Sinteza oktil-β-glukozida u nekonvencionalnom reakcijskom sustavu, katalizirana pomoću enzima β-glukozidaza, izoliranog iz badema

The reaction system for octyl-β-glucoside synthesis catalysed by the almond-β-glucosidase has been characterised. The monophasic octanol saturated with different amounts of water served as a reaction medium. Both the glucose and the activated substrate p-nitrophenyl-β-glucoside were examined as glycon donors. The reverse hydrolysis and the transglycosylation were both used as reaction models for this enzymatically catalysed alkyl-β-glucoside synthesis. The rate of synthesis of octyl-β-glucoside (vS), the rate of hydrolysis, i.e. the glucose formation (vH) and the predicted yield (Y) were determined. The effect of water activity on the synthetic and hydrolytic activity of the enzyme was investigated. Both the rate of synthesis and the rate of hydrolysis increased with the increase of the water activity in the reaction system, showing their maximum values at the water activity close to the saturation level. Thus, the maximum ratio of vS/vH=0.165 was achieved at the water activity of 0.94. The predicted yields were 0.5, 0.75 and 14.19 % and were lower than the actually achieved yields of 19.45, 38 and 36.40 % at the water activities of 0.75, 0.84 and 0.94, respectively. The yield of octyl-β-glucoside in the reverse hydrolysis was only 15.2 %, i.e. 3.25 times lower than the yield obtained in the transglycosylation reaction with the water activity of 0.94. The solubility of glucose in pure octanol was only 1.5 mmol/L at the saturation level of 12 mmol/L in the presence of 10 mmol/L of p-nitrophenyl-β-glucoside, and it increased to 15.5 mmol/L in the presence of octyl-β-glucoside.U radu su ispitani parametri kemijske sinteze oktil-β-glukozida katalizirane pomoću enzima β-glukozidaza. Reakcija je provedena u monofaznom sustavu oktanol/voda. Kao donori glikonske skupine ispitani su glukoza i aktivirani p-nitrofenil-β-glukozid. Enzimska sinteza glikozida odvijala se na dva načina, reverznom hidrolizom i transglikozilacijom. Određeni su sljedeći parametri: brzina sinteze oktil glukozida, brzina hidrolize, tj. nastajanja glukoze i iskorištenje reakcije. Ispitan je i utjecaj aktiviteta vode na aktivnost enzima tijekom sinteze i hidrolize. Utvrđeno je da su se brzine sinteze i hidrolize povećale s povećanjem aktiviteta vode, a najveće su brzine reakcija postignute pri granici zasićenja vodom. Stoga je najveći omjer brzina sinteze i hidrolize od 0,165 postignut pri aktivitetu vode od 0,94. Eksperimentalno dobivene vrijednosti iskorištenja reakcije bile su znatno više od predviđenih: 19,45 % (u usporedbi s predviđenih 0,5 %) pri aktivitetu vode od 0,75; zatim 38 % (predviđeno 0,75 %) pri aktivitetu vode od 0,84, te 36,40 % (predviđeno 14,19 %) pri aktivitetu vode od 0,94. Prinos oktil-glukozida nastalog reverznom hidrolizom bio je samo 15,2 %, tj. 3,25 puta manji od prinosa dobivenog transglikozilacijom pri aktivitetu vode od 0,94. Topljivost glukoze u čistom oktanolu bila je samo 1,5 mmol/L pri granici zasićenja vodom, u prisutnosti 10 mmol/L p-nitrofenil-β-glukozida bila je znatno veća (12 mmol/L), a najveća (15 mmol/L) u prisutnosti oktil-β-glukozida

Uporaba jednostavnih i miješanih reverznih micela za enzimsku sintezu alkilnih glikozida – ekološki prihvatljivih površinski aktivnih tvari

Two types of microemulsion reaction systems: simple and mixed reverse micelles have been investigated as potential bioreactors for a transglycosylation reaction catalyzed by three microbial β-galactosidases: fungal Aspergillus oryzae, yeast Kluyveromyces marxianus and bacterial Escherichia coli β-galactosidase. Several issues such as: the effect of the degree of hydration on the total enzyme activity, the enzyme selectivity towards glycon donors, the interphase quality of simple and mixed micelles and the effect of addition of nonionic cosurfactants on the enzyme activity were all discussed. Both p-nitrophenyl-β-D-galactoside and p-nitrophenyl-β-D-glucoside were used as activated substrates. The total activity of all three examined enzymes showed to be strongly dependent on the degree of hydration of reverse micellar systems and had the highest values at the degree of hydration close to the level of saturation. The highest activity that the A. oryzae β-galactosidase had in simple micelles per mass of protein was 1.8 mmol/(min·mg). The ratio of β-galactosidase and β-glucosidase activities had values above 1 for all the enzymes entrapped in simple micelles, which means that their selectivity towards the p-nitrophenyl-β-galactoside as a glycon donor was strict and higher than that towards the p-nitrophenyl-β-glucoside. This ratio for the A. oryzae β-galactosidase was 1.68. The addition of nonionic cosurfactants had a positive effect on the enzyme activity. Thus, the total activity of A. oryzae β-galactosidase in dioctyl sodium sulfosuccinate/polyethylene glycol (AOT/PEG) mixed micelles with a mass ratio ζ(AOT, PEG)=1 was 1.5 times higher and in AOT/Tween mixed micelles with a mass ratio ζ(AOT, Tween)=0.033 it was even more than 3.5 times higher than the activity of enzyme entrapped in simple micellar reactor. The latter activity was even 4.22 times higher than that of A. oryzae β-galactosidase used as a crude enzyme preparation.U radu su istražena dva tipa mikroemulzijskih reakcijskih sustava, i to jednostavne i miješane reverzne micele, kao potencijalni bioreaktori za transglikozilaciju kataliziranu pomoću tri mikrobne β-galaktozidaze: iz plijesni Aspergillus oryzae, kvasca Kluyveromyces marxianus i bakterije Escherichia coli. Ispitani su sljedeći parametri: utjecaj stupnja hidratacije na ukupnu enzimsku aktivnost, selektivnost enzima spram donora glikonske grupe, kakvoća međufazne površine jednostavnih i miješanih micela, te utjecaj dodatka neionskih pomoćnih površinski aktivnih tvari na aktivnost enzima. Kao aktivirani supstrati upotrijebljeni su p-nitrofenil-β-D-galaktozid i p-nitrofenil-β-D-glukozid. Ukupna aktivnost sva tri enzima u sustavima reverznih micela pokazuje jaku ovisnost o stupnju hidratacije, a najveća je kad je stupanj hidratacije blizu zasićenja. Najveća aktivnost β-galaktozidaze iz A. oryzae u jednostavnim micelama bila je 1.8 mmol/(min∙mg proteina). Omjer aktivnosti β-galaktozidaze i β-glukozidaze bio je veći od 1 za sva tri enzima uklopljena u jednostavnim micelama, što znači da je njihova selektivnost prema p-nitrofenil-β-galaktozidu kao donoru glikonske grupe bila izrazita i veća od one prema p-nitrofenil-β-glukozidu. Taj je omjer za β-galaktozidazu iz A. oryzae bio 1.68. Dodatak neionskih pomoćnih površinski aktivnih tvari pozitivno je utjecao na enzimsku aktivnost. Ukupna aktivnost β-galaktozidaze iz A. oryzae u micelama sastavljenim od dioktil natrijevog sulfosukcinata i polietilen glikola (AOT/PEG) masenog omjera ζ(AOT, PEG)=1 bila je 1.5 puta veća, a u AOT/Tween micelama masenog omjera ζ(AOT, Tween)=0.033 čak više od 3.5 puta veća od aktivnosti enzima uklopljenog u jednostavnim micelama. Aktivnost β-galaktozidaze iz A. oryzae uklopljene u micelama je čak 4.22 puta veća od one sirovog (nepročišćenog) enzimskog preparata

Omjer reakcija transgalaktozilacije i hidrolize različitih β-galaktozidaza što kataliziraju sintezu alkil-β-galaktozida u monofaznim alkoholnim medijima

Three microbial galactosidases, Aspergillus oryzae, Escherichia coli and Kluyveromyces marxianus β-galactosidase, were used as catalysts for transgalactosylation synthesis of alkyl-β-galactosides in single-phased alcohol media. Their selectivity towards different alcohol nucleophiles was quantified by determining the transgalactosylation/hydrolysis ratio in the water/alcohol mixtures containing water in concentrations below the level of saturation. p-Nitrophenyl-β-galactoside was used as a glycosyl donor at a concentration of 10 mM. Both the total reaction rate (transgalactosylation+hydrolysis) and the ratio between the transgalactosylation (alcoholysis) and hydrolysis increased with the increase of water activity. Although the A. oryzae β-galactosidase showed relatively low total activity (3.13 μmol/(min·mg protein)), it exhibited the highest selectivity towards the hexanol nucleophile among the examined enzymes (0.65). The selectivity values in all the examined cases were below one, which implies that the hydrolysis, and not the synthesis, was the dominating reaction. The total reaction rate (transgalactosylation+hydrolysis) was strongly affected by the water activity, and for the specific water activity in the different alcohols, it increased in the following order: n-octanol, n-hexanol, n-butanol.Tri mikrobne galaktozidaze, Aspergillus oryzae, Escherichia coli i Kluyveromyces marxianus β-galaktozidaza, upotrijebljene su kao katalizatori sinteze alkil-β-galaktozida u monofaznim alkoholnim medijima transgalaktozilacijom. Njihova selektivnost prema alkoholnim nukleofilima kvantificirana je određivanjem omjera transgalaktozilacije i hidrolize u smjesama alkohola i vode čiji je udjel vode ispod razine zasićenosti. p-Nitrofenil-β-galaktozid uporabljen je kao glikozilni donor u koncentraciji od 10 mM. Ukupna brzina reakcije (transgalaktozilacija+hidroliza), a i omjer reakcija transgalaktozilacije (alkoholize) i hidrolize, povećavali su se s većom aktivnosti vode. Pritom je Aspergillus oryzae β-galaktozidaza, koja je inače pokazala relativno slabu ukupnu aktivnost (3,13 μmol/(min·mg) protein), imala najveću selektivnost prema nukleofilu heksanola (0.65). Vrijednosti faktora selektivnosti su u svim ispitanim slučajevima bili ispod jedinice, što upućuje na to da prevladava hidroliza, a ne sinteza. Ukupna brzina reakcije transgalaktozilacija+hidroliza) bila je pod jakim utjecajem aktivnosti vode, te je za određenu vrijednost aktivnosti vode rasla prema ovom redoslijedu: n-oktanol, n-heksanol, n-butanol

Utilization of different types of glucose oxidase for reduction of glucose concentration in synthetic grape juice

One of the most promising techniques for oxidation of glucose into a gluconic acid is the utilization of the enzyme glucose oxidase. In order to optimize the process, two types of enzymes were used as catalysts for glucose oxidation in several model synthetic grape juices. The first one is a food grade enzyme Alphamalt Gloxy 5080 from Aspergillus Niger. The other one is pure enzyme from Aspergillus Niger, used as a sole or in a combination with catalase isolated from beef liver. Both the pure glucose solution and the synthetic grape juice were used as substrates for enzymatic pretreatment. The Alphamalt Gloxy 5080 enzyme, used in a concentration of 1 g/L, showed 77.60% substrate conversion of the glucose used in a concentration of 10 g/L. the pure glucose oxidase having concentration of 25 mg/L converted only 1.32% of glucose, while when combined with 15 mL catalase, the conversion was even 49.25%

Utilization of Different Types of Glucose Oxidase for Reduction of Glucose Concentration in Synthetic Grape Juice

One of the most promising techniques for oxidation of glucose into a gluconic acid is the utilization of the enzyme glucose oxidase. In order to optimize the process, two types of enzymes were used as catalysts for glucose oxidation in several model synthetic grape juices. The first one is a food grade enzyme Alphamalt Gloxy 5080 from Aspergillus niger. The other one is pure enzyme from Aspergillus niger, used as a sole or in a combination with catalase isolated from beef liver. Both the pure glucose solution and the synthetic grape juice were used as substrates for enzymatic pretreatment. The Alphamalt Gloxy 5080 enzyme, used in a concentration of 1 g/L, showed 77.60% substrate conversion of the glucose used in a concentration of 10 g/L. the pure glucose oxidase having concentration of 25 mg/L converted only 1.32% of glucose, while when combined with 15 μL catalase, the conversion was even 49.25%.

Simple and Mixed Reverse Micelles as Potential Bioreactors for Enzymatic Synthesis of Alkyl Glycosides – Environmentally Friendly Surfactants

Two types of microemulsion reaction systems: simple and mixed reverse micelles have been investigated as potential bioreactors for a transglycosylation reaction catalyzed by three microbial β-galactosidases: fungal Aspergillus oryzae, yeast Kluyveromyces marxianus and bacterial Escherichia coli β-galactosidase. Several issues such as: the effect of the degree of hydration on the total enzyme activity, the enzyme selectivity towards glycon donors, the interphase quality of simple and mixed micelles and the effect of addition of nonionic cosurfactants on the enzyme activity were all discussed. Both p-nitrophenyl-β-D-galactoside and p-nitrophenyl-β-D-glucoside were used as activated substrates. The total activity of all three examined enzymes showed to be strongly dependent on the degree of hydration of reverse micellar systems and had the highest values at the degree of hydration close to the level of saturation. The highest activity that the A. oryzae β-galactosidase had in simple micelles per mass of protein was 1.8 mmol/(min·mg). The ratio of β-galactosidase and β-glucosidase activities had values above 1 for all the enzymes entrapped in simple micelles, which means that their selectivity towards the p-nitrophenyl-β-galactoside as a glycon donor was strict and higher than that towards the p-nitrophenyl-β-glucoside. This ratio for the A. oryzae β-galactosidase was 1.68. The addition of nonionic cosurfactants had a positive effect on the enzyme activity. Thus, the total activity of A. oryzae β-galactosidase in dioctyl sodium sulfosuccinate/polyethylene glycol (AOT/PEG) mixed micelles with a mass ratio ζ(AOT, PEG)=1 was 1.5 times higher and in AOT/Tween mixed micelles with a mass ratio ζ(AOT, Tween)=0.033 it was even more than 3.5 times higher than the activity of enzyme entrapped in simple micellar reactor. The latter activity was even 4.22 times higher than that of A. oryzae β-galactosidase used as a crude enzyme preparation

Synthesis of Octyl-β-Glucoside Catalyzed by Almond β-Glucosidase in Unconventional Reaction Media

The reaction system for octyl-β-glucoside synthesis catalysed by the almond-β-glucosidase has been characterised. The monophasic octanol saturated with different amounts of water served as a reaction medium. Both the glucose and the activated substrate p-nitrophenyl-β-glucoside were examined as glycon donors. The reverse hydrolysis and the transglycosylation were both used as reaction models for this enzymatically catalysed alkyl-β-glucoside synthesis. The rate of synthesis of octyl-β-glucoside (vS), the rate of hydrolysis, i.e. the glucose formation (vH) and the predicted yield (Y) were determined. The effect of water activity on the synthetic and hydrolytic activity of the enzyme was investigated. Both the rate of synthesis and the rate of hydrolysis increased with the increase of the water activity in the reaction system, showing their maximum values at the water activity close to the saturation level. Thus, the maximum ratio of vS/vH=0.165 was achieved at the water activity of 0.94. The predicted yields were 0.5, 0.75 and 14.19 % and were lower than the actually achieved yields of 19.45, 38 and 36.40 % at the water activities of 0.75, 0.84 and 0.94, respectively. The yield of octyl-β-glucoside in the reverse hydrolysis was only 15.2 %, i.e. 3.25 times lower than the yield obtained in the transglycosylation reaction with the water activity of 0.94. The solubility of glucose in pure octanol was only 1.5 mmol/L at the saturation level of 12 mmol/L in the presence of 10 mmol/L of p-nitrophenyl-β-glucoside, and it increased to 15.5 mmol/L in the presence of octyl-β-glucoside

Transgalactosylation/Hydrolysis Ratios of Various β-Galactosidases Catalyzing Alkyl-β-Galactoside Synthesis in Single-Phased Alcohol Media

Three microbial galactosidases, Aspergillus oryzae, Escherichia coli and Kluyveromyces marxianus β-galactosidase, were used as catalysts for transgalactosylation synthesis of alkyl-β-galactosides in single-phased alcohol media. Their selectivity towards different alcohol nucleophiles was quantified by determining the transgalactosylation/hydrolysis ratio in the water/alcohol mixtures containing water in concentrations below the level of saturation. p-Nitrophenyl-β-galactoside was used as a glycosyl donor at a concentration of 10 mM. Both the total reaction rate (transgalactosylation+hydrolysis) and the ratio between the transgalactosylation (alcoholysis) and hydrolysis increased with the increase of water activity. Although the A. oryzae β-galactosidase showed relatively low total activity (3.13 μmol/(min·mg protein)), it exhibited the highest selectivity towards the hexanol nucleophile among the examined enzymes (0.65). The selectivity values in all the examined cases were below one, which implies that the hydrolysis, and not the synthesis, was the dominating reaction. The total reaction rate (transgalactosylation+hydrolysis) was strongly affected by the water activity, and for the specific water activity in the different alcohols, it increased in the following order: n-octanol, n-hexanol, n-butanol

Preparation of lipases for use in organic solvents

The efficiency of different preparations of lipases was evaluated in organic solvents. Lipases from Humicola lanuginosa, Candida rugosa, Rhizomucor miehei and Pseudomonas cepacia were adsorbed onto the surfactant sorbitan monostearate (Span 60) and the specific activities were compared in hexane to crude powder (used straight from the bottle) and lipase freeze-dried from buffer solution. Lipases adsorbed on the surfactant were "activated" 1.9- to 150-fold compared to the crude lipase. The solubility of the lipase-surfactant preparation in the reaction media was extremely low and the preparation contained aggregates of micrometer size. In further comparisons lipase from H. lanuginosa was freeze-dried in the presence of KCl, crown ethers, immobilised by entrapment into a sol-gel and immobilised on porous polypropylene support (Accurel EP-100). Addition of potassium chloride before freeze-drying of the lipase increased the activity up to 46-fold compared to crude powder. The additive probably worked as an immobilisation matrix for the lipase. When 18-crown-6 was added to the lipase before freeze-drying a 40-fold increase in activity was achieved. In this case a low amount of additive (0.4 mg crown ether/g protein) was needed for activation indicating that specific interactions were involved in the activation. In order to obtain maximal activity, immobilisation on Accurel EP-100 and entrapment into a sol-gel were the best methods to use. The activities were 400- and 320-fold better than that of crude powder. The high activities obtained were due to an improved dispersion of the catalyst in the organic media. The protein loading that could be used when the lipase was adsorbed onto Accurel EP-100 was much higher than what could be used when the lipase was entrapped into a sol-gel. This makes the adsorption technique the best for practical applications. (C) 2002 Elsevier Science Inc. All rights reserved

Competition between transglycosylation and hydrolysis in almond beta-glucosidase-catalyzed conversion of p-nitrophenyl-beta-D-glucoside in monophasic water/alcohol mixtures

Almond beta-glucosidase was used to catalyze the transglycosylation of p-nitrophenyl-beta-D-glucoside to alkyl glucosides, with hydrolysis to glucose as a side reaction. The conversions were carried out in alcohols with varying water contents below water saturation. Both the total reaction rate and the ratio between the transglycosylation and hydrolysis increased with increasing water activity, and at a fixed water activity in the different alcohols, rate and transglycosylation/hydrolysis ratio increased in the following order: 1-octanol < 1-hexanol < 1-butanol. Synthesis of alkyl glucosides by transglycosylation in monophasic alcohol media is thus most favorable for short chain alcohols, and should be carried out at high water content