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Noor Azlina Ibrahim and Norazila Yusoff Faculty of Agro-Industry & Natural Resources, Universiti alaysia !elantan"

ABSTRACT #ater sam$ling have been done at four location of the %o&ing 'ot ($ring and each location have different )ater tem$erature *+,, -,, ., and /,01 2" 3nly sam$les of .,01 and /,01 )ere sho)ed gro)th in nutrient broth" 4ach inoculums have been screened on s5im mil5 agar and t)o isolates *.,a and .,b2 of .,01 sam$les formed clearing zones around the colonies on the $lates" From their $rotease assay, both isolates have identified as a serine $rotease $roducer and from the 6-( r7NA identification, isolate .,a closely related to Bacillus subtilis )hile isolate .,b )as closely related to Bacillus licheniformis" 8hese isolates )ill be used for further studies into the cloning and e9$ression of $rotease gene"

INTRODUCTION :rotease are among the most valuable catalysts used in food, $harmaceutical and detergent industries because they hydrolyze $e$tide bonds in a;ueous environments and synthesize $e$tide bonds in microa;ueous environments *3gino et al., 6<<<2" icrobial $roteases dominate the commercial a$$lications, )ith a large mar5et share ta5en by subtilisin $roteases from Bacillus s$$" for laundry detergent a$$lications" A ma&or re;uirement for commercial a$$lications is thermal stability, because thermal denaturation is a common cause of enzyme inactivation" 8he $otential use of thermostable enzymes in a range of biotechnological a$$lications is )idely ac5no)ledged" 8hermostable $roteases are advantageous in some a$$lications because higher $rocessing tem$eratures can be em$loyed, resulting faster reaction rates due to a decrease in viscosity and an increase in diffusion coefficient of substrates" Furthermore, higher $rocessing tem$eratures )ill also increase the solubility of nongaseous reactants and $roducts as )ell as reduce the incidence of microbial contamination by meso$hilic organisms *3la&uyigbe and A&ele, =,,.2" It is e9$ected that the a$$lications )ill 5ee$ increasing in the future as )ill the need for stable biocatalysts ca$able of )ithstanding harsh conditions of o$eration )hich occurred normally in industry" 4ven though there is no firm evidence to suggest that thermostable enzymes are necessarily derived from thermo$hilic organisms, nevertheless there is a greater chance of finding thermostable $roteins from thermo$hilic bacteria" A )ide range of microbial $roteases from thermo$hilic s$ecies has been e9tensively $urified and characterized" 8hermo$hiles such as Bacillus stearothermophilus *>oonyanas et al., =,,,2, Thermus aquaticus *?abriela et al., =,,@2, Bacillus licheniformis *Ferrero et al., 6<<-2, Bacillus pumilus *!umar, =,,=2, and Thermoanaerobacter yonseiensis *'yenung et al., =,,=2 have been studied for their ca$ability in $roducing thermostable $roteases" >iochemical $ro$erties of the enzymes $roduced from these thermo$hilies have also been )ell investigated" >ecause of their high activity and stability at elevated tem$eratures, the thermo$hilic $roteases can also be used as ideal models for studying thermal stability of $rotein *Rao et al., 6<<A2"

METHODS #ater sam$les )ere ta5en from different locations )ith different tem$erature *8able 62 of %o&ing 'ot ($ring in %o&ing 'ighlands, !elantan" 4ach sam$ling )as done in tri$licates" Bacteria gr !t"" 8he sam$les *6 ml2 )as first inoculated into 6,ml nutrient broth *N>2 medium in a =, ml universal bottle and )ere incubated base on their tem$erature for 6A-=/ hours, )ith sha5en at 6., r$m" I# $ati % & 'r te $(tic )icr rga%i#). 4ach isolates )as screened ;ualitatively for their $rotease $roduction on (5im il5 Agar *( A2 $late and )ere incubated based on their tem$erature for =/ hours" A clear zone around the colonies on the ( A $late gave an indication of $rotease $roducing organism" 8he $ositive isolates )ere then tested for their enzyme activity" Pre'arati % & I% c*$*)#. 8he inoculums )ere $re$ared by inoculating a $ure colony into 6, ml 8ry$ticase (oy >roth *8(>2 in universal bottles and incubated at 6,, r$m in a sha5er incubator for =/ hours at .,01" 8he cells )ere harvested by centrifugation at 6,,,, r$m, /01 for 6, min" 8he bacteria $ellet )as dissolved in saline *,"A.B Na1l2 to give an absorbance reading of ,". at ./,nm" Inoculum *= ml2 )as then inoculated into /, ml 8(> *re$resent .B inoculum size2 and incubated at .,01 for =/ h" 8he culture )as harvested from the medium by centrifuging at 6,,,, r$m and /01 for 6, min" 8he su$ernatant )as then filtered )ith cellulose acetate membrane filter *$ore size, =,Cm2 to obtain the crude enzyme" E%+()atic a##a(#. :rotease activity )as determined by a modification of the method described by Rahman et al", *6<</2" Azocasein *,".B, 6 ml2 )as dissolved in ,"6 8ris-'1l-= m 1a1l =, $' < and $reincubated at .,01" 8he reaction )as initiated by addition of 6,,Cl of enzyme solution, and $erformed at .,01 for @, min" An e;ual volume of 6,B trichloroacetic acid *81A2 )as added to terminate the reaction and the mi9ture allo)ed to stand at room tem$erature for @, min and then centrifuged at 6@,,, r$m for 6, min" 8he absorbance of the su$ernatant )as determined at /.,nm" 3ne unit *U2 of azocaseinase activity )as defined as the amount of enzyme activity that $roduces a change of absorbance *,",,6 $er min2 at /., nm at .,01 under the standard assay conditions" E&&ect & I%"i,it r#. :atterns of inhibition )ere determined using inhibitors such as, $henylmethylsul$honyl fluoride *: (F2, ethylenediaminetetracetic acid *478A2, iodoacetic acid *IAA2, anti$ain, $e$statin, bestatin and elastatin" 8he $rotease $re$aration )as incubated )ith the inhibitors in the ratio 6D6 for @, min at room tem$erature" 8he activity )as determined as above" Bacteria$ I-e%ti&icati %. 8he isolates )ere strea5ed on NA $late and incubated at .,01 for =/ h to get single colonies" 8he colonies on the $late )ere differentiated by the mor$hology of the colony such as size and surface" A single colony )as also $ic5ed for gram staining and the slides )ere observed under microsco$e using 6,,, /,, and 6,,,E resolution" Further identification of the isolates )ere by 6-( r7NA gene identification" Ta,$e .D Numbers of )ater sam$ling from different location of %o&ing 'ot ($ring (am$ling location A2 ($ring >2 #aterfall 12 %o)er )aterfall 72 :ond 8em$erature *012 +, -, ., /,

RESULTS AND DISCUSSION (am$les from .,01 and /,01 )ere able to gro) in nutrient broth medium and t)o isolates from .,01 sam$le *.,a and .,b2 sho)ed $ositive results on ( A $late by forming clearing zones around the colonies" 8he clearing zones re$resent $roteolytic brea5do)n of mil5 $roteins and indication of $rotease secretion" 8he effect of various classes of inhibitors on the $rotease activities )as determined" Inhibitors of 478A, IAA, anti$ain, $e$statin, bestatin and elastatin did not inhibit the $rotease activity *Figure 62" 'o)ever, the $rotease activity )as inhibited by the serine $rotease inhibitor, : (F, )hich resulted 6,,B inhibition at concentration of =,m " 8he results clearly indicated that these enzymes belong to the serine $rotease" Isolates .,a and .,b )as characterized by $hysiological characteristics and gram staining" After =/ h incubation on nutrient agar at .,01, the single colonies a$$eared" 1olonies for isolate .,a )ere small and rough on nutrient agar )hile for isolate .,b, the colonies )ere larger than isolate .,a and smooth on nutrient agar" As for gram staining, the mor$hology sho)ed that both isolates *.,a and .,b2 )ere gram $ositive and straight rods" 8he 6-( r7NA nucleotide se;uence from isolates .,a and .,b *Figure = &@2 has been analyzed using >last from National 1enter of >iotechnology *htt$DFF )))"ncbi"nih"gov2" From the analysis, isolate .,a )as closely related to Bacillus subtilis and isolate .,b )as closely related to Bacillus licheniformis. 8he homology among these bacteria sho)ed a significant value of <<B homology"

Fig*re .D 4ffects of inhibitors *6,m IAA, 6m Anti$ain, =m 4lastatin, 6,m : (F and 6,m 478A2 on $rotease activity"

:e$statin, 6".m

>estatin, =m

Fig*re /0 6-( r7NA se;uence of isolate .,a

Fig*re 1 D 6-s r7NA se;uence of Isolate .,b

REFERENCES >oonyanas (, (u$acho5 (, (uree :, (huitein 1 *=,,,2" :urification and characterization of the highly thermostable $roteases from Bacillus stearothermophilus 8%(@@" :rotein 49$r" :urif" =,D 6/=G6.6" Ferrero A, 1astro ?R, Abate 1 , >aigori 7, (ineriz F *6<<-2" 8hermostable al5aline $rotease of Bacillus licheniformis IR=<D isolation, $roduction and characterization" Appl. Microbiol. Biotechnol" /.D @=+-@@="

?abriela 3, (la)omir 7, Hozef ! *=,,@2" 'igh-level e9$ression, secretion, and $urification of the thermostable a;ualysin I from 8hermus a;uaticus Y8-6 in :ichia $astoris" Protein Expr. Purif" =<D ==@-==<" 'yenung HH, >young 1!, Yu R:, Yu ! *=,,=2" A novel subtilisn-li5e serine $rotease from 8hermoanerobacter yonseiensis !>-6D its cloning, e9$ression, and biochemical $ro$erties" Extremophiles" -D =@@-=/@" !umar 1? *=,,=2" :urification and characterization of a thermostable al5aline $rotease from al5alo$hilic >acillus $umilus" Lett. Appl. Microbiol" @/D 6@-6+" 3gino, '", #atanabe, F", Yamada, ", Na5aga)a, (", 'irose, 8", Noguchi, A", Yasuda, " and Ishi5a)a, '" *6<<<2 :uirfication and characterization of organic-stable $rotease from organic solvent-tolerant :seudomonas aeruginosa :(8-,6" Hournal of >ioscience and >ioengineering" A+D-6--A" 3la&uyigbe, F" " and A&ele, H" 3" *=,,.2" I:roduction dynamics of e9tracellular $rotease from Bacillus s$eciesJ" African Hournal of >iotechnology / *A2K ++- G ++<" Rahman, R"N"L"A, Raza5, 1"N, Am$om, !", >asri, ", Yunus, #" "L", and (alleh, A">" 6<</" :urification and characterization of a heatstable al5aline $rotease from Bacillus stearothermophilus F6" Appl. Microbiol. Biotechnol", /,D A==-A=+" Rao, ">", 8an5sale, A" ", ?athe, "(" and 7esh$ande, M"M" *6<<A2 olecular and biotechnology as$ects of microbial $rotease" icrobiology and olecualr >iology Revie)s" -=*@2D .<+--@.