Zaštitni učinak sastojaka biomase na međufazno djelovanje liaze α-pinen oksida iz bakterije Pseudomonas rhodesiae CIP 107491

α-Pinene oxide lyase is able to catalyze the cleavage of both rings of α-pinene oxide to form cis-2-methyl-5-isopropylhexa-2,5-dienal (isonovalal) with no cofactor requirements. This bioconversion, when carried out under biphasic conditions (water/hexadecane) by Pseudomonas rhodesiae CIP 107491, allows the accumulation of a very high concentration of the product. Nevertheless, the reaction stopped due to the loss of enzyme activity, which has been demonstrated to be an irreversible phenomenon. The enzyme was purified by chromatographic methods to study the reasons of its instability. Bioconversion with pure enzyme showed that a protein precipitation occurred at the liquid-liquid interface, giving rise to a decrease in soluble protein and in residual enzymatic activity. The rate of decrease in soluble protein concentration was not related to the presence of the precursor, which indicated that the inactivation was interfacial, i.e. due to the direct contact of the enzyme with the hexadecane layer. This phenomenon was not present when crude enzymatic extract was used as a biocatalyst. Biomass components present in this extract thus had a protective effect against interfacial phenomena, demonstrating that α-pinene oxide lyase purification does not attract much practical interest for isonovalal production.Liaza α-pinen oksida katalizira cijepanje oba prstena α-pinen oksida bez prisutnosti kofaktora, pri čemu nastaje cis-2-metil-5-izopropilheksa-2,5-dienal (izonovalal). Provedbom takve biokonverzije na granici dviju faza (voda/heksadekan), s pomoću bakterije Pseudomonas rhodesiae CIP 107491, postignuto je nakupljanje velike koncentracije produkta. No, gubitkom aktivnosti enzima dolazi do ireverzibilnog prestanka reakcije. Stoga je enzim pročišćen kromatografijom i ispitani su uzroci njegove nestabilnosti. Utvrđeno je da biokonverzija pomoću pročišćenog enzima dovodi do taloženja proteina na granici faza, a time i smanjenja koncentracije topljivih proteina i enzimske aktivnosti. Smanjenje koncentracije topljivih proteina nije ovisilo o prisutnosti prekurzora, čime je dokazano da je izravni kontakt enzima s površinom heksadekana uzrokovao inaktivaciju međupovršine. Primjena sirovog ekstrakta enzima nije dovela do inaktivacije, pa je zaključeno da sastojci ekstrakta biomase imaju zaštitni učinak na međupovršinu, te da pročišćavanje liaze α-pinen oksida za proizvodnju izonovalala nema praktičnu primjenu

Zaštitni učinak sastojaka biomase na međufazno djelovanje liaze α-pinen oksida iz bakterije Pseudomonas rhodesiae CIP 107491

α-Pinene oxide lyase is able to catalyze the cleavage of both rings of α-pinene oxide to form cis-2-methyl-5-isopropylhexa-2,5-dienal (isonovalal) with no cofactor requirements. This bioconversion, when carried out under biphasic conditions (water/hexadecane) by Pseudomonas rhodesiae CIP 107491, allows the accumulation of a very high concentration of the product. Nevertheless, the reaction stopped due to the loss of enzyme activity, which has been demonstrated to be an irreversible phenomenon. The enzyme was purified by chromatographic methods to study the reasons of its instability. Bioconversion with pure enzyme showed that a protein precipitation occurred at the liquid-liquid interface, giving rise to a decrease in soluble protein and in residual enzymatic activity. The rate of decrease in soluble protein concentration was not related to the presence of the precursor, which indicated that the inactivation was interfacial, i.e. due to the direct contact of the enzyme with the hexadecane layer. This phenomenon was not present when crude enzymatic extract was used as a biocatalyst. Biomass components present in this extract thus had a protective effect against interfacial phenomena, demonstrating that α-pinene oxide lyase purification does not attract much practical interest for isonovalal production.Liaza α-pinen oksida katalizira cijepanje oba prstena α-pinen oksida bez prisutnosti kofaktora, pri čemu nastaje cis-2-metil-5-izopropilheksa-2,5-dienal (izonovalal). Provedbom takve biokonverzije na granici dviju faza (voda/heksadekan), s pomoću bakterije Pseudomonas rhodesiae CIP 107491, postignuto je nakupljanje velike koncentracije produkta. No, gubitkom aktivnosti enzima dolazi do ireverzibilnog prestanka reakcije. Stoga je enzim pročišćen kromatografijom i ispitani su uzroci njegove nestabilnosti. Utvrđeno je da biokonverzija pomoću pročišćenog enzima dovodi do taloženja proteina na granici faza, a time i smanjenja koncentracije topljivih proteina i enzimske aktivnosti. Smanjenje koncentracije topljivih proteina nije ovisilo o prisutnosti prekurzora, čime je dokazano da je izravni kontakt enzima s površinom heksadekana uzrokovao inaktivaciju međupovršine. Primjena sirovog ekstrakta enzima nije dovela do inaktivacije, pa je zaključeno da sastojci ekstrakta biomase imaju zaštitni učinak na međupovršinu, te da pročišćavanje liaze α-pinen oksida za proizvodnju izonovalala nema praktičnu primjenu

Stabilnost glukoza-oksidaze u sporama plijesni Aspergillus niger dobivenih uzgojem na čvrstoj podlozi, te uloga spora u reakciji biokonverzije

The aim of this work is to demonstrate the role of conidial spores as a reservoir of glucose oxidase and their stability as a biocatalyst in the bioconversion reaction for the production of gluconic acid. Solid-state fermentation (SSF) was carried out in fixed-bed column bioreactor for the production of Aspergillus niger spores. Growth parameters, sporulation and kinetics of gluconic acid production were analysed at different time intervals during the course of SSF. Spores of different age (48–216 h) were used as biocatalysts in the bioconversion reaction. Spores harvested at a later period of SSF (196 h) produced high titres of gluconic acid (30 g/L) in the bioconversion medium when compared to the spores harvested at early (48 h) stages of SSF (2.2 g/L). Spores (harvested at 200 h and stored in freezer for 91 days) exhibited the same glucose oxidase activity and served as an active and stable catalyst when compared to the fresh spores, showing that aging (storage) did not affect enzymes present in the spores, which suggested that the spores acted as an efficient enzyme reservoir. Yields close to 93 % were obtained with 98 g/L of gluconic acid production, corresponding to an average productivity of 1.7 g/(L·h). The stability of the enzyme in the spores and the ability of conidia to be stored for a long time without the loss of activity add specific advantage to the bioconversion process.Svrha je rada bila dokazati važnost konidijskih spora kao izvora glukoza-oksidaze, te istražiti stabilnost tih biokatalizatora pri proizvodnji glukonske kiseline. Spore plijesni Aspergillus niger proizvedene su uzgojem na čvrstoj podlozi u kolonskom bioreaktoru s nasutim slojem. Tijekom fermentacije, u različitim vremenskim intervalima, ispitani su parametri rasta, sporulacija i kinetika proizvodnje glukonske kiseline. Kao biokatalizator upotrijebljene su spore različite dobi (48-216 h). Spore dobivene nakon 196 h uzgoja proizvele su biokonverzijom mnogo glukonske kiseline (30 g/L), za razliku od spora prikupljenih nakon 48 h, koje su proizvele samo 2,2 g/L glukonske kiseline. Spore prikupljene nakon 200 h, čuvane 91 dan u zamrzivaču, imale su istu aktivnost glukoza-oksidaze kao i svježe spore, pa su upotrijebljene kao aktivni i stabilni katalizator. Time je dokazano da sazrijevanje (skladištenje) ne utječe na enzime u sporama, te da one mogu biti učinkovito spremište enzima. Pri proizvodnji 98 g/L glukonske kiseline, tj. produktivnosti od 1,7 g/(L·h) postignuto je skoro 93 %-tno iskorištenje. Prednosti su procesa biokonverzije stabilnost enzima u sporama i sposobnost skladištenja konidija duže vrijeme bez gubitka njihove aktivnosti

Stabilnost glukoza-oksidaze u sporama plijesni Aspergillus niger dobivenih uzgojem na čvrstoj podlozi, te uloga spora u reakciji biokonverzije

The aim of this work is to demonstrate the role of conidial spores as a reservoir of glucose oxidase and their stability as a biocatalyst in the bioconversion reaction for the production of gluconic acid. Solid-state fermentation (SSF) was carried out in fixed-bed column bioreactor for the production of Aspergillus niger spores. Growth parameters, sporulation and kinetics of gluconic acid production were analysed at different time intervals during the course of SSF. Spores of different age (48–216 h) were used as biocatalysts in the bioconversion reaction. Spores harvested at a later period of SSF (196 h) produced high titres of gluconic acid (30 g/L) in the bioconversion medium when compared to the spores harvested at early (48 h) stages of SSF (2.2 g/L). Spores (harvested at 200 h and stored in freezer for 91 days) exhibited the same glucose oxidase activity and served as an active and stable catalyst when compared to the fresh spores, showing that aging (storage) did not affect enzymes present in the spores, which suggested that the spores acted as an efficient enzyme reservoir. Yields close to 93 % were obtained with 98 g/L of gluconic acid production, corresponding to an average productivity of 1.7 g/(L·h). The stability of the enzyme in the spores and the ability of conidia to be stored for a long time without the loss of activity add specific advantage to the bioconversion process.Svrha je rada bila dokazati važnost konidijskih spora kao izvora glukoza-oksidaze, te istražiti stabilnost tih biokatalizatora pri proizvodnji glukonske kiseline. Spore plijesni Aspergillus niger proizvedene su uzgojem na čvrstoj podlozi u kolonskom bioreaktoru s nasutim slojem. Tijekom fermentacije, u različitim vremenskim intervalima, ispitani su parametri rasta, sporulacija i kinetika proizvodnje glukonske kiseline. Kao biokatalizator upotrijebljene su spore različite dobi (48-216 h). Spore dobivene nakon 196 h uzgoja proizvele su biokonverzijom mnogo glukonske kiseline (30 g/L), za razliku od spora prikupljenih nakon 48 h, koje su proizvele samo 2,2 g/L glukonske kiseline. Spore prikupljene nakon 200 h, čuvane 91 dan u zamrzivaču, imale su istu aktivnost glukoza-oksidaze kao i svježe spore, pa su upotrijebljene kao aktivni i stabilni katalizator. Time je dokazano da sazrijevanje (skladištenje) ne utječe na enzime u sporama, te da one mogu biti učinkovito spremište enzima. Pri proizvodnji 98 g/L glukonske kiseline, tj. produktivnosti od 1,7 g/(L·h) postignuto je skoro 93 %-tno iskorištenje. Prednosti su procesa biokonverzije stabilnost enzima u sporama i sposobnost skladištenja konidija duže vrijeme bez gubitka njihove aktivnosti

Microbiology and Industrial Biotechnology of Food-Grade Proteases: A Perspective

Najnovija dostignuća u industrijskoj biotehnologiji omogućila su primjenu novih i neotkrivenih mikroorganizama i razvoj poboljšanih postupaka u proizvodnji enzima. To je povećalo prinose enzima i olakšalo razvoj djelotvornijeg industrijskog postupka proizvodnje. U ovom je radu opisana proizvodnja kiselih i neutralnih proteaza, osobito što se tiče proizvođača, postupaka proizvodnje i njihovih poboljšanja, te proizvoda i njegove primjene.Recent developments in industrial biotechnology have resulted in the exploitation of new and undiscovered microorganisms and the devising of improved methods for enzyme production, which have led to increased yields of the enzyme, thus making a viable industrial process feasible. This review tracks the developments in the field of acidic and neutral protease production with regard to the producers, methods of production and their improvement, the product and its applications

Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

International audienceBackground: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results: The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. Conclusions: The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail