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

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