Ex 2 Enzymes

EXPERIMENT NO. 2 ACTIVITY OF ENZYMES OBSERVED IN NEWLY GERMINATED Vigna radiata) ROOTS OF CORN ( Zea mays) AND MUNG BEANS ( Vigna USING COLOR REACTIONS

 February 12, 2016  BOT 120 Ab | Group 5 Mangaln!" #$%&'ln" Ma%!" Xana" P*!&" Maa T%+al!n&a" San P%!" S$al%n% Ma%" Ta%" C!nn% Maaa$  Department of Biology, College of Science, University of the hilippines!Baguio "overnor ac# $%, Baguio, 2600 A,+*a&* Enzymes Enzymes speed speed up chemical chemical processes processes by lowering lowering he aci!aion aci!aion energy energy needed needed o iniiae iniiae he reacion reacions" s" E#amples E#amples o$ enzymes used in his e#perime e#perimen n were hydrolase hydrolasess and o#idoreduc o#idoreducase asess speci$ically amylases amylases and sucrases $or he $ormer and dehydrogenases and caalases $or he laer" This sudy aims o obser!e he aci!iy o$ enzymes on corn roos and mung beans% es $or he  brea&down o$ compounds using color reacion ess% and ideni$y $acors ha a$$ec enzymaic aci!iy"   'or he (odine es% he brigh yellow color o$ he es ube wih corn roo indicaes he hydrolysis o$ sarch and he presence o$ amylase" 'or he Benedic)s Tes% Tes% he appearance o$ an orange  precipiae  precipiae and soluion esed posii!e $or he brea&down o$ sucrose and he presence o$ reducing sugars" 'or he *ehylene Blue reducion es% here was dehydrogenase aci!iy indicaed by he lighening o$ he blue soluion" +asly% $or he caalase enzyme% enzyme% e!oluion o$ gas was no obser!ed wih he boiled poao srips" (n summary% newly germinaed seeds ha!e amylases o hydrolyze sarches% sucrases o brea&down sucrose ino $rucose and glucose and dehydrogenases $or anaerobic phase $or   proper plan growh growh and de!elopmen during during seed germinaion" germinaion" (n addiion% denauraion denauraion o$ enzyme by heaing causes inaci!iy o$ he enzyme li&e caalase"

I. In*!-&*!n Enzymes are $undamenal biomolecules ha ma&e up all li!ing organisms ha li$e would be impossible wihou hem" ,ihou he aci!iy o$ enzymes% biochemical processes end o occur  !ery slowly% slowly% consuming consuming more ime and energy energy in he process% which is no con!enien con!enien and  bene$icial" Thus% enzymes speed up he reacion raes o$ hese processes by lowering he aci!aion energy re-uired o iniiae he se-uence o$ reacions ha happen inside he organism .Taiz and /eiger% 2002" Two o$ he !as classes o$ enzymes as sudied in his e#perimen are .a hydrolases and .b o#idoreducases" ydrolases are responsible in he meabolism or brea&down o$ speci$ic compounds $ound in plan issues .*inic% 200" Among he ypes o$ hydrolases% amylases amylases and sucrases sucrases were e#amined" e#amined" Amylase Amylase $acilia $aciliaes es he endohydr endohydrolys olysis is o$ 1%34464 1%34464 glucosidic lin&ages in polysaccharides conaining hree or more 1%344lin&ed 64glucose unis" (s naural subsrae is sarch and is acion is induced by 27 sarch or solid 8a8O 9 .:pringer% 2009" :ucrase% on he oher hand% is a caalyic enzyme in he hydrolysis o$ sucrose ino glucose and $rucose by brea&ing he O48 .glucose bond .Boye and Arcand% 2012" The oher class o$  enzymes are o#idoreducases% which caalyzes o#idaion reducion reacions and rely on heir  redo# co$acors $or heir aci!iy .Barle% 200" Among he o#idoreducases% dehydrogenases and caalases caalases were he enzymes enzymes sudied" sudied" 6ehydroge 6ehydrogenase nase is an o#idoredu o#idoreducase case ha in!ol!es in!ol!es

nicoinamide adenine dinucleoide .;A6 < and ;A6 in is redo# reacion .=a&ubows&i% n"d"" +asly% caalase is anoher o#idoreducase which is a pero#idase ha caalayzes he brea&down o$  hydrogen pero#ide% a o#ic compound% ino waer and o#ygen which can be uilized by he cell .Boye and Arcand% 2012" The sudy aims o obser!e he aci!iy o$ enzymes such as amylase% sucrose% dehydrogenase and caalase on corn roos and mung beans% es $or he brea&down o$  compounds using color reacion ess% and ideni$y $acors ha a$$ec enzymaic aci!iy"

II. M%*$!+ an Ma*%al+ The e#perimen was di!ided ino wo enzyme ess namely hydrolases and o#idoreducases" (n he es $or hydrolases% amylase and sucrose were esed speci$ically" 'or Amylase% wo es ubes conained 10 ml 0"17 sarch soluion each" The roo o$ wo newly germinaed corn seedlings were placed in one es ube" The ubes were le$ o sand o!ernigh in room emperaure% hen a drop o$ >( was placed on each ube and obser!ed $or color changes" 'or he es $or sucrose% wo es ubes were prepared wih 5ml o$ 1"07 sucrose soluion and $resh corn roos were placed in es ube 1" The ubes were le$ o sand o!ernigh and a$er which% he roos were remo!ed $rom he ube" E-ual !olume o$ Benedic)s soluion was hen placed in he ubes and were heaed in boiling waer bah o es $or presence o$ reducing sugars" (n he es $or o#idoreducases% dehydrogenases and caalase were esed" 'or  dehydrogenases% wo es ubes were $illed wih 0"0017 mehylene blue and 10 grams o$ $reshly germinaed mung beans were placed in one ube" Boh were sealed wih a sopper ensuring ha i was anaerobic and here were no air bubbles" The ubes were allowed o sand o!ernigh and color  changes were noed a$erwards" 'or caalase% wo ubes were $illed wih 5ml o$ 97 hydrogen  pero#ide each" 'resh poao srips were placed in he $irs ube and boiled poao srips were  placed in he second ube" Gas e!oluion a$er 5410 minues were obser!ed"

III. R%+-l*+ an D+&-++!n ydrolases re$ers o a speci$ic class o$ enzyme ha wor&s o conrol he process o$  hydrolysis which is he mainly a chemical reacion ha is caused when a molecule o$ waer wor&s o spli apar anoher molecule .+ehninger e" al% 2005" :ome enzymes in he plan body ha caalyze his reacion are amylases and sucrase" Table 1 shows he resuls o$ he es $or amylase by using he (>( es" :ome polysaccharides ha!e he propery o$ adsorpion $or iodine" These iodine molecules will be rapped in he helical srucure o$ polysaccharides resuling now o a change in color o$ he soluion .amza% 2015" Table 1" 8omparison $or he resuls o$ (odine Tes $or Amylase" :ample

:oluion 8olor  

Tes ube 1 .wih roos o$ corn seedlings

Brigh yellow wih blac& precipiae

Tes ube 2

?ale yellow wih blac& precipiae

The resuls obained showed ha he sample which conained corn roos had brigher yellow color a$er he incubaion ime in comparison o he conrol which has no roos .:ee Appendi#% 'igure @" This indicaes ha some o$ he sarch ha!e been hydrolyzed deermining now he  presence o$ amylase in he newly germinaed corn roos howe!er% he soluion wih corn roos has  blac& precipiae which means ha no all he sarch were hydrolyzed"

An enzyme ha acs as a caalys o hydrolyze carbohydraes is called an amylase" (n  plans% is role is $or brea&ing down sarches" ( also suppors he iniial de!elopmen o$ he plan%  be$ore using he energy $rom phoosynhesis" This enzyme begins heir role in plan de!elopmen as he seed sars o germinae" The ype o$ amylase mos common in plans is alpha amylase ha helps o process basic subsances li&e glycogen and sarch .Baird% 201"

On he oher hand% sucrase is also a hydrolase ha hydrolyzes sucrose o $orm glucose and $rucose" Table 2 shows he resuls ha were obained $or he es o$ sucrase" Table 2" 8omparison o$ he resuls in Benedic)s Tes $or :ucrase" :ample

8olor o$ :oluion

Tes ube 1 .wih corn roos

Orange

Tes ube 2

;o color change

( can be obser!ed ha a$er he boiling process% Tes ube 1 wih corn roos has a color  change $orm blue o orange soluion .:ee Appendi#% 'igure  " This means ha he ube wih corn roos yield a posii!e resul on his es" The principle behind his is ha reducing sugars are o#idized by he copper ion in soluion o $orm a carbo#ylic acid and a reddish precipiae o$  copper .( o#ide .+ehninger e" al%2005" The Benedic)s es was inended o es $or he presence o$ reducing sugars" Based $rom he obained resuls% i can be said ha he roos o$ he newly germinaed corn has sucrase because i displayed a posii!e resul" The sucrase brea&s down he sucrose in he soluion% ha!ing now glucose and $rucose which are reducing sugars" Glucose is needed by cells $or respiraion" ( is used $or respiraion o release energy" :ome is used o  produce cellulose which srenghens he cell wall and proeins li&e enzymes and chlorophyll .BB8% 2013" ,hile $rucose is a ype o$ reducing sugar ha can be $ound in $lowers" This sugar  has a role in pollinaion o$ he plan because i ma&es $lower necar sweeer .:eb% 2015 and ha is he reason why insecs li&e bees and buer$lies are araced o he plan" O#idoreducases are enzymes which caalyze o#idaion4reducion reacions by allowing <  ;A6   or ;A6?< o accep a hydride ion $rom a reduced subsrae% or by allowing ;A6 or   ;A6? o donae a hydride ion o an o#idized subsraeC o#idaion4reducion reacions ypically occur during respiraion .;elson and 8o#% 2005" E#amples o$ enzymes which caalyze such reacions are 6ehydrogenases and 8aalases" Table 9 shows he resuls o$ he es $or dehydrogenases !ia he mehylene blue reducion es" The principle behind his es is ha mehylene blue will ac as a Dsep in reducing agen and donaes ions o o#ygen produced during respiraionC his acion causes he mehylene blue o lose is color and become ransparen .Thornon and asings% 190" Table 9" *ehylene Blue Feducion Tes on 8onrol s" a :ample o$ 'reshly Germinaed *ung Bean :prous :ample

:oluion 8olor  

Tes ube 1 .conrol

deep blue

Tes ube 2

a-ua blue

The resuls obained showed ha he sample which conained mung bean sprous had a clearer% ligher soluion a$er he incubaion ime in conras o he conrol .Appendi# 'ig"10" This indicaes dehydrogenase acion as he mung beans undergo respiraion% releasing o#ygen which will hen be aced upon by he mehylene blue" 'resh mung bean sprous were used as dehydrogenases are mos abundan a his sage" This is due o how dehydrogenases ha!e a maHor  role in he caalysis o$ sored producs during he anaerobic phase o$ seed germinaion .Oi&haena e al"% 2019" 8aalase is anoher o#idoreducase which is ypically $ound in pero#isomes" ( caalyzes he clea!ing o$ damaging  2O2  produced during I4o#idaion ino  2O and O2  .;elson and 8o#% 2005" Table 3 shows he resuls o$ he acion o$ poao slices .$resh and boiled upon he addiion o$ 2O2" Table 3" Tes on he acion o$ caalase in $resh .es ube 1 and boiled .es ube 2 poao slices on 2O2" :ample

Gas E!oluion

Tes ube 1

presen

Tes ube 2

no presen

E!oluion o$ gas was no obser!ed in boiled samples as he enzymes ha!e become denaured and can no longer caalyze he decomposiion o$  2O2 .Appendi# 'ig"11 " Gas e!oluion occurred almos immediaely in $resh samples% indicaing he acion o$ caalase in he poao slices"

IV. L*%a*-% C*% Baird% +" .201" ,ha (s he Fole o$ Amylase in ?lansJ Ferie!ed 'ebruary% % 201 $rom hpKLLwww"wisegee&"orgLwha4is4he4role4o$4amylase4in4plans"hm Barle% ?";" .200" Bioelectrochemistry& =ohn ,iley M :ons% +d" England" Boye% ="% Arcand% N" .2012" Green Technologies in 'ood ?roducion and ?rocessing" :pringer" ;ew Nor&" BB8" .2013" :orage and use o$ glucose" Ferie!ed 'ebruary% % 201 $rom hpKLLwww"bbc"co"u&LschoolsLgcsebiesizeLscienceLadda-aLphoosynhesisLphoosynhesisre!3"sh ml amza" .2015" (odine es $or sarch" Ferie!ed 'ebruary% % 201 $rom hpKLLallmedicalsu$$"comLiodine4es4$or4sarchL =a&ubows&i% " .n"d" O#idai!e FeacionsK 6ehydrogenase and O#idases" Ferie!ed 11 'ebruary% 201 $rom hpKLLbiowi&i"ucda!is"eduLBiochemisryLO#idaionand?hosphorylaionLO#idai!e   Feacions79A6ehydrogenaseandO#idases" +ehninger% A"% ;elson% 6" +"% M 8o#% *" *" .2005" +ehningerPs ?rinciples o$ Biochemisry" '&(    Freeman" *inic% /" .200" ?hysiological roles o$ plan glycoside hydrolases" ?lana%[email protected]K@29430"

Oai&hena% E" E"% AHibade% G" A"% Appah% ="% M Bello% *" .2019" 6ehydrogenase Enzyme Aci!iies in Germinaing 8owpea .igna Qnguiculaa .+ ,alp"  )& Biol& *gric& (ealthcare% +% 9249" :eb% ?" .2015" 'rucose ha!ing an imporan $uncionJ Ferie!ed 'ebruary% % 201 $rom   hpsKLLwww"paleohac&s"comLsugarL$rucose4ha!ing4an4imporan4$uncion41123 :pringer" .2009" Springer (an%boo# of n-ymes& :pringer4erlag" ;ew Nor&" Taiz% +"% /eiger% E" .2002"  lant hysiology& 9rd Ed" :inauer Associaes% (nc"% ?ublishers" :underland% *assachuses" Thornon% "% M asings% E" .190" :udies on O#idaion4Feducion in *il&K The *ehylene Blue Feducion TesR" )ournal of Dairy Science% 1+.9% 2214235"

V. -%+*!n+ /! R%+%a&$ 1" Enumerae he di$$eren $acors ha a$$ec he aci!iy o$ enzymes" Brie$ly describe how hese $acors a$$ec rae o$ enzymaic reacions" a"

Temperaure

The rae o$ enzyme aci!iy increases wih as he emperaure increases because o$ higher  &ineic energy ha leads o collisions beween enzyme and subsrae unil he opimum emperaure% hen $alls o zero because as proeins% hey are denaured" The proein)s aci!e sie may no longer be complemenary o he subsrae molecule"  b" p  p also a$$ecs he rae o$ enzyme aci!iy" Each enzyme has is own range o$ p and a p higher or lower han he opimum would lead o denauraion or a non$uncional enzyme" c" 8oncenraion The more subsrae or enzyme concenraion% he higher he rae o$ enzymaic aci!iy  because o$ increase in collisions" Bu i only increases up o a cerain degree and becomes consan or wha is &nown as he Dplaeau e$$ec because he limiing $acor changes" d" (nhibiors (nhibiors can eiher be re!ersible or non re!ersible" Fe!ersible (nhibiors $orm wea&% non co!alen bonds and resuls o a compeii!e% non compeii!e or mi#ed inhibiion" 8ompeii!e inhibiion happens when an inhibior binds o he aci!e sie o$ he enzyme bloc&ing he binding o$ he subsrae o he enzyme" ;on compeii!e inhibiion is he inhibiion o$ enzymaic aci!iy due o obsrucion o binding because he inhibior bonded anywhere on he enzyme e#cep he aci!e sie" 'or mi#ed inhibiion% a subsrae4enzyme4inhibior comple# is $ormed bu hey are no $uncional" On he oher hand% irre!ersible inhibiors $orm srong% co!alen bonds wih he enzyme or hey denaure i"

2" E#plain why newly germinaed corn and mung bean seeds are sue din his e#perimen"  ;ewly germinaed corn and mung bean seeds were used because hey grow easily and $as

which allows immediae obser!aion and esing" They also represen he monoco and dico pans enabling ideni$icaion o$ similariies beween hem"

Fe$erencesK 'acors a$$ecing Enzyme aci!iy" .2015" (n A +e!el ;oes" Ferie!ed 'ebruary 11% 201 $rom hpKLLale!elnoes"comL'acors4a$$ecing4Enzyme4Aci!iyL13Jree S Taiz% +"% /eiger% E" .2002"  lant hysiology& 9rd Ed" :inauer Associaes% (nc"% ?ublishers" :underland% *assachuses"