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Kakav je očekivani učinak pH na aktivnost gljivične pektinaze?

Kakav je očekivani učinak pH na aktivnost gljivične pektinaze?


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Radim na enzimskom testu za gljivičnu pektinazu. Testirao sam enzim u različitim puferima od pH 1-12,5. Međutim, enzim ima dobre aktivnosti počevši od pH1-10,5.

Da li je moguće imati aktivnost enzima u tako širokom rasponu pH?


Zapravo sumnjam da pektinaza ima tako širok raspon pH vrijednosti u kojem djeluje optimalno. Pretražujući internet pronašao sam dvije brojke koje potvrđuju moje sumnje:

Prvi je iz članka ("Imobilizacija pektinaze adsorpcijom na hitinskom nosaču obloženom alginatom") u kojem se uspoređuje aktivnost nativne i imobilizirane pektinaze pod različitim uvjetima. Drugi je iz tablice podataka kompanije koja prodaje enzim. Oba pokazuju aktivnost preko 50% otprilike između pH 3,5 i 5,5, ali ne pri višim ili nižim pH vrijednostima.


Kakav je očekivani učinak pH na aktivnost gljivične pektinaze? - Biologija

Catalase

Vodikov peroksid (H2O2) je čest nusproizvod metaboličkih reakcija. U visokoj koncentraciji je toksičan pa bi njegovo nakupljanje u stanicama bilo štetno. Većina tkiva, međutim, sadrži enzim katalazu, koji katalizira razgradnju peroksida do vode i kisika na sljedeći način: Reakcija je izuzetno brza. Djelovanje enzima može se lako demonstrirati evolucijom kisika u obliku plinskih mjehurića kada se ekstrakt tkiva koje sadrži enzim dodaje u razrijeđenu otopinu vodikovog peroksida. Kao izvor katalaze koristit ćemo homogeniziranu (mljevenu) pileću ili goveđu jetru. Catalase

  • Catalase - opće informacije Učionice 21. stoljeća
  • Katalaza - izvanredan enzim
  • Enzim toplotne denature
  • Struktura slike i grafikona katalaze
  • Linkovi -

Amilaza

    Reference amilaze:
    • Amilaza - Nezavisna studija Amy Caruana (student na Kean Univ)
    • Amilaza - rezultati iz U. Sjeverne Dakote
    • Enzimi amilaza, temp, pH, koncentracija supstrata
    • Benediktov test za smanjenje šećera - slika rezultata
    • Amilaza Kako djeluje?
    • Amilaza - hidroliza škroba
    • Amilaze pljuvačke - pH
    Pepsin je proteaza koja započinje varenje proteina, razgrađujući ih u peptide i aminokiseline. Pepsinogen, luče ga želučane žlijezde želuca u želudac. Tamo, u kiseloj sredini želuca, pepsinogen se pretvara u pepsin.

    Iako su i pepsin i tripsin proteaze, oni zahtijevaju sasvim različite uvjete kiselosti i alkalnosti za svoje djelovanje.

      Pepsin Reference:
      • pepsin -
      • Pepsin - molekul mjeseca
      • pH optimalan -
      • Pepsin - navodi pH optimum
      Tripsin je proteaza koju pankreas izlučuje u tanko crijevo. Kao pepsin, tripsin razgrađuje proteine ​​u peptide i aminokiseline i stvara se i luči u neaktivnom obliku, tripsinogenu.

      Iako su i pepsin i tripsin proteaze, oni zahtijevaju sasvim različite uvjete kiselosti i alkalnosti za svoje djelovanje.

        Reference tripsina:
        • Opis enciklopedije tripsina
        • Ilustracija traži tripsin u ovoj velikoj datoteci
        • Slike Google pretraživač

        An Analogy

        Pretpostavimo da ste zainteresirani za kupovinu pizzerije i želite istražiti koliko je radnja produktivna, a da sadašnji vlasnik ne zna jer se bojite da će vlasnik podići cijenu. Dakle, umjesto da uđete u prodavnicu i gledate šta se događa i tražite da pregledate knjige koje bilježe troškove i profite, odlučite gledati radnju izvana.

        Promatrajte koliko često kamioni stižu s tijestom za pizzu, dodacima za pizzu (sir, peporoni, itd.) i drugim potrepštinama. Također promatrate koliko često radnici napuštaju radnju kako bi kupcima isporučili pizze.

        U ovoj analogiji, zalihe pizze su reaktanti, a pizze u kutijama koje se isporučuju kupcima su krajnji proizvodi. Radnici u radnji koji oblikuju tijesto, dodaju dodatke i stavljaju pizze u rerne i na kraju u kutije su ekvivalenti enzima.

        Iako zapravo ne vidimo da radnici rade svoj posao, možemo zaključiti da ako trgovina koristi velike količine reaktanata (tijesta i preljeva) i/ili pravi veliki broj krajnjih proizvoda (pica) koje radnici (enzimi) moraju budite veoma aktivni.


        Možda vam se također sviđa

        Beta amilaza se ne nalazi u ljudskom tkivu. irontoenail 8. januara 2013

        Zapravo sam čitao neki dan o tome kako postoje neke prirodne hemikalije koje vas mogu navesti da pomislite da je nešto slatko kada nije. To se dešava kada jedete artičoku, na primjer. Nakon toga mnoge stvari imaju slatki okus (kao što je čista voda, na primjer).

        Pokušavaju pronaći načine da ga koriste kao niskokalorični zaslađivač. Pitam se da li radi sa enzimima kao amilaza. Mada, sa amilazom pretpostavljam da zapravo pretvara skrob u šećer, gde hemikalije samo čine da stvari imaju ukus kao da imaju šećera u sebi. croydon 8. januara 2013

        @Iluviaporos - Zapravo, ne mislim da je ljudima zbog probave rečeno da više žvaću. Mislim, to može važiti za neke ljude, koji uopšte imaju problema sa varenjem, a možda i za hranu koja se ne vari lako, kao što je meso. Ne znam za to.

        Ali uvijek su mi govorili da ljudi trebaju više žvakati hranu jer to usporava cijeli proces jedenja i daje tijelu vremena da vam kaže da je zadovoljno. Uvijek sam mislio da ljudi jednostavno prestaju jesti kada im je želudac pun, ali kada mi je dijagnosticirana insulinska rezistencija, moj doktor je objasnio da tijelo zapravo odlučuje da je sito kada ima dovoljno šećera u krvi.

        Dakle, pretpostavljam da bi i puno žvakanja moglo pomoći u tome, jer bi ubrzalo probavu i omogućilo brži pad šećera u krvi. lluviaporos 7. januara 2013

        Amilaza je razlog zašto kažu da treba da pokušate da temeljno sažvakate hranu pre nego što je progutate. Pljuvačka nije tu samo da vam spriječi isušivanje usta, ona također pokreće probavni proces prije nego što vam hrana čak i dospije u želudac.

        Nekada su preporučivali da ljudi žvaću svaki zalogaj oko 40 puta prije nego što ga progutaju. Ne znam da li je to pravi iznos za koji bi to trebalo da uradite, ali većina ljudi to definitivno ne čini dovoljno.


        Rezultati

        Skrining izolata na aktivnost pektinaze

        Pektinaza ima mnoge primjene u raznim industrijskim procesima ([16] #1170 [17] #1311). Za skrining odličnih sojeva za proizvodnju pektinaze, neki izolati iz primarnog skrininga su tačkasto inokulirani na pektin agar ploče (PAP) u tri primjerka. Dodatna slika je to prikazana detaljnije [vidi Dodatni fajl 1: Slika S1]/(vidi Dodatni fajl 1). Vrijednost Hc od 33 za ovaj izolat bila je značajno viša od vrijednosti za druge izolate (8,87 ± 0,15, sl. 1). Ova bakterija je označena kao izolat B. tequilensis CAS-MEI-2-33, a izvršena je dalja karakterizacija i identifikacija.

        Hc vrijednosti pregledanih sojeva. Vrijednosti su date kao srednja vrijednost ± standardna devijacija (n = 3). Različita slova označavaju značajne razlike od 5%

        Morfološke karakteristike B. tequilensis CAS-MEI-2-33

        B. tequilensis CAS-MEI-2-33 je bila gram-pozitivna bakterija koja je sadržavala spore, a oblik bakterijskih ćelija bio je šiljast. Kada se uzgaja na 37 °C tokom 8–12 h u LB agaru, kolonije od B. tequilensis CAS-MEI-2-33 su bili okrugli, glatki i krem ​​boje, a margina je bila cijela.

        Biohemijska karakterizacija B. tequilensis CAS-MEI-2-33

        Biohemijska karakterizacija B. tequilensis CAS-MEI-2-33 je izveden korištenjem raznih testova, uključujući Voges-Proskauer testove, redukciju nitrata, iskorištenje glukoze, katalazu, pokretljivost, toleranciju lizozima, fenilalanin, želatin, skrob, laktozu, kazein i manitol. Rezultati testova na fenilalanin, skrob i manitol bili su negativni, dok su preostali rezultati bili pozitivni (tabela 1).

        Identifikacija zasnovana na filogenetskoj analizi

        Filogenetsko stablo generirano korištenjem sekvence gena 16S rRNA bakterijskog izolata pokazalo je najveću homologiju (100%) sa B. tequilensis. Nadalje, biohemijska karakterizacija sposobnosti CAS-MEI-2-33 da metabolizira skrob i manitol bila je drugačija od Bacillus subtilis ali sličan onom kod B. tequilensis. Prema Bergeyjevom priručniku za determinantnu bakteriologiju, ovaj soj je dobio ime B. tequilensis CAS-MEI-2-33 ([18] #1309). Konstruirano filogenetsko stablo pokazalo je da ovaj soj ima najbližu genetsku vezu sa B. tequilensis soj P12Pb (slika 2). Stablo je zaključeno metodom spajanja susjeda koristeći softver MEGA 7.0. Brojevi u čvorovima stabla su indikacije nivoa podrške za pokretanje bazirane na analizi spajanja susjeda od 1000 pretpostavljenih replika.

        Filogenetsko stablo zasnovano na analizi sekvence 16S ribosomalne RNK koja pokazuje položaj soja CAS-MEI-2-33 korišćenjem softvera MEGA 7.0. Brojevi u tačkama grananja odnose se na početničke vrijednosti (1000 resamplinga) sa 0,50 kao divergencijom niza

        Toksičnost nikotina za B. tequilensis CAS-MEI-2-33

        TS, sadrži nikotin, koji je štetan za mnoge bakterije, i služi kao poseban izvor ishrane. Prosječan sadržaj nikotina u TS je čak 1900–3800 mg/kg ([1] #1136), što je jednako 76–152 mg/L u mediju za fermentaciju. U ovoj studiji testirano je 500-2000 mg/L nikotina u mediju. Najveća koncentracija nikotina u medijumu za testiranje bila je približno 13 puta veća od koncentracije nikotina u medijumu TS fermentacije. Međutim, rast od B. tequilensis CAS-MEI-2-33 je bio manje pogođen u eksperimentalnim uslovima (slika 3).

        Rast od B. tequilensis CAS-MEI-2-33 pod različitim koncentracijama egzogenog nikotina. Vrijednosti su date kao srednja vrijednost ± standardna devijacija (n = 3)

        Proizvodnja pektinaze

        Proizvodnja visokih titara pektinaze optimizacijom parametara rasta je od primarne važnosti u industriji ([19] #1145). U ovoj studiji implementirana je metoda jedan faktor u vremenu kako bi se optimizirale komponente medija i uvjeti. Enzimska aktivnost je otkrivena upotrebom 3,5-dinitrosalicilne kiseline (DNS) reagensa (Beijing Leagene Biotech. Co., Ltd.) na osnovu proizvodnje D-galakturonske kiseline ([20] #1161). Krivulja za proizvodnju pektinaze tokom fermentacije B. tequilensis CAS-MEI-2-33 je bio ključan za indikaciju optimalne fermentacije. Aktivnost pektinaze se postepeno povećavala do 40 h, osim 32 h, a zatim je počela da opada (Sl. 4a). Najveća aktivnost pektinaze za proces fermentacije koristeći TS kao sirovinu bila je 293 U/mL na 40 h. Parametri kao što je pH fermentacionog medijuma imaju značajan uticaj na rast sojeva i proizvodnju pektinaze. Aktivnost pektinaze (618 ± 9 U/mL, slika 4b) bila je značajno povećana pri pH 7,0 u poređenju sa onim pod drugim uslovima.

        Aktivnost pektinaze B. tequilensis CAS-MEI-2-33 tokom fermentacije. a Aktivnost pektinaze tokom B. tequilensis CAS-MEI-2-33 rast. b Utjecaj početnog pH medija za fermentaciju na aktivnost enzima. c Utjecaj koncentracije stabljike duhana u mediju za fermentaciju na aktivnost enzima. d Utjecaj količine inokuluma na aktivnost enzima. Vrijednosti su date kao srednja vrijednost ± standardna devijacija (n = 3). Različita slova označavaju značajne razlike od 5%

        Nakon određivanja aktivnosti pektinaze sa različitim koncentracijama TS u mediju za fermentaciju, utvrđeno je da je pri koncentraciji od 40 g/L TS i optimiziranom pH, aktivnost pektinaze najveća (730 ± 38 U/mL, sl. 4c). ), uz podršku ANOVA (str = 0.05).

        Količina inokuluma je igrala ključnu ulogu u početnoj fermentaciji. Pod optimalnom koncentracijom pH i TS testirane su koncentracije inokuluma od 1, 3, 5, 7 i 9%. Najveća aktivnost pektinaze (1370 ± 126 U/mL, sl. 4d) sa 3% inokuluma nije se značajno razlikovala od one sa 5% inokuluma. Na višim nivoima, kao što su 7 i 9%, proizvodnja enzima je opala, što bi moglo biti posljedica nadmetanja za hranjive tvari među populacijom bakterija, kao što je uočeno u Thermomucor indicae-seudaticae ([21] #1230).

        Svojstva pektinaze

        Rezultati analize svojstava pektinaze prikazani su na slici 5. pH reakcionog sistema može uticati na aktivnost pektinaze. Aktivnost pektinaze se povećala kada je pH reakcionih sistema povećan sa 6,0 na 10,0, ali ova aktivnost je bila skoro neotkrivena na pH 11,0. Naši rezultati su pokazali da je aktivnost pektinaze od 791 ± 42 U/mL pri pH 10,0 bila najveća zabilježena vrijednost (slika 5a). Potom je istražen utjecaj temperature na aktivnost pektinaze. Aktivnost pektinaze se povećala kada je temperatura reakcije povećana sa 30 °C na 40 °C, dostigla maksimalnu aktivnost na 40 °C, a zatim se brzo smanjila kako je temperatura porasla iznad 40 °C. Aktivnost pektinaze na 40 °C bila je veća od one na drugim temperaturama (slika 5b). Dakle, pektinaza je bila aktivnija pri alkalnom pH i srednjoj temperaturi. Efekti različitih metalnih jona na aktivnost pektinaze prikazani su na slici 5c. Ioni Ag+, Li+, Cu 2+, Ca 2+, Ba 2+ i Mn 2+ posebno su povećali aktivnost enzima, a joni Ag+ su povećali aktivnost pektinaze za 193,95%, što je otprilike 1,94 puta više od one u kontroli . K+, Co 2+, Ni 2+, Mg 2+, Zn 2+, Cd 2+ i Fe 3+ joni, posebno Zn 2+, inhibirali su aktivnost pektinaze. Slika 5d prikazuje termičku stabilnost pektinaze koju proizvodi B. tequilensis CAS-MEI-2-33. Međutim, aktivnost pektinaze se smanjila kada je supernatant bez ćelija stavljen na 60 °C. Aktivnost pektinaze bila je stabilna kada je supernatant bez ćelija inkubiran na 40 °C, međutim, pektinaza nije mogla podnijeti visoku temperaturu dugo vremena.

        Enzimska svojstva pektinaze. a Utjecaj pH supstrata na aktivnost pektinaze. b Utjecaj temperature reakcije na aktivnost pektinaze. c Utjecaj metalnih jona na aktivnost pektinaze. d Temperaturna stabilnost pektinaze. Vrijednosti su date kao srednja vrijednost ± standardna devijacija (n = 3). Različita slova označavaju značajne razlike od 5%

        Djelomično prečišćavanje pektinaze iz CAS-MEI-2-33

        U optimalnim uslovima fermentacije, 2,30 L supernatanta je dobijeno centrifugiranjem bakterija u fermentacionoj tikvici od 3,0 L. Aktivnost pektinaze dostigla je 1771 U/mL, a ukupna aktivnost pektinaze 4,074,513 U (Tabela 2).

        Prema krivulji frakcioniranja amonijum sulfata, odabrana je odgovarajuća zasićenost za soljenje. Kriva frakcioniranja prikazana je na slici 6a. Kada je amonijum sulfat bio zasićen na 70-80%, pektinazna aktivnost precipitata se povećava, dok se supernatanta značajno smanjuje. Kada je stopa zasićenja dostigla 80-90%, aktivnosti pektinaze (pH 10,0, Gly-NaOH) precipitata i supernatanta nisu se značajno promijenile. Ciljni protein je otopljen sa 30 mL pufera (pH 8,0, Tris-HCl) nakon isoljavanja. Zatim je ciljni protein dalje renaturisan sa 55,0 mL pufera (pH 8,0, Tris-HCl) kroz vrećicu za dijalizu. Aktivnost pektinaze bila je 5166 U/mL, a ukupna aktivnost 284,144 U.

        Prečišćavanje alkalne pektinaze iz B. tequilensis CAS-MEI-2-33. a Kriva frakcioniranja amonijum sulfata b Krivulja eluiranja Mini Macro-Prep High-Q ionske izmjene hromatografije c. Kriva eluiranja Sephacryl S-100 kolonske hromatografije d. Djelomično prečišćavanje alkalne pektinaze iz B. tequilensis CAS-MEI-2-33 koristeći TS. M: kreatori molekularne težine 1. Sephacryl S-100 kolonska hromatografija sa pH 7,2 2: Mini Macro-Prep High-Q ionska izmjenjivačka hromatografija sa pH 8,0 3. Koncentrirani rastvor amonijum sulfata za ispuštanje soli

        Nakon dijalize, supernatant je pročišćen Mini Macro-Prep High-Q kolonom za ionsku izmjenu. Rezultati su prikazani na slici 6b. Pektinaza je eluirana sa pH 8,0 Tris-HCl puferom, a kada je kolona gradijentno eluirana sa pH 8,0 Tris-HCl puferom koji sadrži 0-1 mol/L NaCl, pikovi su eluirani. Otkrivanjem aktivnosti pektinaze utvrđeno je da je bio aktivan prvi eluacioni vrh. Prvi eluacioni vrh je sakupljen sa aktivnošću pektinaze od 862,352 U/mL. Ukupna aktivnost je bila 77,611 U. Sephacryl S-100 je uravnotežen sa ultra čistom vodom, uzorkovana i zatim eluirana sa pH 7,2 PBS puferom, sa brzinom protoka od 0,8 mL/min, a efluent je detektovan onlajn UV detektorom na talasne dužine od 280 nm za snimanje vršne krivulje ultraljubičaste apsorpcije (slika 6c). Komponente su sakupljene i korištene za određivanje aktivnosti enzima i sadržaja proteina. Aktivnost pektinaze bila je 13.786 U/mL, a ukupna aktivnost 41.357 U.

        Molekularna težina pročišćene alkalne pektinaze je detektirana elektroforezom u SDS-poliakrilamidnom gelu (PAGE) kako su opisali Mehrnoush et al. ([22] #1306). Molekularna težina pektinaze, koja je bila približno 45,4 kDa, prikazana je na slici 6d. Zatim je proteinska traka izrezana iz SDS-PAGE gela, podvrgnuta LC-MS/MS analizi od strane Shanghai Applied Protein Technology ([23] #1320 [24] #1321) i identificirana pretraživanjem UniProt baze podataka. Dodatna slika o vrhuncu proteinske baze je to prikazana detaljnije [pogledajte Dodatni fajl 2: Slika S2]/(vidi Dodatni fajl 2 za proteinski vrh). Konačno, protein je identifikovan kao pektat liaza, sekvenca je bila K.ASSSNVYTVSNR.N (slika 7). Molekularna težina je bila 45,4 kDa, što je bilo u skladu sa rezultatima SDS-PAGE. Rezultati su pokazali da je enzim bio dobar kandidat za pektat liazu. Nadalje, ova studija je bila novi pokušaj recikliranja i ponovne upotrebe TS u poljoprivrednoj proizvodnji.

        LC-MS/MS analiza proteinskih traka pomoću SDS-PAGE


        Alkalifili

        Na drugom kraju spektra su alkalifili, mikroorganizmi koji imaju pH optimum između 8,0 i 11. Vibrio cholerae , patogeni uzročnik kolere, najbolje raste pri blago bazičnom pH od 8,0, može preživjeti pH vrijednosti od 11,0, ali ga inaktivira želučana kiselina. Kada je u pitanju preživljavanje pri visokom pH, jarko ružičasti halofilni arheon Natronobacterium , pronađen u jezerima soda u Afričkoj dolini Rift, može držati rekord na pH od 10,5 (Sl. 9.37). Ekstremni alkalifili su se prilagodili svom surovom okruženju kroz različite evolucijske modifikacije. Alkalifilne arheje imaju dieter lipidne membrane. Eterska veza je otpornija na hemijsku ili termičku degradaciju u poređenju sa esterskim vezanim fosfolipidima. S obzirom na nedostatak protona u alkalnim sredinama, održavanje protonske pokretačke sile je vjerovatno najhitniji izazov za alkalfile. Jedna od adaptacija alkalifilnih halofilnih bakterija i arheja u jezerima soda i drugim visoko slanim sredinama je evolucija spregnutih transportera i flagela koji iskorištavaju natrijevu pokretačku silu, čime se čuva PMF za oksidativnu i fotofosforilaciju pomoću ATP sintaze. Stanična površina alkalifila ima visoku koncentraciju kiselih (tj. negativno nabijenih) molekula i sugerirano je da djeluje kao "protonski sunđer", omogućavajući bržu bočnu difuziju protona iz ETS-a do ATP sintaze, u poređenju sa brzina difuzije u okolne vode [1] Konačno, alkalofili mogu koristiti Na + /H + antiport da stvore natrijumovu pokretačku silu. Na primjer, alkalifil Bacillus firmus dobija energiju za transportne reakcije i pokretljivost iz SMF-a, a ne iz protonske pokretačke sile. Kao i kod acidofila, geni za izlučene proteine ​​alkalifila su evoluirali da daju enzime koji se odupiru deprotonaciji/denaturaciji i hemijskoj degradaciji pri visokom pH njihove okoline. Ovi enzimi su takođe od interesa za biotehnološke kompanije. Zapravo, deterdženti za pranje rublja, koji su alkalne prirode, sadrže alkalifilne lipaze i proteaze kako bi poboljšali njihovu sposobnost uklanjanja mrlja.

        Slika 9.37. Pogled iz svemira na jezero Natron u Tanzaniji. Ružičasta boja nastaje zbog pigmentacije ekstremno alkalifilnih i halofilnih mikroba koji koloniziraju jezero. [Kredit: NASA]


        Kakav je očekivani učinak pH na aktivnost gljivične pektinaze? - Biologija

        Brzina reakcije na jetru, jabuke i krompir s vodikovim peroksidom

        Svrha ove laboratorije bila je testiranje funkcije enzima u različitim okruženjima. Koncentracija supstrata, temperatura i pH utiču na hemijsku reakciju. U ovoj laboratoriji, enzim katalaza je korišten za razgradnju vodikovog peroksida u manje toksičnu vodu i plin kisika. Koristeći kvantitativno i kvalitativno posmatranje, koncept da enzimi ostaju nakon reakcije potvrđen je od prvog testa. Nakon testiranja jetre, jabuke i krompira zaključeno je da jetra sadrži najviše katalaze. Poslednji test se fokusirao na brzinu reakcije jetre u različitim pH rastvorima. Pokazalo se da je odnos između brzine reakcije katalaze i pH paraboličan sa vrhom blizu neutralnog.

        Ova laboratorija pokrivala je mjerenje efekata promjena temperature, pH i koncentracije enzima na brzine reakcije enzimski katalizirane reakcije u kontroliranom eksperimentu. U ovoj laboratoriji je odgovoreno na pitanja kako faktori okoline utiču na brzinu enzimski kataliziranih reakcija. Na brzinu reakcije enzima uvelike su utjecale promjene temperature, pH i koncentracije enzima. Enzim koji je proučavan u ovoj laboratoriji bila je katalaza. Katalaza razlaže vodikov peroksid, koji je toksičan, na 2 sigurne supstance - vodu i kiseonik, ubrzavajući reakciju. Enzimi poput katalaze su od vitalnog značaja za naše tijelo, jer da se otrovne tvari poput vodikovog peroksida ne razgrađuju u bezopasne tvari u našem tijelu, onda bi otrovale naše stanice. Reakcija koja se odvija je u ovom obliku: 2H2O2 ----> 2H2O + O2


        Metode
        Ova studija je sprovedena u New Tech High @ Coppell pod fasilitacijom gospođe Wootton 8. oktobra 2015. U tri dela laboratorije proučavana je sposobnost razgradnje enzima. U Dijelu A koristili smo 3 ml 3% vodikovog peroksida na komadić jetre da bismo promatrali brzinu reakcije. Kada je to urađeno, uzeli smo ostatke tečnosti i koristili ih na novom komadu mesa da vidimo da li je enzim za višekratnu upotrebu. U dijelu B testirali smo, promatrali i bilježili brzinu reakcije s vodikovim peroksidom na tri supstance: jabuku, jetru i krompir. U dijelu C, promijenili smo pH otopine dodavanjem kapi razrijeđene hlorovodonične kiseline da vidimo da li ona utiče na brzinu reakcije u katalizama na komadićima jetre.

        Kada se supstrat vodikovog peroksida doda supstancama, svaka je izvršila potpuno drugačiju reakciju. Nakon ispuštanja vodikovog peroksida na uzorke jabuke nije bilo naznačene reakcije, pa sam dobio ocjenu 0. Nakon što je ista količina supstrata dodana u krompir, došlo je do blage gazirane i pjenušave reakcije, čime je dobila ocjenu 2. Jetra katalaza je zatim testirana i rezultirala je najvećom reakcijom koja je zbog brzine i veličine dobila ocjenu 5. Nakon testiranja svake supstance, grupa je počela da traži optimalne uslove pH za reakcije. Nakon dodavanja hlorovodonične kiseline u jetru, činilo se da se brzina reakcije smanjuje kako se kiselost povećava.

        Supstrat: vodikov peroksid

        Gledajući naše podatke za prvi eksperiment, možemo zaključiti da jetra sadrži enzim katalaze, dok je vodikov peroksid supstrat. To je tačno jer je u reakciji uočeno da je vodikov peroksid prošao kroz kemijsku promjenu pretvarajući se u molekule vode i molekule dioksida, dok je jetra ostala ista. Budući da je supstrat već prošao kemijsku reakciju, nije mogao proći kroz drugu s novom katalazom, ali stara katalaza je još uvijek imala funkcionalne enzime i stoga se mogla ponovno koristiti. Reakcija je bila vrlo očigledna i brza, što ima smisla s obzirom da je funkcija jetre u organizmu da razgrađuje i pročišćava stanice.

        U drugom dijelu eksperimenta, intenzitet reakcije u rastućem redoslijedu bio je jabuka, krompir i jetra. Uzorak jabuke je jednostavno ugljikohidrat koji je namijenjen kao hrana za sjemenke jabuke i neće imati enzime u sebi, tako da nije uočena reakcija. Krompir će imati poluintenzivnu reakciju jer samo mala koncentracija uzorka krompira čine enzimi, dok ostatak čine škrob i organska jedinjenja. Jetra će imati jaku reakciju jer većinu jetre čine proteini i enzimi koji razgrađuju vodikov peroksid.

        U trećem eksperimentu zaključeno je da se povećanjem kiselosti u supstratu brzina reakcije smanjuje. Naši podaci pokazuju linearni trend, međutim, istraživanja pokazuju da je trend u stvari parabola koja se otvara prema dolje s maksimumom pri pH od 5, kiselosti samog vodikovog peroksida. Budući da smo bili u mogućnosti stimulirati samo okruženje nižeg pH, uspjeli smo dobiti samo 1 smjer nagiba, pokazujući linearne rezultate. Ovo pokazuje da enzimi rade samo na određenom pH nivou. Ako je previsok ili prenizak, to će smanjiti efikasnost i usporiti brzinu reakcija.

        Kroz naše eksperimente ispitana je reakcija između enzima katalaze i vodikovog peroksida. Ponavljanje reakcije s različitim materijalima omogućilo je da se zaključi koncentracija katalaze. Koristeći jetru kao našu kontrolu, mijenjali smo pH otopine u svakom ispitivanju kako bismo testirali brzinu reakcije kako se kiselost povećava. Zaključili smo da kako se pH otopine povećavao, brzina reakcije se povećavala inverzno do pH od oko 5. Greška u našem eksperimentu samo je promijenila specifični pH, ali ukupni rezultat posljednjeg testa ostaje istinit.


        Kostimulacija aktivnosti glikozidaze u tlu i respiracije tla dodavanjem dušika

        Centar za istraživanje kineske mreže za istraživanje ekosistema, Ključna laboratorija za posmatranje i modeliranje mreže ekosistema, Institut za geografske nauke i istraživanje prirodnih resursa, Kineska akademija nauka, Peking, 100101 Kina

        Odsjek za mikrobiologiju i biologiju biljaka, Univerzitet Oklahoma, Norman, OK, 73019 SAD

        Odeljenje za poljoprivredu i nauke o životnoj sredini, Tennessee State University, Nashville, TN, 37209 SAD

        Tiantong Nacionalna terenska posmatračka stanica za šumski ekosistem, Škola ekoloških nauka i nauka o životnoj sredini, East China Normal University, Šangaj, 200062 Kina

        Centar za ekološke nauke i nauke o životnoj sredini, Severozapadni politehnički univerzitet, Xi'an, 710072 Kina

        Državna ključna laboratorija za lesnu i kvartarnu geologiju (SKLLQG), Ključna laboratorija za hemiju i fiziku aerosola, Institut za zemaljsko okruženje, Kineska akademija nauka, Xi'an, 710061 Kina

        Univerzitet Kineske akademije nauka, Peking, 100049 Kina

        Odsjek za mikrobiologiju i biologiju biljaka, Univerzitet Oklahoma, Norman, OK, 73019 SAD

        Odsjek za mikrobiologiju i biologiju biljaka, Univerzitet Oklahoma, Norman, OK, 73019 SAD

        Centar za nauku o Zemljinom sistemu, Univerzitet Tsinghua, Peking, 100084 Kina

        Odeljenje za poljoprivredu i nauke o životnoj sredini, Tennessee State University, Nashville, TN, 37209 SAD

        Dopisivanje: Junji Cao, tel. +86 29 62336233, faks +86 29 62336234, e-mail: [email protected]

        Jianwei Li, tel. +1 615 963 1523, faks +1 615 963 1523, e-mail: [email protected]

        Rui-Wu Wang, tel. +86 29 88460816, faks +86 29 88460816, e-mail: [email protected]

        Tiantong Nacionalna terenska posmatračka stanica za šumski ekosistem, Škola ekoloških nauka i nauka o životnoj sredini, East China Normal University, Šangaj, 200062 Kina

        Centar za globalne promjene i ekološko predviđanje, East China Normal University, Šangaj, 200062 Kina

        Državna ključna laboratorija za lesnu i kvartarnu geologiju (SKLLQG), Ključna laboratorija za hemiju i fiziku aerosola, Institut za Zemljinu okolinu, Kineska akademija nauka, Xi'an, 710061 Kina

        Institut za globalne promjene okoliša, Xi'an Jiaotong Univerzitet, Xi'an, 710049 Kina

        Dopisivanje: Junji Cao, tel. +86 29 62336233, faks +86 29 62336234, e-mail: [email protected]

        Jianwei Li, tel. +1 615 963 1523, faks +1 615 963 1523, e-mail: [email protected]

        Rui-Wu Wang, tel. +86 29 88460816, faks +86 29 88460816, e-mail: [email protected]

        Centar za ekološke nauke i nauke o životnoj sredini, Severozapadni politehnički univerzitet, Xi'an, 710072 Kina

        Dopisivanje: Junji Cao, tel. +86 29 62336233, faks +86 29 62336234, e-mail: [email protected]

        Jianwei Li, tel. +1 615 963 1523, faks +1 615 963 1523, e-mail: [email protected]

        Rui-Wu Wang, tel. +86 29 88460816, faks +86 29 88460816, e-mail: [email protected]

        Državna ključna laboratorija za lesnu i kvartarnu geologiju (SKLLQG), Ključna laboratorija za hemiju i fiziku aerosola, Institut za Zemljinu okolinu, Kineska akademija nauka, Xi'an, 710061 Kina

        Odsjek za mikrobiologiju i biologiju biljaka, Univerzitet Oklahoma, Norman, OK, 73019 SAD

        Državna ključna laboratorija za lesnu i kvartarnu geologiju (SKLLQG), Ključna laboratorija za hemiju i fiziku aerosola, Institut za Zemljinu okolinu, Kineska akademija nauka, Xi'an, 710061 Kina

        Odeljenje za biološke nauke, Univerzitet Arkanzas, Fayetteville, AR, 72701 SAD

        Državna ključna laboratorija za urbanu i regionalnu ekologiju, Istraživački centar za nauke o eko-ekologiji, Kineska akademija nauka, Peking, 100085 Kina

        Centar za istraživanje kineske mreže za istraživanje ekosistema, Ključna laboratorija za posmatranje i modeliranje mreže ekosistema, Institut za geografske nauke i istraživanje prirodnih resursa, Kineska akademija nauka, Peking, 100101 Kina

        Odsjek za mikrobiologiju i biologiju biljaka, Univerzitet Oklahoma, Norman, OK, 73019 SAD

        Odeljenje za poljoprivredu i nauke o životnoj sredini, Tennessee State University, Nashville, TN, 37209 SAD

        Tiantong Nacionalna terenska posmatračka stanica za šumski ekosistem, Škola ekoloških nauka i nauka o životnoj sredini, East China Normal University, Šangaj, 200062 Kina

        Abstract

        Nivoi azota (N) bez presedana taloženi su u ekosistemima tokom prošlog veka, za koje se očekuje da će imati kaskadne efekte na mikrobno posredovano disanje tla (SR). Ekstracelularni enzimi igraju ključnu ulogu u degradaciji organske materije u tlu, a mjerenja njihovih aktivnosti potencijalno su korisni pokazatelji SR. Međutim, veze između ekstracelularnih enzimskih aktivnosti tla (EEA) i SR uz dodatak N nisu utvrđene. Stoga smo sproveli meta-analizu iz 62 publikacije kako bismo sintetizirali odgovore EEA tla i SR na povišeni N. Dodatak dušika značajno je povećao aktivnost glikozidaze (GA) za 13,0%. α-1,4-glukozidaza (AG) za 19,6%, β-1,4-glukozidaza (BG) za 11,1%, β-1,4-ksilozidaza (BX) za 21,9% i β-D-celobiozidaza (CBH) za 12,6%. Povećanja GA su bila očiglednija za dugotrajne, visoke stope, organske i mješovite dodatke N (kombinacija organskog i neorganskog dodatka N), kao i za studije na poljoprivrednom zemljištu. Omjeri odgovora (RR) GA su bili u pozitivnoj korelaciji sa SR-RR, čak i kada su ocijenjeni pojedinačno za AG, BG, BX i CBH. Ova pozitivna korelacija između GA-RR i SR-RR održana je za većinu vrsta vegetacije i tla kao i za različite metode dodavanja N. Naši rezultati pružaju prve dokaze da je GA povezan sa SR pod N dodatkom u nizu ekosistema i naglašavaju potrebu za daljim studijama o odgovoru drugih EEA tla na različite faktore globalne promjene i njihove implikacije na funkcije ekosistema.

        Dodatak S1 Dodatne napomene.

        Tabela S1 Rezultati za pristrasnost objavljivanja.

        Tabela S2 Opis 12 vrsta enzima uključenih u našu preliminarnu analizu.

        Tabela S3 Distribucija metoda dodavanja dušika za različite vrste vegetacije i tla.

        Slika S1 Globalna distribucija eksperimenata sa adicijom dušika odabranih u ovoj meta-analizi. Mapa je kreirana pomoću ArcGIS-a.

        Slika S2 Frekvencijska distribucija omjera odziva (RR) za (a) α-1,4-glukozidaza (AG), (b) β-1,4-glukozidaza (BG), (c) β-D-celobiozidaza (CBH) i (d) β-1,4-ksilozidaza (BX).

        Slika S3 Odnosi između omjera odgovora (RR) disanja tla (SR) i RR-a (a) α-1,4-glukozidaza (AG), (b) β-1,4-glukozidaza (BG), (c) β-D-celobiozidaza (CBH), (d) β-1,4-ksilozidaza (BX), (e) fenol oksidaza (PO), (f) polifenol oksidaza (PHO), (g) invertaza, (h) ureaza, (i) peroksidaza (PER), (j) β-1,4-N-acetilglukozaminidaza (NAG), (k) kisela (alkalna) fosfataza (AP) i (l) leucin amino peptidaza (LAP).

        Slika S4 Odnosi između omjera odgovora (RR) aktivnosti glikozidaze u tlu i (a) brzine dodavanja N, (b) trajanja dodavanja N, (c) učestalosti dodavanja N i (d) veličine uzorka.

        Slika S5 Odnosi između omjera odgovora (RR) aktivnosti glikozidaze i RR disanja tla (SR) za različite metode mjerenja SR.

        Slika S6 Efekti dodavanja N na disanje tla iz prethodnih meta-analiza. Trake grešaka predstavljaju 95% intervale pouzdanosti (CI). Efekat dodavanja N se smatra značajnim ako se CI veličine efekta ne preklapa sa nulom. Veličina uzorka za svaku varijablu je prikazana pored CI. Ova brojka je ponovo nacrtana iz prethodnih meta-analiza koje je objavio (a, b i c) Zhou et al. 2014, (d) Liu et al. 2010 , (e) Lu et al. 2011. i (f) Janssens et al. 2010 . Ra, autotrofno disanje Rh, heterotrofno disanje SR, disanje tla.

        Slika S7 Odnosi između mogućih promjena u mikrobnim zajednicama i fiziologije i omjera odgovora (RR) aktivnosti glikozidaze (a) obilja mikroba, (b) obilja bakterija, (c) obilja gljivica, (d) gljiva/bakterija, (e) mikrobnog ugljenik biomase (MBC), (f) azot mikrobne biomase (MBN) i (g) MBC/MBN. Treseder je sintetizirao odnose između promjena u mikrobnim zajednicama i fiziologije izazvanih dodavanjem N i njihove veze s odgovarajućim promjenama u disanju tla. et al. ( 2008 ).

        Slika S8 (a) Efekti dodavanja N na aktivnosti enzima koji poseduju oksidativni C u tlu. Raspodjela frekvencija omjera odgovora (RR) (b) oksidativnih enzima, (c) fenol oksidaze (PO), (d) peroksidaze (PER) i (e) polifenol oksidaze (PHO). Trake grešaka predstavljaju 95% intervale pouzdanosti (CI). Efekat dodavanja N se smatra značajnim ako se CI veličine efekta ne preklapa sa nulom. Veličina uzorka za svaku varijablu je prikazana pored CI. QB i Qw definirani su u odjeljku Materijali i metode.

        Slika S9 Relationships between the response ratio (RR) of glycosidase activity and the (a) RR of soil total nitrogen (STN), (b) RR of dissolved organic nitrogen (DON), (c) RR of the substrate C:N ratio and (d) substrate C:N ratio.

        Figure S10 Relationships between the response ratio (RR) of glycosidase activity and (a) the substrate pH and (b) the RR of the substrate pH.

        Napomena: Izdavač nije odgovoran za sadržaj ili funkcionalnost bilo koje prateće informacije koju su dali autori. Sve upite (osim sadržaja koji nedostaje) treba uputiti odgovarajućem autoru za članak.


        Sažetak

        Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off. The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of an enzyme. At low temperatures, an increase in temperature increases the rate of an enzyme-catalyzed reaction. At higher temperatures, the protein is denatured, and the rate of the reaction dramatically decreases. An enzyme has an optimum pH range in which it exhibits maximum activity.


        Immobilization of Enzymes and Cells: Methods, Effects and Applications

        Traditionally, enzymes in free solutions (i.e. in soluble or free form) react with substrates to result in products. Such use of enzymes is wasteful, particularly for industrial purposes, since enzymes are not stable, and they cannot be recovered for reuse.

        Immobilization of enzymes (or cells) refers to the technique of confining/anchoring the enzymes (or cells) in or on an inert support for their stability and functional reuse. By employing this technique, enzymes are made more efficient and cost-effective for their industrial use. Some workers regard immobilization as a goose with a golden egg in enzyme technology. Immobilized enzymes retain their structural conformation necessary for catalysis.

        There are several advantages of immobilized enzymes:

        a. Stable and more efficient in function.

        b. Can be reused again and again.

        c. Products are enzyme-free.

        d. Ideal for multi-enzyme reaction systems.

        e. Control of enzyme function is easy.

        f. Suitable for industrial and medical use.

        g. Minimize effluent disposal problems.

        There are however, certain disadvantages also associated with immobilization.

        a. The possibility of loss of biological activity of an enzyme during immobilization or while it is in use.

        b. Immobilization is an expensive affair often requiring sophisticated equipment.

        Immobilized enzymes are generally preferred over immobilized cells due to specificity to yield the products in pure form. However, there are several advantages of using immobilized multi-enzyme systems such as organelles and whole cells over immobilized enzymes. The immobilized cells possess the natural environment with cofactor availability (and also its regeneration capability) and are particularly suitable for multiple enzymatic reactions.

        Methods of Immobilization:

        The commonly employed techniques for immobilization of enzymes are—adsorption, entrapment, covalent binding and cross-linking.

        Adsorption:

        Adsorption involves the physical binding of enzymes (or cells) on the surface of an inert support. The support materials may be inorganic (e.g. alumina, silica gel, calcium phosphate gel, glass) or organic (starch, carboxymethyl cellulose, DEAE-cellulose, DEAE-sephadex).

        Adsorption of enzyme molecules (on the inert support) involves weak forces such as van der Waals forces and hydrogen bonds (Fig. 21.3). Therefore, the adsorbed enzymes can be easily removed by minor changes in pH, ionic strength or temperature. This is a disadvantage for industrial use of enzymes.

        Entrapment:

        Enzymes can be immobilized by physical entrapment inside a polymer or a gel matrix. The size of the matrix pores is such that the enzyme is retained while the substrate and product molecules pass through. In this technique, commonly referred to as lattice entrapment, the enzyme (or cell) is not subjected to strong binding forces and structural distortions.

        Some deactivation may however, occur during immobilization process due to changes in pH or temperature or addition of solvents. The matrices used for entrapping of enzymes include polyacrylamide gel, collagen, gelatin, starch, cellulose, silicone and rubber. Enzymes can be entrapped by several ways.

        1. Enzyme inclusion in gels:

        This is an entrapment of enzymes inside the gels (Fig. 21.4A).

        2. Enzyme inclusion in fibres:

        The enzymes are trapped in a fibre format of the matrix (Fig. 21.4B).

        3. Enzyme inclusion in microcapsules:

        In this case, the enzymes are trapped inside a microcapsule matrix (Fig. 21.4C). The hydrophobic and hydrophilic forms of the matrix polymerise to form a microcapsule containing enzyme molecules inside. The major limitation for entrapment of enzymes is their leakage from the matrix. Most workers prefer to use the technique of entrapment for immobilization of whole cells. Entrapped cells are in use for industrial production of amino acids (L-isoleucine, L-aspartic acid), L-malic acid and hydroquinone.

        Microencapsulation:

        Microencapsulation is a type of entrapment. It refers to the process of spherical particle formation wherein a liquid or suspension is enclosed in a semipermeable membrane. The membrane may be polymeric, lipoidal, lipoprotein-based or non-ionic in nature. There are three distinct ways of microencapsulation.

        1. Building of special membrane reactors.

        3. Stabilization of emulsions to form microcapsules.

        Microencapsulation is recently being used for immobilization of enzymes and mammalian cells. For instance, pancreatic cells grown in cultures can be immobilized by microencapsulation. Hybridoma cells have also been immobilized successfully by this technique.

        Covalent Binding:

        Immobilization of the enzymes can be achieved by creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support (Fig. 21.5). This technique is widely used. However, covalent binding is often associated with loss of some enzyme activity. The inert support usually requires pretreatment (to form pre-activated support) before it binds to enzyme. The following are the common methods of covalent binding.

        1. Cyanogen bromide activation:

        The inert support materials (cellulose, sepharose, sephadex) containing glycol groups are activated by CNBr, which then bind to enzymes and immobilize them (Fig. 21.6A).

        Some of the support materials (amino benzyl cellulose, amino derivatives of polystyrene, aminosilanized porous glass) are subjected to diazotation on treatment with NaNO2 and HCI. They, in turn, bind covalently to tyrosyl or histidyl groups of enzymes (Fig. 21.6B).

        3. Peptide bond formation:

        Enzyme immobi­lization can also be achieved by the formation of peptide bonds between the amino (or carboxyl) groups of the support and the carboxyl (or amino) groups of enzymes (Fig. 21.6C). The support material is first chemically treated to form active functional groups.

        4. Activation by bi- or poly-functional reagents:

        Some of the reagents such as glutaraldehyde can be used to create bonds between amino groups of enzymes and amino groups of support (e.g. aminoethylcellulose, albumin, amino alkylated porous glass). This is depicted in Fig. 21.6D.

        Cross-Linking:

        The absence of a solid support is a characteristic feature of immobilization of enzymes by cross- linking. The enzyme molecules are immobilized by creating cross-links between them, through the involvement of poly-functional reagents. These reagents in fact react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules (Fig. 21.7). There are several reagents in use for cross-linking. These include glutaraldehyde, diazobenzidine, hexamethylene diisocyanate and toluene di- isothiocyanate.

        Glutaraldehyde is the most extensively used cross-linking reagent. It reacts with lysyl residues of the enzymes and forms a Schiff’s base. The cross links formed between the enzyme and glutaraldehyde are irreversible and can withstand extreme pH and temperature. Glutaraldehyde cross- linking has been successfully used to immobilize several industrial enzymes e.g. glucose isomerase, penicillin amidase. The technique of cross-linking is quite simple and cost-effective. But the disadvantage is that it involves the risk of denaturation of the enzyme by the poly-functional reagent.

        Choice of Immobilization Technique:

        The selection of a particular method for immobilization of enzymes is based on a trial and error approach to choose the ideal one. Among the factors that decide a technique, the enzyme catalytic activity, stability, regenerability and cost factor are important.

        Immobilization of L-amino acid acylase:

        L-Amino acid acylase was the first enzyme to be immobilized by a group of Japanese workers (Chibata and Tosa, 1969). More than 40 different immobilization methods were attempted by this group. Only three of them were found be useful. They were covalent binding to iodoacetyl cellulose, ionic binding to DEAE-Sephadex and entrapment within polyacrylamide.

        Stabilization of Soluble Enzymes:

        Some of the enzymes cannot be immobilized and they have to be used in soluble form e.g. enzymes used in liquid detergents, some diagnostic reagents and food additives. Such enzymes can be stabilized by using certain additives or by chemical modifications. The stabilized enzymes have longer half-lives, although they cannot be recycled. Some important methods of enzyme stabilization are briefly described.

        Solvent Stabilization:

        Certain solvents at low concentrations stabilize the enzymes, while at high concentrations the enzymes get denatured e.g. acetone (5%) and ethanol (5%) can stabilize benzyl alcohol dehydro­genase.

        Substrate Stabilization:

        The active site of an enzyme can be stabilized by adding substrates e.g. starch stabilizes a-amylase glucose stabilizes glucose isomerase.

        Stabilization by Polymers:

        Enzymes can be stabilized, particularly against increased temperature, by addition of polymers such as gelatin, albumin and polyethylene glycol.

        Stabilization by Salts:

        Stability of metalloenzymes can be achieved by adding salts such as Ca, Fe, Mn, Cu and Zn e.g. proteases can be stabilized by adding calcium.

        Stabilization by Chemical Modifications:

        Enzymes can be stabilized by suitable chemical modifications without loss of biological activity. There are several types of chemical modifications.

        a. Addition of poly-amino side chains e.g. polytyrosine, polyglycine.

        b. Acylation of enzymes by adding groups such as acetyl, propionyl and succinyl.

        Stabilization by Rebuilding:

        Theoretically, the stability of the enzymes is due to hydrophobic interactions in the core of the enzyme. It is therefore, proposed that enzymes can be stabilized by enhancing hydrophobic interactions. For this purpose, the enzyme is first unfold and then rebuilt in one of the following ways (Fig. 21.8).

        1. The enzyme can be chemically treated (e.g. urea and a disulfide) and then refolded.

        2. The refolding can be done in the presence of low molecular weight ligands.

        3. For certain enzymes, refolding at higher temperatures (around 50°C) stabilize them.

        Stabilization by Site-Directed Mutagenesis:

        Site-directed mutagenesis has been successfully used to produce more stable and functionally more efficient enzymes e.g. subtilisin E.

        Immobilization of Cells:

        Immobilized individual enzymes can be successfully used for single-step reactions. They are, however, not suitable for multi-enzyme reactions and for the reactions requiring cofactors. The whole cells or cellular organelles can be immobilized to serve as multi-enzyme systems. In addition, immobilized cells rather than enzymes are sometimes preferred even for single reactions, due to cost factor in isolating enzymes. For the enzymes which depend on the special arrangement of the membrane, cell immobilization is preferred.

        Immobilized cells have been traditionally used for the treatment of sewage. The techniques employed for immobilization of cells are almost the same as that used for immobilization of enzymes with appropriate modifications. Entrapment and surface attachment techniques are commonly used. Gels, and to some extent membranes, are also employed.

        Immobilized Viable Cells:

        The viability of the cells can be preserved by mild immobilization. Such immobilized cells are particularly useful for fermentations. Sometimes mammalian cell cultures are made to function as immobilized viable cells.

        Immobilized Non-viable Cells:

        In many instances, immobilized non-viable cells are preferred over the enzymes or even the viable cells. This is mainly because of the costly isolation and purification processes. The best example is the immobilization of cells containing glucose isomerase for the industrial production of high fructose syrup. Other important examples of microbial biocatalysts and their applications are given in Table 21.5.

        Limitations of Immobilizing Eukaryotic Cells:

        Prokaryotic cells (particularly bacterial) are mainly used for immobilization. It is also possible to immobilize eukaryotic plant and animal cells. Due to the presence of cellular organelles, the metabolism of eukaryotic cells is slow. Thus, for the industrial production of biochemical, prokaryotic cells are preferred. However, for the production of complex proteins (e.g. immunoglobulin’s) and for the proteins that undergo post- translational modifications, eukaryotic cells may be used.

        Effect of Immobilization on Enzyme Properties:

        Enzyme immobilization is frequently associated with alterations in enzyme properties, particularly the kinetic properties of enzymes.

        Some of them are listed below:

        1. There is a substantial decrease in the enzyme specificity. This may be due to conformational changes that occur when the enzyme gets immobilized.

        2. The kinetic constants Km and Vmax of an immobilized enzyme differ from that of the native enzyme. This is because the conformational change of the enzyme will affect the affinity between enzyme and substrate.

        Immobilized Enzyme Reactors:

        The immobilized enzymes cells are utilized in the industrial processes in the form of enzyme reactors. They are broadly of two types — batch reactors and continuous reactors. The frequently used enzyme reactors are shown in Fig. 21.9.

        Batch Reactors:

        In batch reactors, the immobilized enzymes and substrates are placed, and the reaction is allowed to take place under constant stirring. As the reaction is completed, the product is separated from the enzyme (usually by denaturation).

        Soluble enzymes are commonly used in batch reactors. It is rather difficult to separate the soluble enzymes from the products hence there is a limitation of their reuse. However, special techniques have been developed for recovery of soluble enzymes, although this may result in loss of enzyme activity.

        Stirred tank reactors:

        The simplest form of batch reactor is the stirred tank reactor (Fig. 21.9A). It is composed of a reactor fitted with a stirrer that allows good mixing, and appropriate temperature and pH control. However, there may occur loss of some enzyme activity. A modification of stirred tank reactor is basket reactor. In this system, the enzyme is retained over the impeller blades. Both stirred tank reactor and basket reactor have a well-mixed flow pattern.

        Plug flow type reactors:

        These reactors are alternatives to flow pattern type of reactors. The flow rate of fluids controlled by a plug system. The plug flow type reactors may be in the form of packed bed or fluidized bed (Fig. 21.9B and 21.9C). These reactors are particularly useful when there occurs inadequate product formation in flow type reactors. Further, plug flow reactors are also useful for obtaining kinetic data on the reaction systems.

        Continuous Reactors:

        In continuous enzyme reactors, the substrate is added continuously while the product is removed simultaneously. Immobilized enzymes can also be used for continuous operation. Continuous reactors have certain advantages over batch reactors. These include control over the product formation, convenient operation of the system and easy automation of the entire process. There are mainly two types of continuous reactors-continuous stirred tank reactor (CSTR) and plug reactor (PR). A diagrammatic representation of CSTR is depicted in Fig. 21.9D. CSTR is ideal for good product formation.

        Membrane Reactors:

        Several membranes with a variety of chemical compositions can be used. The commonly used membrane materials include polysulfone, polyamide and cellulose acetate. The biocatalysts (enzymes or cells) are normally retained on the membranes of the reactor. The substrate is introduced into reactor while the product passes out. Good mixing in the reactor can be achieved by using stirrer (Fig. 21.10A). In a continuous membrane reactor, the biocatalysts are held over membrane layers on to which substrate molecules are passed (Fig. 21.10B).

        In a recycle model membrane reactor, the contents (i.e. the solution containing enzymes, cofactors, and substrates along with freshly released product are recycled by using a pump (Fig. 21.10C). The product passes out which can be recovered.

        Applications of Immobilized Enzymes and Cells:

        Immobilized enzymes and cells are very widely used for industrial, analytical and therapeutic purpose, besides their involvement in food production and exploring the knowledge of biochemistry, microbiology and other allied specialties. A brief account of the industrial applications of immobilized cells is given in Table 21.5.

        Manufacture of Commercial Products:

        A selected list of important immobilized enzymes and their industrial applications is given in Table 21.6. Some details on the manufacture of L-amino acids and high fructose syrup are given hereunder.

        Production of L-Amino Acids:

        L-Amino acids (and not D-amino acids) are very important for use in food and feed supplements and medical purposes. The chemical methods employed for their production result in a racemic mixture of D- and L-amino acids. They can be acylated to form D, L-acyl amino acids. The immobilized enzyme aminoacylase (frequently immobilized on DEAE sephadex) can selectively hydrolyse D, L-acyl amino acids to produce L-amino acids.

        The free L-amino acids can separated from the un-hydrolysed D-acyl amino acids. The latter can be recemized to D, L-acyl amino acids and recycled through the enzyme reactor containing immobilized aminoacylase. Huge quantities of L-methionine, L-phenylalanine L-tryptophan and L-valine are produced worldwide by this approach.

        Production of High Fructose Syrup:

        Fructose is the sweetest among the monosaccharide’s, and has twice the sweetening strength of sucrose. Glucose is about 75% as sweet as sucrose. Therefore, glucose (the most abundant monosaccharide) cannot be a good substitute for sucrose for sweetening. Thus, there is a great demand for fructose which is very sweet, but has the same calorific value as that of glucose or sucrose.

        High fructose syrup (HFS) contains approximately equivalent amounts of glucose and fructose. HFS is almost similar to sucrose from nutritional point of view. HFS is a good substitute for sugar in the preparation of soft drinks, processed foods and baking.

        High fructose syrup can be produced from glucose by employing an immobilized enzyme glucose isomerase. The starch containing raw materials (wheat, potato, corn) are subjected to hydrolysis to produce glucose. Glucose isomerase then isomerizes glucose to fructose (Fig. 21.11). The product formed is HFS containing about 50% fructose. (Note: Some authors use the term high fructose corn syrup i.e. HFCS in place of HFS).

        This is an intracellular enzyme produced by a number of microorganisms. The species of Arthrobacter, Bacillus and Streptomyces are the preferred sources. Being an intracellular enzyme, the isolation of glucose isomerase without loss of biological activity requires special and costly techniques. Many a times, whole cells or partly broken cells are immobilized and used.

        Immobilized Enzymes and Cells- Analytical Applications:

        In Biochemical Analysis:

        Immobilized enzymes (or cells) can be used for the development of precise and specific analytical techniques for the estimation of several biochemical compounds. The principle of analytical assay primarily involves the action of the immobilized enzyme on the substrate.

        A decrease in the substrate concentration or an increase in the product level or an alteration in the cofactor concentration can be used for the assay. A selected list of examples of immobilized enzymes used in the assay of some substances is given in Table 21.7. Two types of detector systems are commonly employed.

        Thermistors are heat measuring devices which can record the heat generated in an enzyme catalysed reaction. Electrode devices are used for measuring potential differences in the reaction system. In the Fig. 21.12, an enzyme thermistor and an enzyme electrode, along with a specific urease electrode are depicted.


        Enzyme Reactions: Discussion and Results

        Table 1. Solution concentrations, volumes and observations for Experiment 1: Observing the enzyme reaction.

        Test TubedH2Potato ExtractCatecholZapažanja
        15 ml + 500μl—–500μLSolution turned milky-white to clear. (High substrate)
        25 ml500μl500μlSolution turned yellowish-brown. (High substrate)
        35 ml + 500μl500μl—–Solution is clear, but cloudy-white at the bottom. (Zero substrate)

        *The chemical reaction was observed with the introduction of catechol to the potato extract (tube 2).

        Tabela 2. Solution concentrations, volumes and observations for Experiment 2: The effect of substrate concentration on enzyme activity.

        Test TubedH2Potato ExtractCatecholZapažanja
        15 ml + 500μl500μl500μLSolution turned light, yellowish-brown. (High substrate)
        25 ml + 900μl500μl100μlSolution turned translucent peach. (Low substrate, diluted)

        *The reaction time for tube 1 was the fastest due to the high substrate concentration and lower dH20 concentration.

        Tabela 3. Solution concentrations, volumes and observations for Experiment 3: The effect of enzyme concentration on enzyme activity.

        Test TubedH2Potato ExtractCatecholZapažanja
        15 ml + 500μl500μl500μLThe solution turned from bluish-green to light, yellowish-brown. (High substrate)
        25 ml + 900μl100μl500μlSlightly cloudier than the original clear solution no real change in color. (High substrate, diluted, low enzyme)

        *Lower Dh20 and higher potato extract concentrations allowed for a faster reaction time

        Table 4. Solution concentrations, Buffer pH, volumes, and observations for Experiment 4: The effect of pH on enzyme activity.

        Buffer pHBuffer VolumedH2Potato ExtractCatecholZapažanja
        42ml3ml500μl500μlCloudy white
        62ml3ml500μl500μlTamno žuta
        82ml3ml500μl500μLOrangish-brown
        102ml3ml100μl500μlTranslucent peach

        *The reaction rate increased as the pH increased, with a pH of 6 being the best buffer for catechol oxidase activity. Increasing the pH past 6 showed a decrease in the reaction rate.

        Table 5. Solution concentrations, temperatures, volumes and observations for Experiment 5: The effect of temperature on enzyme activity.

        Test TubedH2Potato ExtractCatecholZapažanja
        1 (0°C)5ml500μl500μlReally light yellowish-brown
        2 (15°C)5ml500μl500μlYellowish-peach
        3 (37°C)5ml500μl500μLOrange-peach
        4 (100°C)5ml500μl500μlReally light peach

        *The fastest reaction rate was observed at 37°C. The colder the temperature (0°C – 15°C), the slower the reaction rate. Enzyme denaturation was observed at 100°C.

        Table 6. Solution concentrations, volumes and observations for Experiment 6: Inhibitor Effects – Inhibiting the Action of Catechol Oxidase

        Test TubedH2Potato ExtractPTUCatecholZapažanja
        15ml + 1ml500μl—–500μlYellowish-peach (Control)
        25ml + 500μL500μl500μl500μlReally light peach
        35ml500μl500μl500μLCloudy, clear-white

        *The fastest, and most pronounced reaction was observed in tube 1 (the solution without phenylthiourea)

        Enzyme Lab Discussion

        For the first experiment, Observing the Enzyme Reaction, it was hypothesized that the enzyme reaction would only occur in the second test tube due to the fact that it was the only tube to contain both the enzyme and substrate. As expected, the solution in tube 2 was the only solution to show the characteristic yellow-brown pigment of benzoquinone production, which was caused by the potato extract converting its catechol into the new product.

        In experiment 2, The Effect of Substrate Concentration on Enzyme activity, the hypothesis was that the tube with the higher substrate concentration would show a faster and more pronounced chemical reaction than the tube with less catechol.

        The hypothesis was supported by the fact that the higher catechol concentration in tube 1 allowed for a similar result to tube 2 from experiment 1, the only difference being that the extra 5mL of dH20 diluted some of the yellowish-brown color observed in the first reaction.

        While there was a chemical reaction observed in tube 2 (experiment 2), it was much slower (with a translucent peach pigment) due to lower a catechol concentration and a higher dH20 concentration. The higher the concentration of catechol, the more benzoquinone that can be produced.

        It was hypothesized in experiment 3, The Effect of Enzyme Concentration on Enzyme Activity, that the higher the concentration of enzyme in the solution, the faster and more pronounced the chemical reaction would be.

        This hypothesis was able to be accepted based on the rate at which the tube with the higher potato extract concentration reacted. Tube 1 had 400μL more potato extract and 400μL less dH20 than tube 2. Because enzymes are biological catalysts that speed up chemical reaction time, the solution in tube 1 quickly changed from a bluish-green pigment, to the yellowish-brown color associated with benzoquinone.

        The lower concentration of potato extract and a higher concentration of dH20 in tube 2 showed no change in color, other than the cloudiness of the potato extract itself.

        In experiment 4, The Effect of pH on Enzyme Activity, the initial hypothesis was that the lower the pH level of the buffer added to the solution, the quicker the reaction rate would be. This hypothesis was not supported by the data observed because higher acidity levels actually slowed the production of benzoquinone – which was the opposite of what was predicted.

        The solution with a pH buffer of 4 remained cloudy white, while the solution with a 6 pH buffer turned yellowish-brown. As the pH increased, the benzoquinone production rate increased. While lower pH buffers proved to be too acidic, more neutral buffers allowed for the best environment for catechol oxidase activity.

        Buffer pH levels higher than 6 showed a slower and less pronounced chemical reaction as well – illustrating the enzyme reaction’s need for neutrality.

        The hypothesis for experiment 5, The Effect on Temperature on Enzyme Activity, was that extremely low temperature would slow the rate of benzoquinone production, while extremely high temperatures would cause the enzymes to denature. This hypothesis was supported by the rate at which the solutions at 0°C – 15°C slowly reacted, and the rate at which the solution at 37°C quickly produced benzoquinone.

        After five minutes at each solution’s designated temperature, the colder solutions barely started to change color, while the warmer temperatures quickly reacted – so much so that at 100°C, the enzymes denatured and the solution began to pale in pigment. Colder temperatures slowed the movement of molecules in the solutions, while warmer temperatures (not including 100°C) allowed for a better environment for catechol oxidase activity.

        For experiment 6, Inhibitor Effects – Inhibiting the Action of Catechol Oxidase, it was hypothesized that the addition of phenylthiourea (PTU) would keep the enzyme reaction from occurring. The hypothesis was able to be accepted due to the fact that the tubes which contained the PTU showed very little change in pigment.

        Tube 1 served as the control, which showed the production of benzoquinone (yellowish-brown color) and allowed for comparison between the three solutions. Considering PTU is a non-competitive inhibitor, tubes 2 and 3 contained solutions that prevented the enzyme from catalyzing the reaction, regardless of whether or not the substrate was bound to the active site.

        The only real issue with any of the 6 experiments was the unsupported hypothesis for the Effect of pH on the Enzyme Activity experiment. I must have tied the preservative nature of benzoquinone with how acidic lemon juice keeps apples from turning brown, so I assumed a low pH would increase the reaction rate. In reality, acidity slows the reaction rate – which is zašto the apples don’t change color.

        In conclusion, these experiments have shown that benzoquinone production can only occur with the presence of both an enzyme and substrate. Factors such as substrate and enzyme concentration, pH, temperature, and the presence of noncompetitive inhibitors can affect enzyme reaction. High substrate concentration will allow for greater benzoquinone production, while high enzyme concentration will speed up the reaction rate – and vise versa.

        In order for enzyme reaction to rapidly occur, it must be done in an environment where the pH is as close to neutral as possible, with the reaction rate slowing in both highly acidic or basic solutions. The same goes for temperature – extremely high or extremely cold temperatures can decrease enzyme reaction rates, or cause the enzymes to denature altogether.

        The introduction of a noncompetitive inhibitor (such as phenylthiourea) allows it to bind to the allosteric site on the enzyme, which keeps the reaction from occurring (regardless of the enzyme or substrate concentration).


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