Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Production of a Thermostable Lipase by Humicola lanuginosa Grown on Sorbitol-Corn Steep Liquor Medium

For thermostable lipase production by Humicola lanuginosa No. 3, a simple optimized medium consisting of (%, w/v): sorbitol, 1.0; corn steep liquor, 1.0; NaCl, 0.5; CaCl₂–2H₂0, 0.01; Silicone Km-70 (antifoamer), 0.2; and whale oil or castor oil as a lipase inducer, 0.3, was used. The yield of the lipase was about 80 — 120U/ml after 25 hr aerobic cultivation at 45°C when the pH was maintained at 7 to 8. The acetone powder preparation of the enzyme was most active at pH 7.0 and 45°C. The enzyme retained 100% activity on incubation for 20 hr at 60°C. The enzyme was able to hydrolyze almost all forms of natural fats tested (14 kinds), coconut oil being the most rapidly hydrolyzed.     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

Production of isomaltooligosaccharides by a-glucosidase immobilized in chitosan beads and by polyethyleneimine-glutaraldehyde treated mycelia of Aspergillus carbonarious

Partially purified a-glucosidase from Aspergillus carbonarious, immobilized on glutaraldehyde-activated chitosan beads in a packed bed reactor, produced isomaltooligosaccharides at a yield of 60% (w/w) using 30% (w/v) maltose solution. Using intact mycelia attached with polyethyleneimine-glutaraldehyde, produced isomaltooligosaccharides at a yield of 46% (w/w) using 30% (w/v) maltose solution. Batchwise reaction stabilities were improved for chitosan beads immobilized enzyme and polyethyleneimine-glutaraldehyde treated mycelia as compared to mycelia without any treatment.     

Entrapment of β-galactosidase in polyvinylalcohol hydrogel

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g⁻¹) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.     

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic Thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Solid state fermentation of Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor for tannase production

Abstract

Production of tannase was performed in packed bed reactor filled with an inert support polyurethane foam (PUF) using Bacillus gottheilii M2S2. The influence of process parameters such as fermentation time (24–72?h), tannic acid concentration (0.5–2.5% w/v), inoculum size (7–12% v/v), and aeration rate (0–0.2?L/min) on tannase production with PUF were analyzed using one variable at a time (OVAT) approach. The outcome of OVAT was optimized by central composite design. Based on the statistical investigation, the proposed mathematical model recommends 1% (w/v) of tannic acid, 10% (v/v) of inoculum size and 0.13?L/min of aeration rate for maximum production (76.57?±?1.25?U/L). The crude enzyme was purified using ammonium sulfate salt precipitation method followed by dialysis. The biochemical properties of partially purified tannase were analyzed and found the optimum pH (4.0), temperature (40?°C) for activity, and K_m (1.077?mM) and V_max (1.11?µM/min) with methyl gallate as a substrate. Based on the SDS-PAGE analysis, tannase exhibited two bands with molecular weights of 57.5 and 42.3?kDa. Briefly, the partially purified tannase showed 4.2 fold increase (63?±?1.60?U/L) in comparison to the submerged fermentation and the production of tannase was validated by using NMR spectrometer.     

Optimization of Enzymatic Hydrolysis Conditions of Caspian kutum (Rutilus frisii kutum)ˮ By-product for Production of Bioactive Peptides with Antioxidative Properties

The enzymatic hydrolysis was performed by Alcalase to recover the fish protein hydrolysate from Caspian kutum by-product (CB). The degree of hydrolysis (DH) was applied for monitoring the hydrolysis reaction of CB. The response surface methodology was applied based on a D-optimal design to perform the optimization process for obtaining the high yield of CB protein hydrolysate. The effect of four independent variables including pH (7.5–8.5), temperature (45–55 °C), time (1–3 h), and enzyme concentration (0.5–1.5% w/w) on DH was studied. The results indicated that the predicted and actual values of the optimum condition had no significant difference. The optimum enzymatic hydrolysis conditions were achieved at pH 8.5, temperature of 55 °C, enzyme concentration of 1.5% w/w, and time of 3 h, which resulted in the maximum value of DH (19.08%). Antioxidant assays including DPPH scavenging and metal chelating activities showed that Caspian kutum protein hydrolysates had antioxidant properties.

     

Production and Characterization of a Biosurfactant from Cyberlindnera samutprakarnensis JP52T

Cyberlindnera samutprakarnensis JP52^T, isolated from cosmetic industrial wastes in Thailand, was found to be an efficient biosurfactant-producing yeast when cultured in a medium containing (2% (w/v) glucose and 2% (v/v) palm oil at 30 °C, 200 rpm for 7 d. The crude biosurfactant had the ability to reduce the surface tension from 55.7 to 30.9 mN/m at 25 °C with a critical micelle concentration (CMC) of 0.046%. Physicochemical analysis of the crude biosurfactant revealed that it had wide ranges of optimum pH and pH stability at 6–9 and 3–10 respectively. It was also thermostable and retained 80% activity even after heat treatment, and it tolerated NaCl at 1.0–10%. Furthermore, it effectively emulsified various vegetable oils with an E24 value of over 80%. A partially purified biosurfactant fraction was analyzed for its structure by MALDI-TOF MS and NMR. This revealed that the biosurfactant mainly contained sophorolipids in C18-(MW 574) and C16-diaceltylated (MW 662) forms.     

Acid and enzymic hydrolysis of chaotropically pretreated millet stalk,acha and rice straws and conversion of the products to ethanol

Successful attempts have been made using acid and enzyme hydrolyses and chaotropic ions to solubilize some common but valuable sources of cellulose which are somewhat inaccessible to useful conventional degradative reagents/systems. Millet stalk, acha and rice straw (15% w/v) were incubated at 50°C for 1 h in 4 m hydrochloric acid saturated in lithium chloride, then hydrolysed for 45–60 s at 100°C, to yield maximally 79.2, 84.0 and 74.7% hydrolysis products for the three straws, respectively. Samples incubated in 1 m hydrochloric acid, containing saturated lithium chloride for 24 h at 27°C, washed thoroughly, freeze dried, then hydrolysed (0.5% w/v) with 0.4% w/v Trichoderma viride cellulase MVA 1284 [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] gave 16.4, 34.4 and 52.8% hydrolysis products for millet stalk, acha and rice straw, respectively. In general conversion of hydrolysed products direct to ethanol may be difficult as yeasts cannot grow in high salt concentrations resulting from acid hydrolyses. Growth response curves for two strains of Saccharomyces cerevisiae show the results of prior removal of chaotropic ions for successful growth of the organisms on the hydrolysates.     

Culture Conditions for the Production of Alkaline Protease from n-Paraffins by a Kabicidin Resistant Mutant No. 5–128B

A novel process for the microbial production of alkaline protease on an industrial scale was successfully established by using a kabicidin resistant mutant, No. 5–128B, derived from Fusarium sp. S–19–5. The most suitable carbon source for producing alkaline protease was n-paraffins (C₁₀~C₁₄) and the effective nitrogen source was dried-yeast cells containing no nucleic acid, the optimum concentrations being 12.5% (w/v) and 7.0% (w/v), respectively. The optimal temperature and initial pH for protease production were 24°C and 6.0, respectively. Under the optimal conditions using a shaker flask mutant No. 5–128B produced 41000 PU/ml of alkaline protease, which corresponded to about 10 times the amount produced by the parent strain. The relation between the high ability to produce alkaline protease and the resistance to kabicidin, a polyene antibiotic, is discussed.     

Hydrolysis of lactose in acid whey using beta-galactosidase immobilized on porous glass particles: preparation and characterization of a reusable catalyst for the production of low-lactose dairy products

Partially purified lactoses (β-D -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus niger, Ladobacillus helveticus, and Saccharomyces lactis were immobilized on diazotized porous glass particles (mean pore diameter, 86.5 nm: particle size diameters, 75–125 μm). In acid whey containing 4–4.5% lactose, A. niger lactase gave the highest activity (89 μmoles lactose hydrolyzed/g glass, min) at 55°C and pH 4.5. Glass-immobilized A. niger laclases (lactase-BG) retained much hydrolytic activity after storage and periodic use for 165 days at 55°C. For values of X greater than 30%, hydrolysis of 0.12M lactose in acid whey by a continuous flow column packed with 2 ml of lactase-BG particles could be correlated by X = 17.2(V/F) + 12.5 where X = lactose hydrolysis, percent of lactose originally present; V = volume of packed bed of lactase-BG, ml; F = flow rate of acid whey, ml/min.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Ultrasound‐assisted dilute acid hydrolysis of tea processing waste for production of fermentable sugar

Lignocellulosic materials that are the most abundant plant biomass in the world have the potential to become sustainable sources of the produced value added products. Tea processing waste (TPW) is a good lignocellulosic source to produce the value added products from fermentable sugars (FSs). Therefore, the present study is undertaken to produce FSs by using ultrasound‐assisted dilute acid (UADA) and dilute acid (DA) hydrolysis of TPW followed by enzymatic hydrolysis. UADA hydrolysis of TPW was optimized by response surface methodology (RSM) at maximum power (900 W) for 2 h. The optimum conditions were determined as 50°C, 1:6 (w/v) solid:liquid ratio, and 1% (w/v) DA concentration, which yielded 20.34 g/L FS concentration. Furthermore, its DA hydrolysis was also optimized by using RSM for comparison and the optimized conditions were found as 120°C, 1:8 solid:liquid ratio, and 1% acid concentration, which produced 25.3 g/L FS yield. Even though the produced sugars with UADA hydrolysis are slightly less, but it can provide significant cost saving due to the lower temperature requirement and less liquid consumption. Besides, enzymatic hydrolysis applied after pretreatments of TPW were very more economic than the conventional enzymatic hydrolysis in the literature due to shorter time requiring. In conclusion, ultrasound‐assisted is a promising technology that can be successfully applied for hydrolysis of biomass and can be an alternative to the other hydrolysis procedures and also TPW can be considered as suitable carbon source for the production of value‐added products like biofuels, organic acids, and polysaccharides. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:393–403, 2016     

Enzymatic decrease in the phenolic content of canola meal using concentrated aqueous or aqueous/hexane slurries

An enzymatic process to decrease the phenolic content in canola meal was investigated. The new method was based on the addition of an enzyme preparation from the white-rot fungus Trametes versicolor to concentrated meal-buffer slurries. This approach eliminated the extraction of the valuable meal components such as proteins and carbohydrates. Two systems were considered: (i) slurries with canola meal concentrations higher than 33% [w/v]; (ii) slurries with canola meal concentrations equal to or less than 12.5% [w/v] with n-hexane as the main component of the continuous phase. The concentration of sinapic acid esters decreased by 99% after a 1.5, 2 and 3 hour long treatment of the meal with an initial moisture content of 75% at 90°C, 70°C and 50°C, respectively. The process was carried out at temperaturs as high as 110°C. Both the enzyme and the moisture concentrations influenced the enzymatic process and their action was coupled. The concentration of oxygen strongly affected the process. The enzymatic process was able to be carried out in the presence of hexane as the main component of the continuous phase. The optimum temperature for such a process was 30–40°C, At 30°C, after 1 h of treatment, the meal phenolic content was decreased by 97%. The water uptake by the meal was diminished in the presence of hexane.     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

ULTRASONIC-ASSISTED PRODUCTION OF ANTIOXIDATIVE POLYSACCHARIDES FROM Crassostrea hongkongensis

The beneficial effects of oyster extract against various disorders and diseases induced by oxidative stress have aroused great interest. In this article, ultrasonic-assisted enzymolysis was employed to produce polysaccharides of Crassostrea hongkongensis (CHP) and their antioxidant activity was investigated. A single-factor experiment and then a four-factor, three-level Box–Behnken design were first used to optimize ultrasonic extraction for polysaccharides. On the basis of ridge analysis, the optimum conditions are obtained as ultrasonic treatment time of 24 min, power of 876 W, temperature of 49°C, and material–solvent ratio of 1:6 (w/v). It is found that ultrasound pretreatment before protease hydrolysis was a great help to improve CHP yield and purity, especially more favorable with flavorzyme, neutrase, alcalase, and pepsin. Furthermore, the polysaccharide fraction, which was obtained by ultrasonic pretreatment and then alcalase hydrolysis at the conditions of 3000 U/g, 55°C, pH 8.0, for 4 hr, exhibited an obvious scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (98.48 ± 0.55% and 99.20 ± 0.12%, respectively) and a lenoleic acid peroxidation inhibition effect (85.48 ± 0.65%) at a concentration of 5.0 mg/mL. These results reveal the potential application of CHP in functional food and nutraceuticals.     

An efficient method for the preparation of protoplasts fromTrichoderma viride

Conditions for an efficient high-yield procedure for the preparation of protoplasts fromTrichoderma viride have been determined. The optimum yield of protoplasts was obtained using 15–18-h-old unbranched mycelia, 0.7 mol/L KCl in phosphate buffer (pH 6), and 5 % (W/V) of lyophilized snail gut-juice enzyme. The conversion of mycelia to protoplasts was complete within 40–60 min incubation at 30 °C.     

Hydrolysis of rice bran oil using an immobilized lipase from Candida rugosa in isooctane

The kinetics of enzymatic hydrolysis of rice bran oil in isooctane by immobilized Candida rugosa lipase in a batch reactor showed competitive inhibition by isooctane with a dissociation constant, K1, of 0.92 M. Continuous hydrolysis of rice bran oil was performed in recycling, packed bed reactor with 4352 U of immobilized lipase; the optimum recycle ratio was 9 and the operational half-life was 360 h without isooctane but 288 h with 25% (v/v) isooctane in rice bran oil.