Characteristics of immobilised β‐glucosidase and its effect on bound volatile compounds in orange juice

In this study, bound volatile compounds were isolated and extracted with Amberlite XAD‐2 resin and then hydrolysed by free or immobilised β‐glucosidase. The released bound volatiles were analysed by GC‐MS. In addition, the optimisation of immobilisation method on sodium alginate and the characteristics of immobilised β‐glucosidase were studied. The results showed that crosslinking‐entrapment was the best method. The optimal conditions of this method were as follows: sodium alginate concentration 3.5%, glutaraldehyde concentration 1%, crosslinking time 3 h, immobilisation time 2 h and CaCl₂ concentration 3%. The optimum temperature for β‐glucosidase (65 °C) was decreased by 10 °C after immobilisation, while the optimum pH values for free and immobilised β‐glucosidase were both at pH 5.0. The K_m values of free and immobilised β‐glucosidase were 14.89 and 0.59 m , respectively. In total, thirteen and six bound volatile compounds were detected in orange juice hydrolysed by free and immobilised β‐glucosidase, including benzenic compounds, terpenic compounds, hydroxy esters, C₁₃‐norisoprenoids and alcohols.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

Properties and applications of β‐glycosidase from Bacteroides thetaiotaomicron that specifically hydrolyses isoflavone glycosides

To modify the glycan part of glycosides, the gene encoding β‐glycosidase was cloned from Bacteroides thetaiotaomicron VPI‐5482. The cloned gene, bt_1780, was expressed in Escherichia coli MC1061 and the expressed enzyme was purified using Ni‐NTA affinity chromatography. The purified enzyme, BTBG, showed optimal activity at 50 °C and pH 5.5. Interestingly, this enzyme did not have any hydrolysing activity on ordinary β‐linkage–containing substrates such as xylobiose, lactose and cello‐oligosaccharide, but specifically hydrolysed isoflavone glycosides such as daidzin, genistin and glycitin. Compared to a commercial beta glucosidase, BTBG selectively hydrolysed isoflavone glycosides in soybean extract mixture solution. These results suggest that BTBG may be a specialized enzyme for the hydrolysis of glycosides and that the substrate specificity of BTBG is applicable for the bioconversion of isoflavone glycosides in the food industry.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Characterization of an extracellular β‐d‐fructofuranosidase produced by Aspergillus niveus during solid‐state fermentation (SSF) of cassava husk

β‐d ‐Fructofuranosidases are biotechnologically important enzymes produced by various organisms. Here, Aspergillus niveus produced an extracellular β‐d ‐fructofuranosidase during SSF of cassava husk. This enzyme was purified 8.5‐fold (recovery of 5.2%). A 37‐kDa protein band was observed after 8% SDS‐PAGE. Native molecular mass is 91.2 kDa. Optimal temperature and pH of activity were 55°C and 4.5, respectively. The enzyme was stable at 50°C for 1 hr, and 80% of its activity was retained after 1 hr at pH 8.0. The enzymatic activity was improved by Mn²⁺, was resistant to most solvents, and was inhibited by Triton X‐100 and Tween 20. K_m and V_max with sucrose were 22.98 mM and 120.48 U/mg of protein, respectively. With Mn²⁺, these values were 16.31 mM and 0.30 U/mg of protein. The enzyme did not hydrolyze inulin and for this reason can be considered a true invertase. Thus, A. niveus β‐d ‐fructofuranosidase holds promise for invert sugar production.

Practical applications

β‐d ‐Fructofuranosidase is an enzyme that can be applied to different industrial sectors, especially food and beverage industries. It is responsible for the hydrolysis of sucrose and yields an equimolar mixture of D‐glucose and D‐fructose, named as inverted sugar syrup, with broad applications in the confectionery industry. The Aspergillus niveus enzyme hydrolyzed only sucrose here and can be considered a true invertase, showing its potential for application to invert sugar production. Besides, the use of cassava husk for enzyme production means an interesting utilization route of this agroindustrial residue. Thus, characterization of this enzyme is an important step for identification of its potential for practical applications.     

Production of feed enzymes from citrus processing waste by solid‐state fermentation with Eupenicillium javanicum

Bingtang sweet orange processing waste was utilised to produce four feed enzymes (Endoglucanase, β‐glucosidase, pectinase and xylanase) by the solid‐state fermentation (SSF) with Eupenicillium javanicum. The factors related with SSF including moisture content, temperature, initial pH, time, carbon source (0.5 g), nitrogen sources (0.05 g), inorganic mineral salts (0.1 g) were investigated separately. The corresponding optimal condition was: moisture content 80% (w/w), temperature 30 °C, natural pH, time 96 h, wheat bran 0.5 g, (NH₄)₂SO₄ 0.05 g or NaNO₃ 0.05 g, CaCl₂ 0.1 g. The L₉(3⁴) orthogonal experiment results showed that the optimal condition for producing above multiple enzymes was: moisture content 80% (w/w), temperature 30 °C, wheat bran 1 g, (NH₄)₂SO₄ 0.05 g, NaNO₃ 0.05 g, CaCl₂ 0.1 g, fermentation time 96 h and natural pH. Under this condition, the average activity of Endoglucanase (CMCase), β‐glucosidase, pectinase and xylanase by E. javanicum could reach 46.80, 49.64, 51.87 and 106.42 U g^?1, respectively, which was significantly higher than those in single factor experiments. Our present results demonstrated that E. javanicum could also be an effective and useful fungus for multienzyme preparation especially for β‐glucosidase and xylanase from citrus processing wastes.     

Preparation of lactose‐free milk by using salt‐fractionated almond (Amygdalus communis) β‐galactosidase

β‐galactosidase was isolated from almond (Amygdalus communis) extract by ammonium sulfate precipitation. Almond proteins precipitated by using ammonium sulfate and then dialysed exhibited 5.3‐fold purification of β‐galactosidase, and the yield of enzyme preparation was 96.5%. The partially purified β‐galactosidase exhibited pH and temperature optima at pH 5.5 and 50 °C, respectively. The enzyme was significantly stable against heat, pH, calcium and magnesium ions and D ‐galactose. The almond β‐galactosidase preparation exhibited over 89% activity even after 2 months storage at 4 °C. Hydrolysis of lactose in milk and whey was performed in a stirred batch process by using this enzyme preparation. These observations indicated that the hydrolysis of lactose increased continuously with time. The enzyme could hydrolyse 94% of lactose in buffer solution and whey whereas 90% of lactose hydrolysis was achieved in milk. The main aim of the present study was to prepare lactose‐free milk, which must be free from contamination, and the process should be inexpensive. Copyright © 2007 Society of Chemical Industry     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

α‐Amylase immobilization on functionalized nano CaCO3 by covalent attachment

In this study, α‐amylase was immobilized on glutaraldehyde activated silanized calcium carbonate nanoparticles by a using covalent binding method. The surface modified nano calcium carbonate (CaCO₃) were characterized using FTIR and SEM. Immobilization yield was found as 199.43 mg/g of calcium carbonate nanoparticles. The maximum activity was observed at pH 6.5. The immobilized enzyme had a higher activity at elevated temperature (50–90°C) than the free one. Reuse studies demonstrated that the immobilized enzyme could reuse 25 times while retaining 18.2% of its activity. Free enzyme lost its activity completely within 15 days. V_max values for the free and immobilized enzymes were calculated as 10 and 0.35 mg/mL/min, respectively.     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

Vanilla curing under laboratory conditions

A laboratory model curing is described in which the cured vanilla beans are analysed for enzyme activity and aroma. The activity of the enzymes was highest in green beans. β-Glucosidase (β-Glu) could not be detected after 24 h of autoclaving. Peroxidase (PER) and protease (PROT) activity decreased, but were still present (20%) after 29 days. Phenylalanine ammonia lyase (PAL) survived autoclaving, but was not detected later in the process. Beans that were scalded for 20 min at 80 °C showed no detectable β-Glu and PAL activity, but PROT and PER were still active. Under traditional curing conditions glucovanillin (GV) and glucovanillic acid (GVA) were hydrolysed to vanillin and vanillic acid, respectively. Upon scalding for 20 min at 80 °C the concentration of glucosides was still high (after 16 day: GV 2000 ppm, GVA 700 ppm). This may be an indication that the normal scalding leads to inactivation of a non-specific glucosidase, while the prolonged scalding also inactivates a specific glucosidase.     

Effects of malting on β‐glucanase and phytase activity in barley grain

The effects of malting on β‐glucan and phytate were investigated in one naked and one covered barley by a full factorial experiment with three factors (steeping temperature, moisture content and germination temperature) each with two levels. Analysis of total content of β‐glucan in the malted samples showed small changes after steeping at the high temperature (48 °C), while steeping at the lower temperature (15 °C) gave a significantly lower content. This trend was even stronger for β‐glucan unextractable at 38 °C. Analysis of the activity of β‐glucanase for the samples steeped at 15 °C showed a strong increase over the time of germination, while those steeped at 48 °C had a much slower development. The other two factors influenced the outcome to a small extent, mainly because the steeping temperature was the most important factor overall where any changes in β‐glucan and β‐glucanase were observed. When β‐glucan was extracted at 100 °C, a larger yield was obtained, and this was influenced by the steeping temperature in a much stronger way than for β‐glucan extracted at 38 °C. Determination of average molecular weight for β‐glucan extracted at 100 °C gave a lower value for samples steeped at 15 than at 48 °C. The design did not have any large effects on phytate degradation and phytase activity. However, it indicated that selective control of the enzymes might be possible, since phytase activity was barely affected by the parameters studied, while β‐glucanase was heavily affected. © 2002 Society of Chemical Industry     

Immobilization of amyloglucosidase on polystyrene anion exchange resin I. preparation and properties

Amyloglucosidase (exo‐1,4‐ α‐D‐glucosidase, E C 3.2. 1.3) was coupled to glutaraldehyde activated Indion 48‐R (a cross‐linked macroporous anion exchanger) by Schiff base reaction. The bound enzyme exhibited 60–70% activity of the free enzyme. Substrate concentrations as high as 32% (w/w) liquefied tapioca starch could be quantitatively converted into 96–98% (w/w) dextrose in 24 h at 50°C and pH 4.5. Though immobilization lowered the temperature optimum to 50–60°C from 65°C for the free enzyme, it increased the temperature stability. However, there was no change either in the pH optimum or pH stability after immobilization. In batch operations, the immobilized preparation showed a half life of 32 and 12 days at 50°C and 60°C respectively.     

Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Chaperone‐like activity of β‐casein and its effect on residual in vitro activity of horseradish peroxidase

In this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone‐like activity of β‐casein under elevated temperature. It was shown that β‐casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between proteins. Incubating HRP (0.1 mg mL^?1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL^?1 β‐casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β‐casein on HRP disappears when pH is below the isoelectric point of β‐casein. It was also proven by light scattering studies that β‐casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries such as enzyme manufacturers and food industry.