Search

Zaradi negativnih vplivov uporabe fosilnih goriv je potrebno raziskati načine pridobivanja obnovljivih virov energije. Sem spadajo biogoriva, pridobljena iz lignocelulozne biomase. Za obdelavo lignocelulozne biomase so potrebni encimi, ki katalizirajo razgradnjo lignoceluloznih polimerov, med katere spadajo glikozid hidrolaze. Nove encime iz okolja lahko med drugim pridobivamo z metagenomskim pristopom. Ta temelji na analizi celotne DNA mikrobne združbe vzorca iz nekega okolja, izbiri ustreznih genov ter izražanju teh genov v heterolognih ekspresijskih sistemih. V magistrski nalogi smo analizirali 5 zapisov, ki domnevno kodirajo encime, vpletene v razgradnjo lignoceluloznih substratov ter jih poskusili heterologno izraziti v različnih sevih bakterije Escherichia coli. Med tarčnimi geni, ki smo jih poskusili heterologno izraziti smo uspešno proizvedli 40 kDa veliko ksilanazo iz družine glikozid hidrolaz 10. Tarčni gen smo vnesli v ekspresijski sev E. coli in sprožili njegovo izražanje. Potrdili smo ksilanolitično aktivnost produkta tarčnega gena, na ksilanu in na odpadnih pivskih tropinah. Ugotovili smo, da je encimska aktivnost tako grobega encimskega pripravka iz bakterijske kulture kot očiščenega tarčnega encima na pivskih tropinah približno dvakrat višja pri 25 °C kot pri 37 °C. Da bi neposredno preverili biotehnološki potencial nove ksilanaze za pretvorbo pivovarskih odpadkov v bioplin, smo izvedli tudi test biometanskega potenciala. Primerjali smo proizvodnjo biometana oz. bioplina iz pivskih tropin predobdelanih z encimom Y006 z neobdelanimi pivskimi tropinami. Due to the negative effects associated to fossil fuel use more research needs to be done on renewable energy sources. Biofuels synthesized from lignocellulosic biomass represent one of the alternatives to fossil fuels. Enzymes that catalyze the degradation of lignocellulosic polymers are crucial for processing of lignocellulosic biomass to bioenergy. Glycoside hydrolases represent the most important group of these enzymes. New enzymes from the environment can be obtained without isolating new bacteria by using the metagenomic approach. This approach is based on the analysis of the whole DNA isolated from an environmental sample, bioinformatic analysis of the genes and/or expression of target genes in a heterologous expression system. In this work we analysed 5 ORFs encoding putative enzymes involved in lignocellulose degradation on compost metagenome, which we expressed in various Escherichia coli strains. Among these putative enzymes, we successfully expressed a 40 kDa xylanase from glycoside hydrolase family 10. In addition, we also tested biotechnological potential of the target enzymes for valorization of brewer's spent grain. The activity of crude as well as isolated enzyme on brewer’s spent grain was approximately two times higher at 25 °C then at 37 °C. Finally, we conducted a biomethane potential test (BMP), and compared methane production from brewer's spent grain that was pre-processed with Y006 and methane production from unprocessed brewer's spent grain.

Proizvodi za posebne prehrambene potrebe, kao što su keksi i kruh za oboljele od dijabetesa i/ili celijakije, često imaju lošija senzorska svojstva u odnosu na konvencionalne proizvode. Kako bi takvi proizvodi istovremeno bili nutritivno vrijedni i senzorski prihvatljivi za potrošača, potrebno je provesti ispitivanje njihovih senzorskih svojstava. U ovom radu su ispitana senzorska svojstva keksa bez šećera i bezglutenskog kruha s dodatkom posija prosa i ksilanaze primjenom testova deskriptivne i hedonističke analize te nizanja po preferenciji. Proso je iznimno vrijedna žitarica čije se posije koriste za obogaćivanje hrane, jer ne sadrži gluten i djeluje hipoglikemijski. Najveći nedostatak posija prosa je to što daje proizvodu nepoželjan gorak okus i time smanjuje njegovu prihvatljivost. Enzim ksilanaza se koristi u pekarstvu jer poboljšava obradivost i stabilnost tijesta. Rezultati deskriptivne analize bezglutenskog kruha i keksa bez šećera pokazali su da su posije prosa uzrokovale pojavu tamnije boje, povećanje gorkog i naknadnog gorkog okusa te smanjenje tvrdoće. Nepoželjni gorki okus smanjen je kod uzoraka s ksilanazom tretiranim posijama prosa. Hedonistička analiza pokazala je porast prihvatljivosti kruha s dodatkom ksilanazom tretiranih posija prosa u odnosu na kruh s netretiranim posijama te je testom nizanja po preferenciji zauzeo prvo mjesto. Kod keksa bez šećera hedonističkom analizom i testom nizanja po preferenciji pokazalo se da najbolje ocijenjen sveukupni doživljaj ima kontrolni uzorak, a keks s dodanim posijama je bio u kategoriji 'neznatno mi se sviđa'. Products for special dietary needs, such as biscuits and bread for people with diabetes and / or celiac disease, often have poorer sensory properties compared to conventional products. In order for such products to be both nutritionally valuable and sensory-acceptable to the consumer, it is necessary to conduct a test of their sensory properties. In this research, the sensory properties of sugar-free biscuits and gluten-free bread with the addition of millet bran and xylanase were tested using descriptive and hedonistic analysis and a sequencing by preference. Millet is an extremely valuable cereal whose bran is used to enrich food because it does not contain gluten and has a hypoglycemic effect. The biggest disadvantage of millet bran is that it gives the product an undesirable bitter taste and thus reduces its acceptability. The enzyme xylanase is used in baking because it improves the workability and stability of the dough. The results of a descriptive analysis of gluten-free bread and sugar-free biscuits showed that addition of millet bran caused a darker color of the product, increased bitter and subsequent bitter taste, and decreased hardness. Undesirable bitter taste was reduced in the sample with xylanase-treated millet bran. Hedonistic analysis shows an increase in the acceptability of bread with the addition of enzym treated millet bran compared to bread with untreated bran, and it took first place in the sequence test by preference. Hedonistic analysis and sequencing by preference of sugar-free biscuits showed that a control sample had the best rated overall experience, while the biscuit with bran was rated as 'slightly liked'.

Uporaba fosilnih goriv je zaradi sproščanja toplogrednih plinov vse večji okoljevarstveni problem, zato je pomembno iskanje novih, trajnostnih in okolju prijaznejših virov energije. Biogoriva druge generacije imajo velik potencial za razvoj trajnostne ekonomije. Pridobivajo jih z razgradnjo in fermentacijo lignocelulozne biomase, ki je najbolj razširjen in cenovno ugoden obnovljiv substrat. Za razvoj proizvodnje biogoriv je ključno iskanje novih encimov za razgradnjo biomase, saj ti zaenkrat zaradi visoke cene, premajhne učinkovitosti in slabe stabilnosti v procesnih razmerah še ne dosegajo povpraševanja. V magistrski nalogi smo v metagenomski DNA komposta identificirali ksilanazo iz družine GH10 z dvema vezavnima domenama za ogljikove hidrate (CBM). Tarčni gen (xylM_3) smo prenesli v ekspresijski sev in analizirali izražanje produkta pri različnih pogojih. Ugotovili smo, da se produkt tarčnega gena optimalno izraža ob dodatku 0,75 mM IPTG, največ aktivnega encima pa dobimo po 20-urni inkubaciji pri 18 °C. Tarčni encim smo poskušali očistiti z afinitetno kromatografijo (Ni-NTA) v nativnih in denaturirajočih pogojih, vendar izoliranega nismo uspeli pridobiti v aktivni obliki. Kljub vsemu smo z analizo encimskih frakcij ugotovili, da je encimski ekstrakt aktiven v temperaturnem območju med 20 in 60 °C in pri pH 5-8, najvišjo aktivnost pa dosega pri temperaturi 50 °C in pH = 6. Pokazali smo tudi, da encimski ekstrakt lahko razgrajuje bukov ksilan in arabinoksilan. Aktivnost encimskega ekstrakta na ksilanu se je povečala ob dodatku 1, 5 in 10 mM K+ in 1 mM Co2+ ter 0,2 mM detergenta Triton-X 100. Ob dodatku ostalih testiranih ionov in kemikalij nismo ugotovili sprememb v encimski aktivnosti ali pa se je ta znižala. The use of fossil fuels is an increasing environmental problem due to the release of greenhouse gases. It has therefore become increasingly important to find new sustainable and more environmentaly friendly energy resources. Second-generation biofuels have great potential for development of bioeconomy as they are obtained by decomposition and fermentation of lignocellulosic biomass, the most widespread and affordable renewable substrate. In recent years a lot of effort has been put into finding novel enzymes, which are the key for the development of cost-effective biofuels production. In the following work, the product of a gene encoding putative xylanase (xylM_3), obtained from metagenomic library of compost microbial community was heterologically expressed and biochemicaly characterized. Bioinformatic analysis of the target gene revealed an enzyme with a single catalytic site belonging to glycoside hydrolase family 10 (GH10) and two carbohydrate-binding modules (CBM 2 and CBM 60). Enzyme was heterologically expressed in E. coli BL21 (DE3). The optimum conditions for the enzyme production were determined. Largest amounts of active enzyme were produced at 0.75 mM IPTG, after 20 hours incubation at 18 °C. The enzyme extract was active in the temperature range between 20 and 60 °C and pH 5-8, with the highest activity measured at 50 °C and pH = 6. The activity of the enzyme extract on beechwood xylan and arabinoxylan was confirmed. The addition of 1, 5 and 10 mM K+, 1 mM Co2+ and 0.2 mM Triton-X 100 increased the xylanolytic activity of the enzyme extract. On the other hand, the enzyme extract retaind the original activity in the presence of of 20 % formic acid, while the addition of other tested organic solvents (methanol, ethanol and acetone) reduced its activity.

Metagenomski pristop iskanja novih biotehnološko zanimivih encimov temelji na pregledu celokupne DNA kompleksnih okoljskih vzorcev, čemur sledi kloniranje in izražanje tarčnih genov v dobro poznanih in z vidika gojenja nezahtevnih ekspresijskih sevih. Med iskane encime uvrščamo tudi glikozid hidrolaze, ki razgrajujejo kompleksne rastlinske polisaharide. V magistrski nalogi smo se osredotočili na izolacijo in karakterizacijo produkta izbranega gena (xyl6) z zapisom za domnevno glikozid hidrolazo iz družine 10 (GH10), identificiranega v metagenomu komposta. Izolirali smo encim z molekulsko maso približno 40 kDa, z aktivnostjo na substratih bukovem ksilanu in arabinoksilanu. Temperaturni optimum delovanja izoliranega encima na bukovem ksilanu v 50 mM K-fosfatnem pufru s pH 8 je pri temperaturi 50 °C. Ugotovili smo, da kovinski ioni Co2+, Fe3+, Mn2+ ali Ca2+ v koncentracijah 5 mmol/L povečajo aktivnost preučevanega encima, v koncentracijah 10 mmol/L pa jo zmanjšajo. Aktivnost encima popolnoma inhibirajo ioni Cu2+ v koncentraciji 10 mmol/L. Aktivnost tarčnega encima se zmanjša tudi v prisotnosti NaCl v koncentracijah nad 100 mmol/L, ob prisotnosti 10 % (v/v) metanola, etanola ali acetona ter 20 mM EDTA ali SDS. Prav tako encim ni stabilen pri temperaturah nad 50 °C ter v kislih pogojih (pod pH 6). Povečano aktivnost preučevanega encima smo zaznali v prisotnosti reducentov, 2 mM DTT ali 10 mM ß-merkaptoetanola. Ugotavljali smo tudi kinetične parametre tarčnega encima pri optimalnih pogojih delovanja na bukovem ksilanu in ocenili vrednosti Vmax na 787,2 ± 12,9 μmol/min/mg, Km na 1,8 ± 0,3 mg/mL in kcat na 526,2 ± 17,4 s-1. Pokazali smo tudi, da je ksilanaza Xyl6 sposobna razgraditi tudi s hemicelulozo bogate materiale, kot so pšenični ali ječmenovi otrobi, zaradi česa je potencialno biotehnološko zanimiv encim. Metagenomic approach seeks novel enzymes beneficial to microbial biotechnology through the screening of metagenomes from complex environments, with crucial steps: cloning and the expression of selected genes in unpretentious bacterial or other types of strains. Glycoside hydrolases degrade complex plant polysaccharides and belong to a group of one of the most demanded industrially relevant enzymes. In this thesis we focused on isolation and characterization of putative GH10 identified in compost metagenome (xyl6). We successfully isolated xylanase with molecular mass approx. 40 kDa and activity against beechwood xylan and arabinoxylan. Recombinant xylanase revealed optimal xylanolytic activity against beechwood xylan at 50 °C and pH 8. Xylanolytic activity of Xyl6 increased in the presence of 5 mM and decreased in the presence of 10 mM Co2+, Fe3+, Mn2+ or Ca2+. The latter concentration of Cu2+ completely inhibited the enzyme activity. Enzyme activity also decreased when exposed to NaCl above 100 mmol/L. Exposure to 10 % (v/v) methanol, ethanol or acetone, as well as 20 mM EDTA or SDS was shown to decrease xylanolytic activity of Xyl6. Furthermore, enzyme was not stable at a temperature above 50 °C or in acidic conditions (below pH 6). Xylanolytic activity increased when exposed to reducing agents such as 2 mM DTT or 10 mM ß-mercaptoethanol. The recombinant xylanase exhibited Km and Vmax values of 1,8 ± 0,3 mg/mL and 787,2 ± 12,9 μmol/min/mg for beechwood xylan at optimal conditions, and turnover number (kcat) value of 526,2 ± 17,4 s-1. Enzyme also proved successful in the degradation of natural hemicellulose-rich substrates, such as wheat or barley bran, thus has great potential for future use in industrial applications.

Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Purified recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100 % active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the first report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran., Utvrđeno je da soj bakterije Bacillus tequilensis BT21, izoliran iz morskog sedimenta, može proizvesti izvanstaničnu ksilanazu. Gen xynBT21 za kodiranje ksilanaze kloniran je i eksprimiran u bakteriji Escherichia coli, gdje je kodirao protein molekularne mase od 23,3 kDa, koji sadržava 213 aminokiselinskih ostataka. Optimalna aktivnost pročišćene rekombinantne ksilanaze postignuta je pri temperaturi od 60 °C i pH=6. Enzim je bio izuzetno stabilan pri alkalnim pH vrijednostima. Pri pH=7 aktivnost mu je bila 100 % tijekom 24 sata, dok se tijekom inkubacije pri pH=8 i 9 aktivnost enzima povećala. Ksilanaza iz B. tequilensis imala je alkalnu pI vrijednost od 9,4; a pripada obitelji glikozilnih hidrolaza 11. Ispitano je djelovanje ksilanaze XynBT21 na ksilan iz bukve i ksilooligosaharide. Njihovom hidrolizom dobivena je pretežno ksilobioza (X2) uz manju količinu ksiloze (X1), zbog čega je zaključeno da je XynBT21 vjerojatno endoksilanaza. Enzimskom hidrolizom pšeničnih mekinja potvrđeno je da ksilanaza može proizvesti ksilobiozu na toj podlozi. Ksilooligosaharidi, osobito ksilobioza, imaju snažna bifidogena svojstva, pa se sve češće primjenjuju kao prebiotici. Ovo je prvi rad koji opisuje primjenu nove ksilanaze iz morske bakterije B. tequilensis BT21 za oslobađanje ksilobioze iz pšeničnih mekinja.

U probavnom se sustavu biljojeda nalaze vrlo učinkoviti mikroorganizmi što razgrađuju celulolozu i hemicelulozu iz sažvakanog biljnog materijala, te ih opskrbljuju hranjivim tvarima. Osim protozoa i gljivica, razgradnji otporne (hemi)celulozne biomase u buragu znatno pridonose i bakterije. U fokusu je ovog preglednog rada opis enzimskog sustava triju predstavnika bakterija iz buraga što proizvode celulazu, i to: Ruminococcus flavefaciens, Prevotella bryantii i Pseudobutyrivibrio xylanivorans. R. flavefaciens je poznat po proizvodnji jedne od najkompleksnijih enzimskih struktura, pa bi se mogao upotrijebiti za dizajn enzimskog kompleksa za razgradnju stanične stijenke biljaka. S druge strane, bakterije Prevotella bryantii i Pseudobutyrivibrio xylanivorans proizvode jednostavne, slobodne i vrlo aktivne ksilanaze. Bakterija P. xylanivorans ima i probiotička svojstva, pa se može upotrijebiti za proizvodnju bioplina i kao dodatak krmivu. Ispitivanje je genoma i proteoma bakterija što razgrađuju celulozu i hemicelulozu usmjereno na identifikaciju novih enzima, koji se nakon kloniranja i ekspresije u odgovarajućim domaćinima mogu upotrijebiti za izradu vrlo aktivnih rekombinantnih hidrolitičkih mikroorganizama, te primijeniti u različitim biotehnološkim procesima., Herbivorous animals harbour potent cellulolytic and hemicellulolytic microorganisms that supply the host with nutrients acquired from degradation of ingested plant material. In addition to protozoa and fungi, rumen bacteria contribute a considerable part in the breakdown of recalcitrant (hemi)cellulosic biomass. The present review is focused on the enzymatic systems of three representative fibrolytic rumen bacteria, namely Ruminococcus flavefaciens, Prevotella bryantii and Pseudobutyrivibrio xylanivorans. R. flavefaciens is known for one of the most elaborated cellulosome architectures and might represent a promising candidate for the construction of designer cellulosomes. On the other hand, Prevotella bryantii and Pseudobutyrivibrio xylanivorans produce multiple free, but highly efficient xylanases. In addition, P. xylanivorans was also shown to have some probiotic traits, which makes it a promising candidate not only for biogas production, but also as an animal feed supplement. Genomic and proteomic analyses of cellulolytic and hemicellulolytic bacterial species aim to identify novel enzymes, which can then be cloned and expressed in adequate hosts to construct highly active recombinant hydrolytic microorganisms applicable for different biotechnological tasks.

The aim of this study is to develop a new technology for making traditional Lithuanian non-alcoholic beverage kvass from fermented cereals by extending the spectrum of raw materials (extruded rye) and applying new biotechnological resources (xylanolytic enzymes and lactic acid bacteria (LAB)) to improve its functional properties. Arabinoxylans in extruded rye were very efficiently hydrolysed into oligosaccharides by xylanolytic complex Ceremix Plus MG. Using Ceremix Plus MG and LAB fermentation, the yield of arabinoxylooligosaccharides and xylooligosaccharides in beverage was increased to 300 and 1100 mg/L, respectively. Beverages fermented by LAB had lower pH values and ethanol volume fraction compared to the yeast-fermented beverage. The acceptability of the beverage fermented by Lactobacillus sakei was higher than of Pediococcus pentosaceus- or yeast-fermented beverages and similar to the acceptability of commercial kvass made from malt extract. The results showed that extruded rye, xylanolytic enzymes and LAB can be used for production of novel and safe high-value non-alcoholic beverages., Svrha je ovoga rada bila razviti novu tehnologiju proizvodnje tradicionalnog litvanskog bezalkoholnog napitka (kvas) iz fermentiranih žitarica pomoću novih sirovina (ekstrudirana raž) i biotehnoloških materijala (ksilanaze i bakterije mliječno-kiselog vrenja), a radi poboljšanja njegovih funkcionalnih svojstava. Kompleks ksilanaza Ceremix Plus MG uspješno je upotrijebljen za razgradnju arabinoksilana iz ekstrudirane raži. Nakon razgradnje arabinoksilana i mliječno-kiselog vrenja prinos je arabinooligosaharida u dobivenom napitku porastao na 300 mg/L, a ksilooligosaharida na 1100 mg/L. Napici fermentirani s pomoću bakterija mliječno-kiselog vrenja imali su niže pH-vrijednosti te manji volumni udjel etanola od napitaka fermentiranih s pomoću kvasaca. Napitak fermentiran s pomoću bakterije Lactobacillus casei imao je bolju prihvatljivost od onog fermentiranog s pomoću bakterije Pediococcus pentosaceus ili kvasca, a sličnu onoj komercijalnog kvasa dobivenog iz slada. Rezultati pokazuju da se ekstrudirana raž, ksilanaze i mliječno-kisele bakterije mogu uspješno primijeniti u proizvodnji novog, zdravstveno ispravnog bezalkoholnog napitka dodane vrijednosti.

The Plackett-Burman screening design (PBSD) and Central composite rotatable design (CCRD) were used to optimize the fermentation parameters for enhanced xylanase production from Aspergillus flavus DFR-6 in submerged cultivation. The PBSD demonstrated that the positive main effects of yeast extract, wheat bran, Tween 80 and NaNO3 were significant at 10 % level of significance. The interactive effects of these factors were deduced using CCRD and finally, the production medium was optimized using Design-Expert software. The optimized medium conditions with 97.2 % desirability and with respect to `minimum organic nitrogen source’ were (in g/L): NaNO3 4.09, K2HPO4 0.5, MgSO4·7H2O 0.25, KCl 0.25, FeSO4 0.005, wheat bran 24.99, yeast extract 10 and Tween 80 0.21 mL/L, pH=5.0. The enzyme was produced in 50 mL of medium working volume using 500-mL conical flask at 35 °C with 1 % (by volume) of inoculum size. The production titre of xylanase under aforementioned conditions was 31.4 U/mL after 6 days of static fermentation which is approx. fourfold higher than obtained from the unoptimized medium., Da bi se optimirali uvjeti submerzne fermentacije te povećala proizvodnja ksilanaze iz Aspergillus flavus DFR-6, primijenjeni su Plackett-Burmanov i centralno složeni dizajn. Plackett-Burmanov dizajn pokazao je da su pozitivni učinci dodatka kvaščeva ekstrakta, pšeničnih mekinja, Tween 80 i NaNO3 statistički značajni na razini od 10 %. Međusobni učinci tih faktora isključeni su pomoću centralno složenog dizajna, a zatim je podloga optimirana pomoću „Design Expert“ softvera. Utvrđen je odgovarajući (97,2 %) sastav optimirane podloge s minimalnom količinom organskog dušika, i to: 4,09 g/L NaNO3; 0,5 g/L K2HPO4; 0,25 g/L MgSO4·7H2O; 0,25 g/L KCl; 0,005 g/L FeSO4; 24,99 g/L pšeničnih mekinja; 10 g/L kvaščeva ekstrakta i 0,21 mL/L Tween 80 (pH=5,0). Enzim je proizveden u 50 mL podloge, s 1 % inokuluma, u Erlenmeyerovoj tikvici od 500 mL, na temperaturi od 35 °C. Pri tim je uvjetima, nakon 6 dana fermentacije bez miješanja, dobiveno 31,4 U/mL ksilanaze, što je otprilike 4 puta više nego na neoptimiranoj podlozi.

To efficiently produce xylanase for food processing industry, a gene encoding xylanase II (XynII) from Aspergillus usamii has been cloned into the vector pPIC9K and integrated into the genome of Pichia pastoris KM71 by electroporation. By means of minimal dextrose (MD) plates and PCR, the recombinant P. pastoris strains (His+Muts) have been obtained. Activity assay and SDS-PAGE demonstrate that XynII was extracellularly expressed in P. pastoris with the induction of methanol. In shake flask culture, the xylanase activity was up to 1760 U/mL, with the specific activity of 3846.83 U/mg. The optimal pH and temperature of the recombinant XynII were pH=4.0 and 50 °C, respectively. The xylanase was stable below 50 °C and within pH=3.0–5.0. The molecular mass of the recombinant protein was estimated to be 21 kDa by SDS-PAGE. This enzyme had Km of 4.55 mg/mL, vmax of 15.15 mM/s and kcat of 455 s–1. Its activity was increased by EDTA and Ca2+ ions, but strongly inhibited by Mn2+ and Fe2+ ions. This is the first report demonstrating the possibility of mass production of A. usamii protein using P. pastoris., Da bi se uspješno proizvela ksilanaza za primjenu u prehrambenoj industriji, gen koji kodira ksilanazu II (Xyn II) iz plijesni Aspergillus usamii kloniran je u vektor pPIC9K i elektroporacijom integriran u genom kvasca Pichia pastoris KM71. Rekombinantni sojevi P. pastoris (His+Muts) uzgojeni su na podlogama s minimalnom količinom dekstroze te izdvojeni pomoću PCR metode. Procjenom aktivnosti i molekularne mase enzima (SDS-PAGE metodom) utvrđeno je da je dodatak metanola inducirao ekstracelularnu ekspresiju Xyn II u kvascu P. pastoris. U pokusu na tresilici izmjerena je najveća aktivnost ksilanaze od 1760 U/mL i specifična aktivnost od 3846,83 U/mg. Optimalna pH-vrijednost za aktivnost rekombinantne Xyn II bila je 4, a optimalna temperatura 50 °C. Ksilanaza je bila stabilna na temperaturama nižim od optimalne, te u rasponu pH=3-5. Pomoću SDS-PAGE metode procijenjena je molekularna masa enzima od 21 kDa. Michaelis-ova konstantna iznosila je Km=4,55 mg/mL, maksimalna brzina reakcija bila je vmax=15,15 mM/s, a kinetička konstanta kcat=455 s-1. Aktivnost enzima potaknuta je dodatkom EDTA i Ca2+ iona, a inhibirana ionima Mn2+ i Fe2+. U radu je prvi put opisana mogućnost masovne proizvodnje proteina iz A. usamii s pomoću kvasca P. pastoris.

The xylanase production by a new Cochliobolus sativus Cs5 strain was improved under submerged fermentation. The xylanase was induced by xylan and repressed by glucose, sucrose, maltose, xylose, starch and cellulose. Highest enzyme production (98.25 IU/mL) was recorded when wheat straw (4 % by mass per volume) was used as a carbon source after 120 h of incubation. NaNO3 increased xylanase production 5.4-fold as compared to the control. Optimum initial pH was found to be 4.5 to 5. The C. sativus Cs5 strain grown under submerged culture in a simple medium proved to be a promising microorganism for xylanase production., Submerznim uzgojem novog soja nitaste gljive Cochliobolus sativus Cs5 poboljšana je proizvodnja ksilanaze, potaknuta ksilanom, a potisnuta glukozom, saharozom, maltozom, ksilozom, škrobom i celulozom. Upotrebom pšenične slame (4 %, m/V) kao izvora ugljika, nakon 120 sati inkubacije postignuta je najveća proizvodnja enzima (98,25 IU/mL). Dodatak NaNO3 povećao je proizvodnju ksilanaze 5,4 puta u usporedbi s kontrolnim uzorkom. Optimalna početna pH-vrijednost bila je od 4,5 do 5. Utvrđeno je da se za proizvodnju ksilanaze odlično može upotrijebiti soj C. sativus Cs5, submerzno uzgojen u podlozi jednostavnog sastava.

Xylanase was produced by Aspergillus niger LPB 326 cultivated on lignocellulosic substrate composed by sugarcane bagasse and soybean meal in solid-state fermentation. The effects of various variables were observed and optimized by applying statistical experimental designs. The best xylanase activity was obtained in a medium containing 10 g of sugarcane bagasse and soybean meal in the ratio of 65 and 35 %, respectively, moistened to 85 % of initial water content with a nutrient salt solution composed of (in g/L): CuSO4 0.4, KH2PO4 1.5 and CoSO4 0.0012, and incubated for 4 days at 30 °C. Under these optimized conditions, a xylanase activity of 3099 IU/g of dry matter was obtained., U radu je istražena proizvodnja ksilanaze na lignoceluloznoj podlozi od otpadaka šećerne trske i sojine sačme s pomoću plijesni Aspergillus niger LPB 326. Ispitan je utjecaj različitih varijabli pa je proizvodnja optimirana primjenom statističkog eksperimentalnog dizajna. Najbolja aktivnost ksilanaze postignuta je upotrebom podloge s 10 g otpadaka šećerne trske i sojine sačme u omjeru 65:35, navlaženoj do 85 % početne vlažnosti otopinom hranjivih soli sastava (u g/L): CuSO4 0,4; KH2PO4 1,5 i CoSO4 0,0012; inkubiranoj tijekom 4 dana pri 30 °C. U tim uvjetima postignuta je aktivnost ksilanaze od 3099 IU/g suhe tvari.