Subtili sin from psychroph iI ic antarct ic bacteria has been well characterized in co mparison to otherenzy mes2(,. Hydrol ytic enzy mes arc essential for process ing the huge quantiti es of orga nic matt cr. mainl y in marine habita!. Bacterial consortia with different but compl ementary ex trace llu lar hydrolytic enzy me profil es act on organic particles to rapidl y so lubili ze maj or macromolecular constituents . Hydrolyti c enzy me activities such as protease, phosphatase, glucos idase and other enzy me activities are responsible for enzy matic fractionati on of ca rbon, nitrogen, and phosphorous containing organic wastes and the production or these enzymes is mainl y attributed to attached I . I I ' '7 lLlctena popu atlons- .
Bacteria present in marine snow ha ve ce ll specific ec toenzy me activities several -fold o r magnitude hi gher than bacteria present in the surrounding water. The
phenomenon is indispensab le for optimum growth even in the presence of low-quantities of nutri ents in thei r I b· ?X-10 S I I I la Itat - - . evera enzy mes are a so t;nown to he pro-duced ex tracellularl y by psychrophilic microorganisms. The enzy me, alkaline phosphat<\.~e is prod uced to supplement their metabo li sm with organic c- rbon subst rate. which is also a fina l product of phos phoester hyd rolys is. Further the hi gh phosphatase activity of bacteria is an indication of hi gh phosp horous flux in these microenvironments .
Effect OjLOlI1 Telllperolllre on Memhrone COl1lpositioll Plants, animals, and microorgani sms predominantl y
incorporat e mono-unsaturated and poly unsaturat ed fatty ac id s into the phospholipid fraction of their ce ll membrane at low growth temperalLl res. Seve ral detailed investigat ions ha ve been undertaken to eluc idate t he effect of low temperatures Oil ratty ac id unsaru rari on of membrane lipids in psyc hrophiles. Mono- unsaturated and po lyun saturated fatty acid containin g li pids ha ve been implieatedmainl y in the maintenance of liquid crystalline state of ce ll membranes. The mod ul ati on of membrane liquid crysta lli, ' <;(ate of its constituent lipids is termed as homeov isco us adaptation. The phenome non is essential for the membrane to carry out its usual ce llu lar functions like so lute transport. nutrient uptake, assembly of transport prote in s. and for the stahilit y or membrane bound enzy mes. The im portance of a temperature induced synthesis of desaturase has been revea led in Bacillus Illegaterilllll. At 10w temperatures, fatty acid sy nthase II acti vi ty increases several fold in comparison to synthase I act ivit y. Substantial amounts of short-chain mono- un saturated and di-un saturated fatty acid sy nthes is occurred in psyc hrophili c VilJrio isolates grown at low tempera tures. Another well known PUFA 20:5 has been observed to be sy nthes ized by a marine Flexi/mcter sp II. In one of the previous in vesti
gati ons , several strain s of Vihrio IIwrillllS grow n at 2°C were analysed for their PUFA's cO Ill Pos iti on of ce ll me mbranes. The sy nthes is o f doc ohexaenoic acid (22:6), and eicosapentaenoic acid (20:5) in these bacteria has been found to oceur predominant ly in response to low growth temperatures. Bacterial strains cO lltaining docohexaenoic acid di splayed hi gher growth tempera
ture but at less than 20°e. Whereas the strain s with eicosa pentaeno ic ac id ha ve di sp la ye d a maxim ulll
growth between 20- 25°C (ref. 32). Ac ti ve sy nthes is or PUFA's is a characteri st ic feature of deep-sea mi croorganisms. The sy nthes is of large amoun ts of di -unsa tu -
RAMA 'A 1'1 (Ii.: BIOTECH OLOGICAL APPLICATION OF PSYCHROPHILES I) I
1~lted fatty acids ( 18:2) has been reported in Vihrio sp.
Ev idence supporting this phenomena also has been provided by co ld shock ex periments in mesophiles as well
as in psychrophili c bacteria and yeas t "·'~. Microorgan
isms w ith high quantiti es of unsaturated fatt y ac ids are
less susceptible to damage occurring at culture storage
temperature as low as minus ~m°c.
Cold-Shock Pm/eills ond Their Role in Cold
Tolerall ce
The major inducible proteins such as co ld-shock pro
teins (Csps), co ld accl imati on proteins (Caps), heat ac
climati on proteins (Haps) and heat shock proteins (Hsps) are likely to pl ay an essential role in their co ld adaptation process.\) . The inducti on of co ld-shock pro
teins namely CspA. CspB. Cspc. CspD, and CspE has
been studi ed, in detail, mainl y in Ecoli. These proteins show amino ac id sequence homology w ith each other"\('.
T he co ld- shock protein CspA (F IO.6) protein has been des ignated as major co ld-shock protein in bacteria due to its high leve l of ex press ion (200- fold increase) fo l
low ing a shift down from 37° to 10°C (ref. 36). The cold
shock protein CspA is a 70 amino ac id protein encoded
by the cspA gene. In the same famil y, there are other
genes responsible for the synthesis of co ld-shock pro
teins namely CspB.CspC, an I CspD w ith amino acid
res idues of 7 1. 6lJ . and 74, respecti vely. Further these
protei ns show 79. 70 and45 per cent seq uence homology with CspA prote in . The recentl y reported CspE protein
with 69 amino acid res idues has shown 70 per cent
homology with the l1lajor co ld-shock protein . Bes ides the co ld stress response in several mesophi I ic bacteria
has been shown to in vol ve the same gene products for
ce ll adaptation . Co ld shock proteins, in general , ha ve
been proved to eli cit transc ripti onal and translational contro l in prokaryotic as well as in eukaryotic organ
isms. Illitiation of translation appears to be the most thcnnoscnsiti ve step but elongation of the proteins can occur uninterrupted ly at low temperature as revealed by Friedman el (fl . l7. IX . It has also been demonstrated that
the ce ll growth stops below 7.8°C onl y due to the inabilit y to initiate protein synthesis. The inducti on of
co ld-shock proteins increases from 2 to 10 fold after
lowering the growth from 37° to 27°C'\h These prote in ~ also include NusA which is required for terminati on and anti- tennination of tran sc ripti on. Initiation factor 2 binds to the charged tRN A 'IlIC' to the initiation site 01"30S
ri bosomal subuni t for maintaining the translation proc
ess. Its ability to combine w ith polynuc leotide phospho-
ry lase has been found to be necessary in the degradati on.
of mRNA . In addition to these fact ors the role of tri gger factor
(TF) in co ld-shock response in mesophilic E coli has also been described'H. Thi s protein ex hibits prol y l isolllerase activit y and has been implicated in co ld adaptati on of bacteria . The express ion of T F was found to occur i ll response to cold-shock suggesting that it is distinct from heat-shock protein repertoire. It shows affinity to GroEL and enhances its binding to unfolded protei ns of the ce ll. Based on these observati ons, presumabl y, T F plays a major ro le in protein folding and also in the degradati on
ofmisfolded polypeptides. Theexpress ion of the protei n was found to increase after initi al co ld-shock and during
storage of ELOli at 4°C. The pattern of expression
close ly resembles w ith that of the well studied co ld-. ,\ I}
shock protem response' .
Protein Fo/ding and Inlra ll /()/eclI/or Inle r({ctiolls
Several factors have been described to contri bu te to
the fl ex ibility of enzymes in psychrophi les. Flex ibil it y of a protein conformati on is often known to play a major role in dec iding the therlllosenisiti v ity and catal ytic efficiency of enzymes. The ri gidity of a protein mole
cules is mainl y reduced through changes in intramolecll lar interacti ons. According to recent in vest igati ons
protein surface properti es like low hyd rophobicit y and high hydrophilic contribute to the stabi lity of enzymes in psychrophiles. Decrease in hydrophob icit y is a-;
cribed to the presence of low number of pro line res idues. However, increased hydrophili cit y is caused by the incorporati on of more charged amino acid res idues to forlll an extended surface loops . Nevertheless the decreased
content of isoleucine, arginine, and at times the tota l content of arginine+l ys ine suggests that hydrophili cit y can also be achieved by subst itution of amino acid
'd h I I I d . . I ~ () ~ I C . res t ues ot er t lan t l e c large amIno actc s . . ertal1l
psychrophilic enzymes are completely devo id of disul phide bonds, is also attributed to the protein fl ex ihilit /'
In spite of all these generalisations the data are insuff ic ient to explain conclu sively the ro le of vari ous interactions in enzy me stability in psychrophiles. Aspartare transcarbamy lase produced by a psychrophilic st rain of antarctica di splayed 26 per cent or it s optimum enzyme
acti v it y at O°C~2 . Some of the kineti c properti es and
thermal stabi I ity of t he enzyme resembled transcarbamylase of mesophili c Ecnli . It indicates that these enzymes can achieve ca talytic effi c iency throu gh specific changes in the cataly ti c site in add ition to c h ,lIl g('~
. ~UI that occur In the enzyme mo lecule .
.I SC I IND RES VOL 59 FEBRUARY 2000
Further, diverse type of enzy mes were found to be responsible for co ld-adaptati on apart from fatty acid desaturases a~d co ld shockproteins as well as co ld ac
cl i mation protei ns. was con fi rmed by the transposon mutant analysis . Goverde el of. ~ 'i , have demonstrated the
role of an exoribonuclease. polynucleotide phosphorylase (PNPase) in co ld adaptation. The enzyme P Pase mediates gene expression through the .degradati on of mRN A in the ce ll . Arter an initial cl eavage by other
cndonucleol yt ic enzymes the:r to 5' exonuc lease acti vit y llf P Pase i:- responsible for the degradati on of IllRNA in eubacteria . However, in E.co/i, two types of
:r to 5' exonucleases i.e. RNase II and PNPase, were
found to be responsible for exonuclease acti vity. A lso,
studies have revea led that the loss of thi s acti v it y in conditi onall y lethal mutants was found to be detrimental to the growth of E.co /i strain at low temperatures.
Ycrs illi({ elllemco/ili("(l a psychrotrophic iso late from a tran sposon mutant library was found to depend on the
enhanced ex press ion of PNPase and its ml< A for eo lcl
adapt;lti on at 5°C. The co ld inducible promoter con
tai ned an ATTGG sequcnce characteri sti c of co ld proilluters~h Although inconclusi ve generalizati ons
ha ve been made by vari ous aut hors some of the factors which are responsihle for co ld adaptation are: (i) Pres
ence of suhstantia l amount of pol yunsaturated fatty acid:- in the lipid Illoieti es of ce ll membranes~7 .4 X , ( ii )
Illodifi cations in enzy mc structure resulting in high speci fi c ac tivity at low- temperatures i .e. , low Kill and hi gh ,uhstrate turno ver. This phenomenon is being refl ected in the ac t i vat ion energies of psychrophi I ic P.jll/orescells
w hich showed activation cnergy values o f 10-38 .0 k.l /mol whereas the mesophili c protease showed 60.0 k.l /mol. ( iii ) modifica ti ons in the D A replicati on, tran-.. d I ' I . ,. I II ~I) 'iO scnptlon. an trans atlon macl lnery 0 t l e ce " .
Biotechnological Aspects
1\ rC/IUe/ £n::.yllies os NOII(' / Bioc({lO/ysls
Several groups are active ly engaged in the exp loitation of thermophili c ;! nd hyperthermophilic enzymes from archae in developed countri cs. Enzymes of ex trel1lophiles known as ex trelllo7,Yllles can be empl oyed in \'ariou:- fields ranging from the production of bulk chcmical:- to hea lthcare products. Ex tremozy mes can optimall y functi on at both cxt remes of temperature and pH. On t hc other hand. some microorganisms thri ve at hi gh sa lt concentrations and, therefore. their enzymcs exhib it ;1 natural tendency to work at high osmolalit y. Thennm:Y llles refe rs to enzy mes which are produced by
thermophi les and hyperthermoph il es. There enzymes
are optimall y acti ve between 60- 125°C. The tempera
ture characteristic of these enzymes makes them attrac
tive in various biotechnologica l applications, They ha ve
been used in molecular biology and one of the successfu l
sto'ries is attributed to DN'A polymerase of ThCrI1ll1S
aquati clls and its wide use in po l ymera~e chain reacti on.
Detergents and starch hydrolysis for the product ion of
glucose and high fructose corn sy ru p using ~-amy l asc
and glucose isomerase respecti ve ly. Several of them
ha ve been proposed for the synthes is of organic chemi
ca ls in addition to diagnostic kits , waste treatment , pulp
and paper-milling and in the preparat ion of animal
feed'i. Thermostable enzymes ha ve been shown to be
resistant to various chemicals and can be functi onal in
organic so lven ts in the absence of water, thereby leading
to many des irable properties. Proteases of Archae arc
known to hydrol yze highl y res istant proteins like keratll1
for the producti on of am ino acids . Alcohol dehydro
genase produced by Th crllloallo cro/Jill/ll brock ii is cur
rentl y used in biosensors for the detection o f alcohol ' !
and in the synthes is of chiral compounds" . The enzyme
is act i ve at high solvent concentration and 6()°C. One of
the great advantages of using enzy mes in organic sol
vents is to reverse the hydrolytic reaction occurring in
aqueous media. Enzymcs which hydro lyze esters , pep
tides, and oligosaccharides in water can synthesize the
same compounds in organi c so lvent s. Biosensors pn>
pared with thermophilic cn zyl1lc .~ do show rcmarkab le
stabi lity when exposed to va ri ous chemica l reagent:
during immobi li zation procedure for biosensor and stor
age during transportati on in tropi ca l countries. Thermo
stable enzymes may be used as ant ibody conjugates in
immunoassays, However the main constraint at prcsent
is their prohibitive cos t of production and reluctance to
use innovati ve methods. Hydrogenases are predominantl y found in hyperther
mophilic fermentati vc archae. In view of the depleting
energy reserves biophotol ys is of water by solar energy
has been inves ti gated to li berate hydrogen. Mel/wllo
bacTeriulII IhernlOW/lOlroph i c lIIlI produces hyd ro
genase 'i ~. Immobilized enzy me processes based on these
enzy mes show an enormous operat ional stab ilit y mak
ing a bioprocess cos t-effecti ve. Higher y ields of en
zymes can be obtained with minimal loss of activit:
duri ng puri fi ca ti on'i"'.
RAMANA £'1 (fl. BIOTECHNOLOGICAL APPLICATION OF PSYCHROPHILES 9]
Use of' Psychrophile Enzymes in Food Processing Indust rv
Psychrophili c mi croorgani sms and thei r enzy mes ha ve a wide range of appli cations in dairy and food industry. Psychrophilic milk coagulating enzy mes have the advantage of controlled case in coagulati on for maintaining the quality of whey resulting from cheese industry which can be used in other processes. The enzyme activity in whey ca n be destroyed by pasteuri zati on. The commercial mi crob ial rennet available in the market in developed countri es with the brand names Marzyme, Rennilase 50TL. and Moelilase are products of colel act ive microorganisms. Another interestin g appli cati on
of co ld ac tive enzy mes is in the form of~-ga l ac tos idase.
Lactose hyd rolys is in milk and whey to ga lac tose and glucose results in increased solu bility, digestibility, and
sweetness of milk. Usua ll y, ~-galactosidase obtained from mesophilic strains of Klu vermyces and Aspergillus stra in s are ac ti ve at re lati ve ly hi gher temperatures, i.e.,
~O-4()°C, and the milk has to be processed in conventionalmeth ods for at least 4 hours for complete hydrol ysis of lactose. These conditi ons increase the chances of microbia l contamination during the process. With the
use of thermolabile ~-ga l actos i dases hydrolysis of lac
tose can be carried out at 50 _10°C in about 16-24 h.
Us ing the co ld active ~-galactosidase 70-80 per cent of product yields can be obtained, which is much hi gher in comparison to the processes obta ined usin g enzy me from meso philic mi croorgan isms . A commerciall y availabl e ga lactos idase is in use for the purpose. The commercial co ld ac ti ve neutral protease is mainl y obtained from Bacillus suhrilis and being marketed under the commercial name eutrase. The enzyme is known to increase the fl avour intensity with red uction in the ripening time from 4 to Imon.
Psychrophili c microorgani sms are able to produce various enzymes of industrial im portance. Neutral proteases from psyc hrophilic bacte ria are being used in cheese maturation . Po lymer degrading enzy mes such as amylases, pullul anases, xy lanases, and proteases are empl oyed in food processing. Proteases with low optimum temperature and hi gh pH are being marketed under the commercial names Savinase, Maxaca l, and Opticlea n.
Source of' No rurul Piglll ents Carotenoids arc present in va ri ous mi croorgani sms
and they play an important ro le in protecting the photosy ntheti c machinery of the organ ism from photo-oxida-
tion. Several bacteria of antarctic origin can also produce pi gments and mainly belong to the Flecw/Jacillus5
1> , Pseudomonas".7, and Micrococcll.l''''x. As there is grow in g tendency to use natural pigments, bacterial pigments of different hues and colours may prove to be handy and renewable source for food processing industry.
Hydrolysare of'Biol11as.I'es As Feed Stock The extracellular production of Lal11inaria sp decom
posing enzy mes were partly characteri zed in marine bacterial iso lates belonging to the genera AlreroJ11ol1us sp, Pseudomonas sp, Moraxellu sp. and Flavo!Ja, " teriun/'() These enzymes were found to be hi ghl y active agai nst several marine po lysaccharides like alginate, fucoidan , and cellulose. The bacteri a population largely consists of psychrophilic Vibrio sp whose optimulll
growth temperature ranges from 50 _20°C. In the deepsea, bacteria and cyanobacteri a that take part in the
biodegradation of phytodetritus between 20_ 15°C and 3t
a depth of> 4500 m601>1. The enzymes responsible for
decomposition were identifi ed as a lginase and fll
coidanase at IS°C. Hydrolytic acti vi ty is a common feature of marine bacteri al populations as well as other mi croorgan isms. In future, thi s may help in producing sin gle ce ll protein and liquid fu el, after hydrolysis of enormous amounts of sea-weeds and aquati c plant biomass.
Several types of psyc hrophili c microalgae have been reported from antarctica and other cold habitats. Because of their and inex pensi ve growth requirements substrates comprising solar li ght and other inorganic compounds present in marine waters can be used for biochemical production such as vitam ins, carotenoids, pi gments po lysaccharides, protein , and foods.
Lipids as Food Addiri ve.l' Microbial lipids containing polyunsaturated fatt y ac
ids (PUFA 's) are reco mmended to increase nutriti onal va lue of food products and as additives in cos metics and as startin g substrates fo r the preparat ion of pharmacell ticals(12 Polyunsaturated fatty ac ids are commonly found in marine microorgani sms6
' . Probably these organi sms produce PUFA 's in response to low temperature of marine habitats.
Li pids ext rac ted from psyc hroph i I ic antarctica bacteri a and marine algae mainl y cons ist of C I 6 and CI S unsaturated fatty ac ids. The marine algae AnadY()fIll'IIC' .I'relLara can synthesize 16-22 carboo containing unsatllrated fatty ac ids possessing as much as four conjugated
lJ-l .I SCIIND RES VOL 59 FEBRUAR Y 200()
double bonds6-1 . T he synthes is and modifica ti on of fa tt y
ac ids mainl y occurs in chl oroplas t and in the endopl asmic reticulum of these eukaryotic microorga ni sms(''' . A group or psychrophil ic se;l- ice derived bacteri a l strains ;Ire known to prod uce polyun sa turated fatt y ac id s such
; I ~ eicosapent aeno ic ac id (20:5w3) and arac hidonic acid
(20:4w6) . Bacteria belonging to the fam il y Flovo/mcleri((ceu arc a lso known to synthesize a range of volatile fatt y ac id containing lipids in additi on to algae.
Sillg/(' Cell Prolein/i"()J1/ Chilill Chi tinase (EC 3.2. 1.14) rrom Serrati a marcescens
QMB 1466 was attempted to hydrol yse large quantiti es or she ll fi sh chi tin i nt 0 so lu h Ie monomers such as N -acety l glucosa mine and it s conversion to singly ce ll pro te in . The process inc ludes size redu cti on, deprote inati on. and demi ner;ili zati onllh T he hydrolysis or chitinmay also be carried out usin g chitin frolTlmarine bacteria since chiti -
I ~ I " (,7 nase pro( ucers are preva ent In ma ri ne waters .
US£' oj' Hw /ro/vt i f' £ 11 ;'.1'11/ (' .1' ill PII /p /ndllsl ry
Debleachin g o r paper mill pulp has extensively studicd by many in vesti gators. Art er ce llulose xy lelll is the mos t ahunda nt biopo lymer in nature . The main sugar cO lllponent or xy lan is D- xy lose. Heillicellulose con~ i ., t ~ or a .~e ri es or Ill.: teropo lyselcc haridcs that .inc lude glucan s. ma nnans. arab inans, and xy lans. Microbi a l enzylllL:S dcgrad ing hClll ice liuloscs have great industri al app licatio ns. Cell ulase frec he ill ice ll ulases arc use rul in the pu lp and paper ind ustry ror bleaching of kra rt plll p('~'(,'} Xylanases are usefu l in the mod ifi cati on or pulp during paper makin g anel 1'0 1' recyc ling of waste pape r. Wi th the eliscovcry o r morc andlllore vari eties or hL: lll icc liulases it woul d be possible to appl y them comIllerc iall y in the pulp industry. Alka line xy lanascs arc pan ic ul;II'ly prererrcd in pu lp 1ll;lk ing proccss or p;lper indus try. Incrcasing so lubilit y or xy lan at hi gh pH helps in incrcasin g thc hydro lys is or xy lan by thcse enzy me. Xylanascs arc large ly produced by a lka liphiles or thc ge nu s 13((cillllS. Alka lophi le /J{/ c i llllS strai ns opti ma ll y grow at hi gh pH and the ir xy lanases also d isp lay optiIllUIll acti vit y at pH > 9.0. B{/ci llllS .l'Ifhl ili .l' xylanase hydro lyses lip to 3X.O per cc nt or thc subs trate with low produ c t i n h i bit io n co n s t ;In t s in co m pa ri so n to l'rich()r/£, J'/I/({ virir/{/ (' xylanse preparati on. However. xylanase pruduct inn by pscy roph iles has not been atte ':ll pt ed. Some o r the ;il ka liphilic /Jucill llS sp xylanase
showed hi gher opt illlu l1l pH and temperature (60°C). In spi tl' o r a ll these deve lopillents xy lanases or thermophilic a lka liph iles ;Ire prefe rab le to wit hstand hi gh pul p
temperature of 60°C and pH > 9.0. Zakaria el u/n have
reported the synthesis of ~-man nana~e in a psyc hrophilie stra in of F /a vo /)({Cleri /lll/ . The enzyme sy nthesis occu rs in the presence o r I per cent gua r gum and it wa~
abl e to hyd rolyse efri c ientl y the same substrate opti
ma ll y at 35°C. At 10°C t he enzyme di sp layed 25 per cent of its optimal acti vity .
Mi croorgani sms producing a8etyl esterases whi ch are fun cti onall y and phys iologicall y simila r to lipases III
combinati on with u-glucou ronidase~ are required in the pulp mak ing process to obtain max imulTI xy lelll hydrolysis. Bio-pulping/enzymatic pulping for xylan hydro l y si ~
and cellul ose deri vatizati on a lso requires the cfloperat i ve act i vi ty or es t e ra~es in the enzy mat ic degrada t ion or
70 acety l xy lans .
Use oj' £ n ;,vllle.l' i ll B iodelergenl.l'
The concept of utili zing enzymes ~ t a rted in 19 13 by Otto Rohm has deve loped now in to a maj or commercial exploit ati on of proteases, li pases, amylases, and ce ll ulases as detergent add it i ves 7 1. Remarkabl y, most of these enzy mes are being produced rrom microbia l sources inc lud ing bacteria, yeas t. and fungus or geneti call y cngineeredll1i croorgani sms. These en7.ymes arc marke ted at present throughout wo rl d under seve ral bra nd names. Thesc enzy mes arc optill1a ll y acti ve between pH 9.0-11 .0 . Genetic engineerin g is wide ly used to increase enzyme yie lds from wil d mi crobial strain s which arc uneconorl1i ca l fo r large-scale prod ucti on. Subti li sin type
of proteases and u - amy lases ha ve bee n round to he suitable for detergent industry. Alpha amylases arc useful not onl y as add it ives o f lau ndry detergents hut they are incorporated into powder and I iqu icl formul at ions 1'0 1'
indu stri al andmachinc dish-washin g. Protease, li pase, aillylase, and cL: liul ases have bee n
widel y report ed in psyc hrophili c microo rgani sms and they Illay prove very use fu l 1'0 1' low tempera ture washing. The principl e of the so call ed "greener" and mil der chemi cals detergents is based on enzymes di splaying hi gh activity at low temperatures. Cold was hing te lll peratures are preferred in several countri es. Amylases and other enzy mes which arc stab le in the presence or protease prove to be preferable 1'0 1' kitchen ware washing. With the d iscovery or nove l enzy mes in pscy m philic, in rUtll rC, it w ill be p()s ~ i b l c to ro rm ulatc detergents 1'0 1' vari ous purposes. Ps)'c hrophil ic ~ li b l ill e
p rot ea se~ report ed in t hc' ant a rct ic P.I'('{f{/() " {() IIU.I' sp and AC/'{J ,,{()lIu .I'· Ityrlmph i /u strain can he utili zed a~ deterge nt add itive2x
. Enzy mes with 1ll;lx imull1 ac tiv it y at
RAMANA ('I o/.: BIOTECHNOLOGICAL APPLICATION OF PSYCHROPH ILES 95
alkaline pH and stable at moderate ly higher temperatures are hi ghl y useful as detergent additives:12
Psychroph iles As A SOllrce of' Pharnwceuticals Discovery of secondary metabolites from marine mi
croorganisms hJS been drawing much attention for the last three decades. As a consequence, several strains of bacteria , streptomyces, and fungus have been reported to produce anti funga l, anticancer, and anti-tumor agents. It is ev ident that most of these mi croorgani sms belong to the category of psychrophi les as they have been isolated from deep-sea sed iments, marine waters and gut flora of aquatic an imals and plants72 The bacterial genera that produce these agents are: Streptol1lvces, Altero/1/011£1,1'. [3u c illlls. Mic{'ococc l/s . Ae{'ol17o l1 as. Fhll'o/)ucleriulI l. Moru .. rel/o, Pseudoll1o//as, and Vihrio
with growth temperature between -3 to 3()°e. Aquatic plants and animals are hi ghl y prone to infestation by pathogenic microorgani sms. Surface bacterial sy mbi osis is an essenti al adaptive and protective mechan ism in fres h water and marine sed iments. An Alterol11olws 'iP has been reported to synthes ize 2,3-indolinedi one (isatin ). Experiments have amply demonstrated that thi s co mpound protects the shrimp Palaell1o/7 macrodact,\'IllS from a pathogeni c fungu s L(/genidilllll ccrLLillectes. Similarl y. another strain of AlterolllO//as sp, intimately assoc iateci with the marine sponge Holichondria okac/o. prod uces a tetracyc lic alkaloid namely alterimide A T".
There is a wide scope for the discovery of novel bi ologicall y act ive cOlllpounds in marine mi croorgani sms. By learning their structural intri cac ies the development of new pharmaceutical prociucts mi ght be possibl e in future. Studi es have also indicated that marine stra ins of Morctxel/({ sp ancl Flol'olxlcleril/lI1 sp can prod uce anti -
. I I . . I 74 7S TI . vlra ane anti-tumor agents. respecti ve y " . le anti -tumor polysacc haride has been iclentifi ed as narinactin . Pathirana et (117
(, isolated L-quinovose class of esters from marine actinolllycetes. Unfortunately, bio logica ll y active co mpounds from marine microorgani sms have not been exp loited. Jensen and Fenica l have adequately emph asized on the potentia l of marine microorganisms77 Similarl y. antarcti c bacteri al isolates may yield several of these metabolites. since most of the genera also occ ur predo minantl y in antarctic hab itat. To our knowl edge, so far no attempt has been made to screen antarcti ca these bacteria for the production of biologica ll y active compound s.
Sha ve r and Schi ff et (/1. 7 K have demonstrated the usefu lness of protease and amylase mi xture from BacilIllS slIhtilis in removing the dental plaque. Lipases are
mainly used as stereo-spec ifi c catal ysts and in the biotransfonnati ons of various hi gh va lue compouncis
I tOI' d I . I 7')-X I SUC 1 as avounng agents an p larmaceutlca s . Concentrates of polyunsaturated fatty acids of the type 3-3 are obtained by the hyd rol ys is of fish oil l ". Fatty acids such as gadoleic acid, erucic. and nervoni c acids are obtained by the hydrolys is of seed oil sX:1 . Lipolytic enzy mes are widely empl oyed for the extracti on of these hi gh va lue nutropharmaceutics. Lipases have been studied mainly from antarctic Moraxel/a 5. 17 , Pseudomo/7(/s
and Bacillus sp and Serratia liquefaciens isolated frorn blue green algal matsK-l . Lipases from psyc hrophil ic bacteria are preferred because of their unique positionJI specifity for fatty acids not found in mesophili c enzy mes. The enzy me frolll a psyc hrotrophic Pselldo-1I10nas strain was found to ex hi bit 1,3 - pos iti onal spec ifi city towards tri glyceride substrates like triolein. Owing to their low activat ion energies, i.e. 11 .2 and 7.7 kcailmol , cold-acti ve lipases are catalyticall y more efficient at low temperatures in compari son to mesophil ic enzymes. The enzyme was found to be activated by organic solvents like methanol and dimethyl sulphoxide from 0-30 per cent (vo l/vo l). Enzy mes which show stability in organic so lvents are biotechnologica ll y important to carry out enzymatic reacti ons in organic so lvents. Thi s as pect of bi otransformati on is gaining great importance to increase reaction rate and to ex tend the substrate spec ific ity of enzy mes . Use of organic so lvents as a medium of reaction aids in solubili sing the CO I11-
pounds which are in so luble in water. In the bioconversion of water insoluble compounds such enzymes arc quite useful. Bacteri a able to grow in the presence of high concentrations of organic solvents have been reported from deep-sea sediments with an optimulTl
growth temperature of lODe. They have been ident i fied as genera Flavobacterium. Bacil/lls sp, and Arthro/Ju( 'tel' and can withstand hi gh concentrati ons of benzene. to luene, and p- xy lene8S Recentl y, it has been found that immobilized enzymes in organic so lvents are efficient in terms of their velocity to catalyse biochemica l transformation to obtain biologica ll y active compounds.
Trehalose (u-D-gi ucopyra nosy I u-D- glu copy ranoside) is a non-redu cing di saccharide widespread among bacteria. The sugar is economi ca ll y important because of its function as cryoprotectant to stabili ze protein s and protection of anima l ti ssues . The sugar finds various app lications such as sweetener, stabilizer of frozen foods, in cosmetics and as a drug additi ve. Trehalose is formed by an enzyme trehal ase present in
()h J SCI I 0 RES VOL 59 r EBRUARY 2000
several psychrophilic and thermophilic bacteria. Psychroph i I ic microorgan isms synt hes ize t he sugar to protec t the cell rrom desiccation. heat, and os moti c shock.
USCS III' Buclerio/ 1('(' NlIdeul ill~ ;\~ellis
There arc seve ral uses ror ice-nu c lea ting agents (I A) produced by bacteria . They are being used in artiricial snow making, in the prod uction o r ice cream:-. and ot her frozen foods. In immunodiagnostic kits as a conjugate to antibod ies and as a substitute 1'0 1' silver iodide in c loud seeding. Among severa l organisms, rN A from bacteria have attrac ted Illuch prominence OWIll ~ !O
thei I' abi I ity to form ice nuclei at relat i vel y hi gh templ.:l alUre in cOlllpa ri son to other sou rces. The subj ect has been rev iewed in detail hy Lindow (' I u/.X6
.X7
PselldolllOrws
"."ri,lg l/ {' . / 'SCI f(/o/IIOIIUS .fI//oreS(·ells. PS(! IIdol1lOlf(lS
I'iridijl(/\'{/. E/'\\'illi(/ herhic()/u. and X(/I7I//() IIWIWS C(flll
/1 ('slri .\ are the we ll known producers or INA . Further, :-,ollle marine strain:-. of PsclIdllll/()lIusjlllo r(!scells have bee n ~ h own to produce IN As . Most or the bacterial strain~ u~ed ror producing IN A are psyc hrophilic in flatu re. Severa I properties or IN A reselllble psychrophi lie enzymes . Particularl y. I A~ are thermolabi le Illolec ul e:-.. For many practical uses, ice nucleati ng agents rrom bacterial sou rces are preferable, since mi croorganisms ca n he illlProved by reco mbinant D A techno logy and they can he mass produced in bior'eactor:-.. The unique propert y or hacterial ice-nuclea ting age nt~ is that they arc least soluhleeve n at hi gh temperalure .~ in comparison to plant and insect agentsxx . An
optilllum growt h temperature or2{)OC is required 1'01' the :--y nthes is or INA in hacteria such as P.svrillgo (!. The icc iluc lea ting temperature as we ll as the sy nthesis o r 1 A is hi ghly influenced by h,lc terial growth temperature.
INA produced at 2()OC preserve the propert y or icc nuc leati ng act i vi ty at hi gher temperature. In t he case 1'J!lIorc.\·('el1 .\·x'l. l 's{,lIdolll()1I0S s,'/'ill g (/c is an 0 th e I' widely occurring hacteri ,iI epiph yte. The IN A is produced in the rorm or an oute r membrane prote in . Large aggregates or I A ca n orient wa te r molec ul es into c ryst, illattice mimicking the nat ural ice particles and leading to a cascade or ice crystal rorlllati onxh
US(' ill F enll (!lIll1lioll Indllsll'\'
Me:-.ophili c yeasts which contain un saturated ratt y ,Ic itis in membran ous lipids hilve been round to be res is
tan t between -~m to -2{) dc. normall y pre fe rred fo r the ir ~torage in baking :Jnd other rood process in g industries·JI ,.
I-or \\ ine productio n, re rment ation at 6-HuC has been round tn reduct' the inhibitory errcct oj ethanol 011 cell
meillbrane of yeast ce ll s'l l. Speci fically, cryotolerant yeas t strains ha ve been select ive ly obtained . Sparkling wine is produced Ll s ing the yeast strain s which . arc tole rant to hi gh alcohol concen trati ons.
Applicatioll ill Micm/Jio/ L eochillg
Currentillicrobiall eaching operati ons in vo lve ox idati ve so lubi I ization or copper and uran iUIll ores. Leachin g: operati ons in teillperate cOll ntries have to be carried ou t at very low aillbi ent temperature. The temperature dependen t variati ons of microbial acti vity has been noticed to affec t the ore leac hing operati ons by Ahonen and Tuovinan'12 . Microbial leaching operation from sulphide
ores was studied at low temperature between 4°-:n °c. Probably the process in vo lves psychrophilic microorgani sms and illlprove llle ills in thi s area wou ld be possi bl e by e mpl oying pure cu ltures or the bacteria or antarcti ca origin or from other co ld hab itat s.
Usc ill Biorel71ediclliol1
In add ition to the ir abi I ity to bi ode~rade various CO Ill
pou nd s in the habitat they can be proillising in bi orelllediation or several pollutants at low-temperatures. The occ urrence ofbiodegradative psychrophil es is re lative ly more in contaillinated s ites where the aillbi ent tempera ture overl aps with the optilllum growth temperature or these mi croorganisms. Psychrotrophic hydroca rhon ckgrad ing bacteria ha ve been isola ted rrolll oi I poll uted sites or Onta ri o and Quebec and it ha:-. been noticed that the occurrence of hyd rocarbon degradi ng Illicroorga niSllls is low from relat ively pristine environmental habi tats'll. The bacterial strain s were fo und to Illinerali ze dodecane, hexadeca ne. naphthal ene, and toluene at 10\\
temperatures and it has been ev ident in laboratory and fi e ld experiments , utili zin g specific bacteri a l cultures. Some of the degradati ve genes responsib le 1'01' ca taboli sm or naphthalene (nd oB) and tolue ne (xy IE. todC I) have al so been detec ted in psyc hrotrophic bacte ria using mol ec ular tec hniques sllch ;IS sou thern hyhridi zatinn and polYllle rase chain reactiun'I-I. P:-.yc hrophilic ba-:teria that can degrade hi gh Illo lecular we ight hydroca rbons at low temperature were desc ribed earl ie r'I' .
A psychrotrophic bacteriulll , Rhodo('()cclI.I sp. stralll Q 15 has been studied ror its ability to deg rade n-alkane~ and diesel fuel at low temperature. The strain mineralize short chai n alkanes li ke dodeca nc anci hexadecane .It hi gher rat es than the lon g chain hyd rocarhons such ;1:-'
octacosane and dotri acont ane (() "' -,'i °Cl. Also the ~l raln
has been able to Ill llleralize at ho th branched a lkane~ and
substitut ed cyclohexalle~ PI'L'sl'nt iL di c~el (lil () (' ) III
R!\M ANA 1'/ fit. rl lOTECHNOLOGICAL APPLICATION OF PSYCHROPH ILES
addit ion. minerali zati on of hexadecane at SoC was noticed in hydroca rbon contaminated and pri stine so il
. . I . R/ / '!6 microcosms uSin g tle organis m IO{ OCOCCIIS . The psyc hrotrophic bacterium Rhor/ococclIs sp. strain Q 15 that ca n degrades n-alkanes and diese l fuel at ~o_S oC
was show n to contain the gene responsible for the producti on of al iphati c aldehyde dehydrogenase wi th DNA sequ ence rese mblin g the mesophilic R.ery fhmpolis
thcA genc.
i\l7aemlJ ic Digesfio/l o(Orgonic W(fSf eS
The obli gate anaerobes whi ch convert organic acid s to CH.j and CO2 (methanogens) are hi ghl y sensiti ve to low temperature and pH levels. Psyc hrophilic anaerobic bacteria are not ex tensi ve ly in vesti gated in co mpari son to aerobi c bacteria. ow ing to their very low growth rates and cumbersome isolati on procedures . However, there are some reports that describe the isolation of psyc hrophili c anaerobi c bacteria from antarcti ca habitat. Vari ety of organic wast es can be degraded to innocuous prod ucts by an,:erobi c degradat ion. It is one of the potential routes for methane generati on in co ld habitats. Further. anaerobic di ges ti on has assumed much importance in the treatment of vari ous tox ic chemi cals and hi gh stn~ngth industrial wastes. Iso lation of psychrophilic anaerob ic mi croorganisms is of immense importance to suppl ement the anaerob ic diges ti on process to he ca rried out at low- temperatu res . At present , Llll aerobi c di gestion of sewage sludge, cow dung, and other animal wastes is treated by adapted mixed microbial
. 'J7 . . consortia . Nevertheless, microbial growth and meth-ane production is ve ry limi ted at sub-ambient tempera
tures (~S OC). The rate of methan ogenesis can be Increased several fo ld by low temperatures adaptation of methanoge ns The process can be made poss ible by sel ec ti ve enri ch ment of psyc hroph i I ic methan ogens through long term laboratory trial s. Hence the basic understa ndin g of these microo rgan isms is essential for i mpro vi ng the performance of the methanogen ic processes. Bes ides methanogens. bacteria with the abi lity to reduce sulphate ha ve been isolated and identified as
t) N
/) e.w/(orhopa/lls " ({CIIO/{flllS ' . Few report s have re-vealed the importan ce and the ro le of methan ogens in antarct ic habitat. In one of these studies, a psychrophi I ic strain of M ef//{/l7ogenilllll/i'ig id lllll sp nov., was iso lated from Ace lake, antarctica whi ch grows optimally at
I soc. The organi sm was found to produce methane from hydrogen and carbon di ox ide. The growth rate of psychrophilic methan ogens is ve ry low (0.24 d·l)with a td
of 2. 9d()() . S imi larl y, an acetoc lasti c psychrophi I ic strai n of M ethwwcoccoides /Jur{ol1i from Ace lake, antarct ica. has been repo rted J(X).
D el1.itrijicofiol1 afDrinking Water Sources
The presence of hi gh nitrate concentrations in water has been a major problem in many countries inc ludill p: the US, Canada, and Eu rope. Drinking'water containin p:
0 ,- quantities exceeding 10-50 mg/l are harmful. The most widely used practi ce for the remova l of NO\- is bio logica l denitrification. Most of the denitri fi cati on
processes are carried out at about 'Wc. Ho lI () and C k l UI I d ' b' I I .. ,.. . za 0 , eva uLlte micro ILl C en Itn Icat lon process at
12°C and the maximum specific nitrate remova l rate observed was n o mg NO \-/h /g cell s. The nitrate re
mova l capac ity of the reactor at I (j°C was SO to 60 kg NO,-/m' /d. To increase the treatment effi ciency at these temperatures, immobilized microbial ce ll based bi orcac tors such as packed bed, and fluidi zed bed are being used. Also, a fu ll sca le denitrifi cation unit usin g the
same process has been operated between ' 20 and 18°C. Temperature decrease of few degrees may redllce the process effi c iency substant wll y whi ch emphas ises t hc need to use co ld acti ve deni trifying bacteria . The denitri fi cation effici ency has been found to reduce hy abollt ~3 per cent while the maximum denitrification rate ~It
18°C recorded was 2.24kg NO,-/m' lcl'll . However. report does not conform to the presence of psychrophil ic nature of these microorganisms. However, it appears that microbial population consists of co ld -adapted mesophi I ic bacteria funct ion in g at lowe r temperatu re probab ly with lower denitrificati on rates. The rate or denitrification in these cases can be enhanced hy employ in g psyc hroph il ic bacteria iso lated from permanentl y co ld habitats.
Biological Amendment of Psychrophiles and Genetic Engineering
With the adven t of polymerase chain reacti on, va ri ous genes responsible for biodegradati on and metabolism ha ve been ide ntifi ed and charac te ri zed from psyc hrop hil es. Recombinant D A tec hnique .~ have been proved to be of great utility in the e luc idation of properties of crenarchaeota whi ch cannot be culti vated under laboratory conditions. Two subtili sins prod uced by a marine iso late of Bacillus T A3Y and TA4 1 stra in ha ve been purified and the genes responsible for the ir synthes is are identified as Subt I and subt2. T he enzyme displayed a hi gh catalytic effici ency at low and moderate temperatures. However, the enzy me was hi ghl y ther-
.I SCIIND RES VOL)<) FEBR UARY ::W(){)
mosensiti ve as revealed by it s fl ex ibility and 3-D protein
conrormati on analys is. The gene responsible for a-amylase producti on in a psychroph i I ic strai n of A /lemmol1({s
ha /op/all cl is has been c loned in mesophili c E.coli to
probe its protein ro lding mechanism and ef fect host ce ll
phys iology on enzy me ac ti v it l~ . Studies have shown
that the enzyme expressed was simi lar in every aspect.
T he hetero logous expression system prov ides a means
ror large-sca le producti on or co ld ac ti ve enzy mes in a cost-effec ti ve manner. T he erfect of in tramolecular in
terac ti ons th at increase the ri gidity of the protein Ll:lck
bone have been inserted by sit e directed mutagenesi~ , d
asses~ the impact or fl ex ibilit y on thermosensiti vity or
the enzy me. T he ri gidit y has been increased by creating
add iti ona l sa lt-bridges, aromatic interact ions and by in-. I 'f'" fl' . I(P - I()~ creaslIl g t le a1 Inlt y or ca clum Ions - .
Ext remozymes or psychrophil es can be mass pro
duced w ithout growing the ac tual culture. Gene c loning
or psychrophilic enzymes may enable them to prod uce in pu re rorm by modern met hods or protein ex press ion
i ll hete ro logous hosts such as I:'. coli using simple med i; i
components. M ost o ften it is dirf icul t to rind an enzy me
wit h propert ies matching w ith the react ion conditi ons. Thererore, to mod iry the enzyme for a spec ific task
bio-amendment or rat ional des ign has been carried out.
As a result , xerozy rnes or enzymes roreign to the nature
have been created us ing chellli cal and protein engineer. I ' 10:; Ing teclnlqucs .
Mesophili c bacteri a can ex press enzymes of psychro
phi lic bacteri a w ithout dras tic change in their catalyt ic
prnpert ies. It has been shown that cion ing or I ipasc genes
i llt o mcsophi lic . E.co/i pr ' :-,e rvcd its low temperature
ac ti vit y an d thermolabile property or co ld acti ve bacte
ria . Hcte rologous ex pression or genes rcsponsible 1'0 1'
co ld act i ve enzymes in mesoph i les. t hcrerore, offers a convenient means or econom ical producti on of co ld
. I I ' . I I' . 10(, enzymes In arge-sca e tor commercia app Ica tlOns .
On the same ground. over ex pn::ss ion of a psycl1r()ph i I ic
(X- amy lase gene was de lllonst r;lted in a mesophilic strain or E.Coli. The st ructura l inve. tigations revealed
tha t the ]-D conronnalions and ac t ivity or the enzymc were :-.imilar w ith thc wild type enzyme. For the produc
tiOIl or enzyme" hetero logoLl ," expre:-,:-,ion systems such a, in I:'.r'oli. an opt imum tcmperature has to be determined in order to obta i llmaximum growth ort he organ
i."1ll <1I111 also 10 I11d lnlaill st"bil i ty or the ex pressed cll/.ymc . T illiS. isolat ion or nove I p:-.ychroph i I ic micro
orga ni ,~ ms would widen the gene diversi ty requi red for
oht ;li nin!,! usefu l chcmicals through recombi nant DNA
technology. M ethods like site-direc ted mutagenes is and
prote in engineering, have been fo ll owed to increase thc
catal yti c effi ciency or enzymes at ex tremes of tempera
ture, pH, and in organic so lvents. Crellarc/we ll l1l .1'.1'111-
hiosulI1 an uncult i vated psychrophi I ic microorganism
belonging to the kingdom Crenarchaeota has been re
ported by Schleper el a /1°. Culturing or thi s microorgan
ism under laboratory conditi ons has not been pos. iblc
and it s phenotypic characterizati on was carried ou t
so lely using molecular techniqucs and al so dependi ng
on it: d istribution in co ld and temperate hab itats . After
isolation and ampliri ca ti on of DNA and RNA. the ge
netic materi al has been ex pressed in meso ph i I ic hos t:-,
for elucidat ing properti es of their gene products. The
in vesti gat ions carried out under simi lar lines, revealed
the presence of a DNA polymerase in C. sv lllbioSII IJI and
it was found to be thermolab i Ie w ith rap id loss or ac ti vity
at 4()°C.
Protein enginee ring ha:-, been most II seful in under
standing the complex relat ionship between protell1
stru ctu re and runct ion al low temperat tlrt'. T he techn iq uc
has orfe red an effecti ve means to unders tand and prcdict
the contribution of cova lent and ionic interact ions for
protein stabili ty, fl ex ibility, and enzyme act ivit y.
A sparta te carbamoy l transferase hus been studied
relat i ve ly more w ith respec t to its molecul ar :-. tructure
and enzymology, since th is is one or the mu lt imeric.
all osteri c enzyme prcsent in psychrophi I ie, as we ll a:-,
psyehrot rophic bac teri a. Attempts have been made t()
unrave l the changes that could occur in enzy me activity
and cont ro lling mechani sm after its clnning in heterolo
gous express ion systcms. The genes res pons ihle ror
ATCase in a Vihrio st rain 269] hilvc heen clolled III
mesophili c E.co/i stra in ')~ .
Conclusion T he appli ca ti on or ex trel11ophil es, in general, anci
psyc hrophiles, in parti cular. in industri al processc:-,
opens up new era in biotechnology. Eac h group has
unique biochemica l realUres which can be exploi ted for
use in bi otechnological industri c:-.. The main rC;I:-,on ror
se lect ing enzy mes rrom thi:-, group of lI1icroorga lli slll:-' i :-,
their high stabi l ity and reduced r isk or contaminati(in at
low temperalUrcs. Due to thei r UllllSWtl properti es thc"c
enzy mcs are expec ted to f ill the gap bctween hio log ical
and chem ica l processes. However the biotechnology or
extrel110plliles is sti ll in its inrancy but ha~ important and
rar reaching implicat ion.
RAMAN A 1'1 (I/.: BIOTECHNOLOG ICAL APPLICATION OF PSYCHROPHILES lili
References Fukulla~a I\J & Ru ssc ll N J . .I Cell Microh io/ , 136 ( l liliO)
1660.
2 Z()hcll C E. Morille M icmhi ll/. (,h r(lI/ ica Ho /. ( 1942) 136,
:I MOI'i t;1 R Y . IIw ll'1'io/ Ne\ '. 29 ( l li75 ) 144.
-+ Jay .I M . //11 .1 Fllod Micmhio/. 4 ( 19X7 ) 25 .
" Fcller G. Lonhiellne T . Dcro,lnn l' C. L ihioull c C & Beeulllcll .I V . .I /ho/ (,h(,lII. 2(,7 ( 11)92) 52 17.
(, DCJlr;ld;1 P & I3rcllchley .I E. A/III/ 1~'/ll'iro/l Micro/; io l. (,.,
( 1(97) 2lJ2X.
7 Rcddy G S N. R ; lj a~o Jla l all G & Shivaji S. FL'MS Mic/'()/Jiol LI'II. 122 (11)1)4) 21 1.
X Woocsc CR . Kandler 0 &. Wheclis M Z. f' roc N!II/ AC<ld Sci
USA. 1i7 (11)1)0 ) 4576.
l) Stcltcr K O. FCMS i\I/iuhio/ Nel'. I Ii ( lli%) 14li,
10 Sehlcpe r C. Swa llson R V. Mathur E .I & Delon~ E F . .I /laele('( ol. 179 ( I t)l)7 ) nm.
II l3 ow lll ;1Il .I P. M cCo llllll()n S A. Hrown M V. N ichol;ls 0 S &. McM eekin .I A. A/'l' l b ll'iro ll Microh illl . (,3 ( I l)li7 ) 306X.
12 J UIl~C K. Gosnik .I J. Ilopps H G &. Stalcy .I T . .'irsl AI'Jl I
Micw/'io l. 2 1 ( 1')l)X) 30(1.
1.< Mitchel P. Yell 1-1 C &. M,nhemeicr P r. AJlI'/ b ll'i roll Micm-
hilll . 49 ( 1I)X5) 1:1:12,
1-+ Gounot AM . .I AI'I'I B((e l" ri lll. 7 1 ( l li91 ) 3X6.
I 5 Mar~cs in R & Schinner F . .IlJ((cl('('i(}l. 33 (ll)li4) I .
16 r ellcr G. Th iry ~. A rpi ~ ny .I L . Mer~eay M & Gcr(by C. Fl,M."; Mierohiol LI'II. (l(, I I li9()) :nli ,
17 S i ll ~ h L. Alal11 S I & Duhcy P. I 'mc .)\'1111' SlIswill /)('1 ' CIII 'i ro ll
\vild Lili'. ICclllre I'm Ell v ironl11elll ,ti Mana~l'lIlc ll l. Ujjain . India ). I X- 2 1 Deccmher I () ()7 . PI'. X3.
I X Lovc l;lIl ti .1. GUlshal1 K . K ;lsmir J. f)renla P &. I3rel1chicy .I E.
I')
20
2 1
,1
25
27
AJlI'I I-:II\ 'imll Miel'f)hiol. (,() ( 1')l)4) 12.
V1 ;lrti nez J. Smith D C. Sl cw;lrd Ci F &. 1\ I.al11 F. Af/III11
Mil' mhillll~·CII I. 10 ( I ')% ) 221.
Kohori H. Sulli van C \V &. Shi/Y;1 H. Pm I' N((ll l l c({(1 S, 'i USII. X I ( I ')X4) 6(,l) I .
Il allll'l'Ci . Tm/ll I (,hl'III/ ~· . (,X I 1')l)O) 133 .
Harashim,l A . T ak,ld ;1 Y &. Fukllll a~a ' . /li llsei /lioin/II IIII lJ i{)e lwlII . (,() I 11)% ) 1324 .
LllPpCS R. Dc\'( 1S ' . W illL-m S. 13,IrthcleillY P &. l\1at ; I ~ lI c R r . .lI'/nclIl .• n ( I')()() ) 27(,.
Ru sse ll P. I lel'\l i ~ P. Pc llL-rill N B. I r~en s R I .. rvl;l i-- i .I S &. . 1;1I11CS T. l-FMS ivI icw li i (}1 I: ('()I . 53 ( 19XX ) 101 .
n ovc r ,I N. AI' I II t-:11I 'il'llli i'v/ir'('o/Jiol. (,0 ( 11)t}4) 174,
" cl ln C. Ll'i1U S~y (i &. ( jc rday ( ' , 111'1'1 LII I'i w II Micm/Jill l. (,4 ( I ')l) ~ ) I 1(, .'.
Z< 1i-- i lr ia M M . i\sh iuchi 1\1 Y;!nl ilmllio S &. Ya~ i T. li it)\'( i lii ll il 'l'l lll ll/ /lIOI'lWIII , (,2 ( 1')l)X) (,) 5.
V ilkrci V. (,h c ,~" 1 .1 P. Cnd;l )' l' &. Vanheculllcil .1. l 'ml l' lI( \ , ; , 6 i 19
'n ) 2-+(,2
21) l\.il l'IIl' I !\ 1 I\: Ilnlllll l i . Il/uri ll t' 111 (>1. 11 .\ I 19')2 ) :1 41 .
3() Smith 0 C. Simon M. Al ldredge A L &. Azam F. Nil/III'£' , 359
( 1992) 47 .
3 1 Perry J J. C((/1 J Micro/Jiol. 43 ( 11)t}7) X41 .
32 Hamamoto 1'. Takata . Kudo T & Horikoshi K . FEI'vIS Microli iol Lell. 129 ( 1995 ) 5 I.
33 Suutari M &. Llilkso S . .1 Cell Micwhio/. I3X ( 11)')2) 445 ,
34 Suulari M & Laakso S. AI'I)/ 1,'III 'im/l Micmliio/ . 51i ( 1992)
233X .
35 Michel V. Lchou x I. Dcpret G. A ngl;ldc P. Lahc1ic.l &. Hehralil l M . .I IJ{(claiol. 179 ( Ilit}7) Tn I .
3(, Joncs P G. Bogelen R f\ V & Neidhardt F C. .I1J((claill l. l(it)
( 1(87) 2()l)2.
1,7 Goldstcin.l . Polill N S & Inouye M . PI'IIC NOlI Amd Sci USA .
X7 ( 1990) 2X3.
3X Friedman H. Ponzy Lu . & Alexa ndcr Ri ch, .I M ol liilll . (; I ( I 'nl) 105 .
3t) Kantlrnr 0 & Ciol dhe r~ A L. I I('(}(' Nil/I ;lcwl ,)"i USA. 94 ( 19(7) 497X.
40 Fcll er G & Gerd;lY C. Cd/ll lll r Moln Liti' Sci. 53 ( 1<)1) 7)
X30.
41 Gerikc . D,lnson ~.1. Ru ssell N .I &. H ()u~h [) W. !:'tll · .l Ii ioclwlIl. 248 ( 19li7) 49.
42 Sun K . Camartlella L. Dipri sco G & Hervc Ci. FCMS MilTo/Jiol
LPII. I('4 ( lliliX)375,
41 Narinx E. Bai se E &. Gerday C. Prllll'ill 1: 11,<.:. IU ( I I} I) 7)
1271 .
44 Gcrday C. A itta leb M . Arpi ~ n y .1 L. Bai se E. Chess;1 .1 P. Garsoux G, Pctrescu l &. FellerG . Ii({)c/t 1'1 II liiol,/n' A( 111 . 1342 ( 11)97) 11 9.
-+5 Ciov<.:rdc R L .1 . Vcld .1 II I. Kusters .1 (i & M Olll F R. ,\.{" I Mil'('o/Jiol. 2X ( 199X) 55.1
4(, Milra S. K im H & Bce l ~ hor<.:r D II. M olt' (vlicl'tliliol . 19 (1 ')9h ) 321) ,
47
49
5ll
5 1
54
'is
5{]
Ru sscll N J. TIllS. (March , 19X4 ) l OX .
Ru ssc ll N .J &. Fuku naga . {-'EMS Micm/Jio l N,' \ . 75 ( 1<)<)1)) 17 1,
D;til u~c .1 .1 . H;Ullamol (l T. Horikoshi K . M ('rit a Y i\ I. Stelter K & McCl()~kcy J A. J IIO('/l' ri ll l. 179 ( I ()(n ) I l) I X.
L I T cana A. Br,mdi A . i-i ti c(J nl i\ll. Spu ri o R. LI POl l & l i uaicr/i C O. Pmc Nail Acad Sci USA. XX ( Ili9 I ) I ()1)lJ7 .
Vi <.: illc C. Burdett 0 S &. 7.eik us .l G. Iholl 'r /lllni ,1111( NI'l . 2 ( I')% ) I .
Slili th M D & O lson C L. I I I(II/" { CiII ' II( , 47 ( I 'n5 ) 1074 .
.I oncs J 13 & Beck .I r. Tn h Chl'lli. I II ( 197(, ) I ()7,
Gr;II' E G & Thaucr R K . FhHS Li' ll . 136 ( 19X I ) I (,5 .
Sharp R .1 &. Munster M .I. cil l 'd in Mi('w/Jc.1 ill I: 1'/ 1'1'111 ,
LII\ 'i roll llll 'lI l.I' . vol 17 hy R i\ Herhert &. G A Cotld ( /\ cadclll ic' Press Inc . London) I <) X(,. 2 1 'i .
McGlli rc A J.Fr;lI lI.Ill' II ;ll P D. c !\IcM cck in T 1\ . Sn/ il /' /'/ (vl ic'('() /Jiol. 9 t 1')X7) 2(,5 ,
57 Shivai i S. Shyamalil R;II' N. S;li , rcc L . Shelll V. Rl'dd\' (i S i\ & I3 lt a r~a \' a P 1\ 1. AI'I ,I I"II\ 'i /'()/( fI /i('{'(lh in l. 55 ( llJ X') ) 767.
I()() J SC I IND RES V O L 59 FEBRUARY 20()()
.'iX Shi v; l.ii S. ShY;lIl1al;I I~;lo . Saisn:e L. Shelh V. Reddy G S N & Bh;lrgava PM . ./Iliosci. 13 ( 19XX) 4O'J.
'i ') Uchida M & K;l\vamura T. '/ M{/ri!w lIiol"c/llwl. 2 ( 1<}<}5) n.
()O Hoppe II G. Kim S .I & Gocke K. ;\ /I/ I! / :'III 'imll Micmliiol. 54 ( I<)XX) 7X4.
() I Lnchte K & Turl ey C M. NlIIl/n' . 3.B ( 19XX) () 7· ()<) .
()2 M;ilcila r X. Reyes II R. Garc ia II S. Il ill C G & Aillundson C H . / :·II~ Micmlii(}/ ·/ i'l"/lIwl. 14 (1<)<)2) 426.
()~ Ahlgren G. Guslafsson I Il & Boherg M . ./ PII."wl. 21\ ( 1<)92 ) .,,7.
()4 Mikhailllv;1 M V. Belilis D L. Wisc 1 L. Cicrwick W II. lorri s .I ;lcoh R S. Li/lit/.I . 3U ( 1<)9'i) 5X~.
()5 Tholilpson G i\ . /li(}cll"11I lIio!,lI."s Acill. I3U2 ( 19l)() 17.
()() Cosio I G. Fisher R A & CIiToad P A. '/ Food Sci. 47 ( 19X2) <)01 .
()7 Reicha rdt W. Micmli iol /:mlogr. 15 ( 19XX) ~ I I.
()X P;liec M G. Bernier R & .Iur;lsek I .. lI ioleclllllll /I ioellg. 32 ( I<)XX) 2.,,'i .
()l) Mora F. Contat .I . Barnoud F. 1~la F 8<. Noe P . ./ IVood S, ·i Tn/lIIl1l. (, ( I I)X() 147.
70 Hiely P. MacKen/ie CR. Puls.l & Schneidcr II. .//lioll'cllll ol.4 ( I<)X() 7:1 1.
7 1 i\ lallllos H. C!II'III /Ild. (, ( 1<)<)0) I X'i .
72 Ishid;1 M. Saito M. Kililura S & Nagayalila F . ./ Mllrill" /lioll'cllllol. 3 ( 1<)%) 262.
7.', Shigell1mi II. B;lc I /\ . Yazawa K. Sasaki T & KobaY; lshi .I . ./ (}rg Clleill. 57 ( 1<)<)2) 4~ 17.
74 Ci.orenes R. JoI're .l T & Bosch i\. ClIlI./ M icmli io/. 35 ( I <}X9 )
1015.
7'i UIl1C/;IWa H. Okalili Y. Kuru s;lw;1 S. Ohnuki T & Ishizawa M. ./ Allliliiol. 36 ( 19X.1)47 1.
76 Palhir;lna C. Dwit R. Jcmcn P R & Fenica l W. Telmlwdmll Lell .. n ( 1<)<) I ) 7()() I .
77 Jensen P R & Fenical W . AIIII 1<1'1 '. Microliiol. 41\ . (1994)
559
7 Shavcr K J & Schi IT T. FOllrlr- .I·"I 'elllll Me"l /111 Assoc /)l'1Il H,'s. Houston. Texas USA. 1969.
7') W;il low G. Lovell ST. Magyal' C. Svinger A. Szilagy A. Z;lvodsky P. Ringe D & Petsko Ci A. /Jroll'ill LlIg. J() ( 1997) 6()'i .
XO Ilarwood.l. Trl'lIds /liocll"111 Sci. 14 ( 19X9) 125.
X I Macr;IC A R & Halllillond R C. /Iio!t'c/II/II/ Cm(' lic 1:'lIg HI'I '. 3 ( I<)X5) 19.1 .
X2 Lawson L D & Illlghes B (i . Il ioc/wlII /Iio!'''rs Nes COIIIIII I/II. 152 ( I<)XX ) ~2X .
X~ Princcn L H 8<. Rothrll s .l i\ . .1 ;\/ll ()il CIIl'IIl SoC, 51 ( I<)X4)
2X I .
X4 1{;1I11;ln;1 K V. SIlIl/il'.l· Oil II{/CI,' /'i{/ll:'lI~nll£,.I' IIII 'o/J'('(1 ill LOll ' 1,'II I/Il'/'{/{IIU' I liod" gmr/fIl ioll. PhD thes is. Ji waj i Uni versity. Gwalinr. Indi;1 19<)7.
Wi K:ilO C. Inoue i\ & Iinrikoshi K . TIIITl:'lH. 14 ( 19%) ().
X6 Lindow S E. cited in IJh\'IO/I(fI"ogl'llic IJm l,{//'\'OII'S by M S
Mount and G H L acy (i\C: ldCllli c Press. Londun) 19x2. ~"':i.
X7 Lindow S E. AIIII H£'I ' IJ/,yIO/Wllllilogr. 21 ( I '>X3a) ."6.,,.
XX G;lreth .l W. /1i(}[l'Cllllol Cellel /:'lIg Rl'1'. 5 ( 19X7) 107 .
X<) Ohala H. Sa iki Y . Tan ishit ;1 .I & Toku Y;l ln;1 T. ./ /-"1'1'1/ /('111 Tl'c/II III I. (,5 (19R7) (lIn .
9() BcnitL T. Maria J. R:llni re/. G . Cas trejon F & Codon A C. /IiOlI'c/llwl Prog . 12 (19l)() 14<).
91 Suhden R E. Winc Ye; lst: Selcc tion ;Ind M()d iri C< lti on. in Yeast Str, lin Sclection. edited hy C J Panch;iI (Marcc:1 Dc k~ c r. Ne\\' Ymk) 199 1. pp. 11 ~- 1 "'7.
<)2 Ahoncn L & Tuovincn 0 II. A/I/II /;'II I'II'!III Mi,'/'"li iol. ::'7 (199 I ) I3X.
9." Wh yte L G. Grcer C W &. Inni ss W E. Gill .I Micmllilll. 42 ( 1996) 9<) .
94 Xu Y. Zhang Y F. Liang Z Y. Vandecastcelc M. Lcg ra in C & GlandorlT . Microhilliogr. 144 ( 199X) 14.,, 5.
9'i Whyte L G. Hawari J. Zhou E. Bourhonnic: rc L. Inniss W I: & Grec r C W. A/I/" f:'! /I 'imll Mi(·roliiol. (,4 ( I 99x) 257X.
lJ6 Leahy .l G & CollI'eli R R. Miemhillll« '!' . 54 ( I l)I)() 3():; .
In Singh L . MaurY:l II S. J{;llllan;1 K V 8.:. Alalll S I. /Iilln" Ti ', ·llIwl. 53 (I 9<).'i) 147.
l)X Isaken M F & Teske A. ; \ /'e" Mi, '/'" liilll . I(,() (I l)l)() ) I (,O.
l)1) Franzlll:1Il PD. Liu Y . l3alkwill D L & Aldri ch H C. 1111.1 Sn I/o ('[{'/' iol. 47 ( 1997) IO()X.
100 Fr;1I1zm:1Il P D. Springcr N. Lud w ig W. Conll';IY E. DelllaC:lrio & Rohde M. Sy.I'l A/I/Ii Microliiol. 15 ( 1l)l)2 ) 57:1.
101 Hollow J & Czako L. /\CllI lJi()[l'c/llwl. 7 ( 1')X7) 4 17.
102 Fell cr G & Gerday C. C('1/ Moll'<' Lifl' Sci. 50 ( 19')X ) 116.".
1m Fcller G. Narinx E. Arpigny .l L. Aittaleh M. B;li s E. C;cnicol S & Gerday C. FI;MS rV/iemhiol N,'!'. I X ( I l)%) I Xl) .
104 Nari nx E. Bai se E & Gerday C. i-'ml('ill /;·lIg . II) ( 1')ln) 127 1.
1O'i N icdlell1;1Il S L. Cril H,'I' /I i()[('clllwl. 4 ( 19')() '273 .
I D6 Feller G. Thiry M. i\rpign)' .1 L & Gerday C. (;" lIe. 1!12 (I')l) I )
II I .
ID7 Hoshino T. Ishizaki K . Saka illolo T. Kun et;1 H. YUl11()to I & Matsuynl1la H. Lell A/I/II Microliilll. 25 ( 1<)<)7 ) 7().
lOX Margesin R & Schinncr F. FeMS Micmhill / L('ll. 79 (19')1)
257.
10<) Ray M K . Silaralllalllma T. Gandhi S & Sh ivaji S. A/I/Ii Hill 'i ron Micmliio/. 116 (1 994) 'i'i .
liD Sai Ram M. Singh L. AI: II11 S I & Agarwal 1 K . IVorld ./ l\I!icmhiol /lj()[n/llwl. IH ( 1<)94) .,, 56.
I II Choo D W. Ku riha ra T. Suzuki T. Soda K & Esaki N. 1\/1/1/
b'i'i roll Microhiol. 64 ( 1l)<)X) 4X(1.
112 Baral A & Fox P F. Food C"eII/. 51\ ( 1997) .,,:; .
11:1 Singh L. Sai Ralll M. Ag: llwal M K & AI;lIn S I. ./ Cell A/I/II Microbiol. 4() ( 1<)94) .".,,9.
11 4 Aghajari N . Feller G. C;erd .IY C & Haser R. Pmll' ill Sci. 5 (1 996)2 12S.
115 Kimu ra T & Horikos li i K . Ag/'i lJi,,1 C!1l'1Il. 53 ( 19X9) 296."
RAMANA 1' / lI!. : BIOTEC HNOLOG ICA L A PPLICAT IO OF PSYC HROPH IL ES 101
116 Fel lcr G. Thicry L. Dcroannc. Liholili c C. I3cc llrncn J &
Gcrd;IY c. .1 iJiIJ! CiII'1I 1. 2li7 ( I ()9(1 ) 5:21 7.
11 7 Kirnura T &. S;lit;l111a K 1-1 . S/lIre!1. 42 ( 1990) 4()].
IIX AlvarCi. M. Zcc lcn.l P. M 'lini"roid Y . Rcnticrdclrllc F. M anial
.I A. Wynns L . Wicrclwa R K &. Macs D . .I !lio! Chl'lIl. 273
( 1l)()X) 2 1 ()1) .
Il l) Pi crrard A. Lcdcnt P. Docquiere .I D. Fcller G. Gerday C &
Frere .l M . Ft"MS MicrohiIJ! LeI/a.\". I li I ( 199X) 3 1 I .
120 Whyte L G. BOllrbonniere L & Greer C W. AflfI! ! :'I11 'im ll
Micl"lliJ io! . li3 ( 1997) :1719.
12 1 Okll Y: l1 11<1 1-1 . 'cno A. Enari D. M orita N & Ku sano T. Ardl
MicmiJio!. ) lit) ( 199H) 21) .
122 Tok llY:ll1la T. Y oshida N. Matslli shi T. T 'lk:ih ;l~hi R . Kanchhira T & Shinoh;lr;1 M . .1 Fl'l"In iJ iol'ngng. X3 ( 191) 7) 377.
123 b id'l M . Saito M . Kimura S & NagaY:II11a F . .1 Mllrin l' UiO/l'clt
no!. 3 ( 1996) 262.