Purification and isolation of β-glucans from barley: Downstream process intensification

The purpose of this work was to study the purification and isolation (downstream process) of the β-glucan extracted from barley in an ultrasound assisted extraction (UAE) process. The co-extracted starch (average concentration 5.2 ± 0.1 g/L) was hydrolyzed by means of α-amylase. The optimization of the hydrolysis length, temperature and enzyme dose led to removal efficiencies higher than 90% (9 min hydrolysis length and enzyme doses of 100 μL/g at 55 °C), compared to the traditional hydrolysis processes (1 h at 95 °C). In a second step, a significant intensification of the process has been achieved by dosing the enzyme during the UAE step (7 min at 55 °C), resulting in a starch removal of 90%.Dextrins and other oligosaccharides were formed as a consequence of the enzymatic hydrolysis. In order to separate these low molecular weight molecules from the β-glucan (239 kDa), an ultrafiltration process (polysulfone membrane, nominal MWCO 100 kDa) was tried in a tangential flow cell: diafiltration successfully eliminated more than 45% of the oligosaccharides present in the liquid, providing a significant increase in the concentration of β-glucan and with the possibility of improving the percentage of elimination.The combination of these three technologies (UAE, enzymatic hydrolysis and diafiltration) allows getting high purity β-glucan concentrates (greater than 70%).     

Production of fermentable sugars from enzymatic hydrolysis of pretreated municipal solid waste after autoclave process

A ligno-cellulosic concentrate from municipal solid waste (MSW) obtained after an autoclave separation process was investigated for its potential as a feedstock to produce fermentable sugars for ethanol production. A maximum enzymatic hydrolysis conversion of 53% of the cellulose and hemi-cellulose was found using a particle size range of 150–300 μm hydrolyzed in a 100 ml buffer solution containing 6 wt% lingo-cellulosic MSW concentrate with 90 mg cellulase at pH 4.8 held at 40 °C for 12 h. The hydrolysis rate leveled off at longer hydrolysis time and with increased substrate concentration and was related to enzymatic access to substrate. Lower hydrolysis rate at smaller particle size indicates that the grinding process may change the surface chemistry or morphology of the fibers making them less available for enzyme access. A drop in the hydrolysis rate was observed for the particles above 300 μm associate with the longer diffusion time for the enzyme into the fiber particles. The findings indicate that 152 L of ethanol could be obtained from a ton of lingo-cellulosic concentrate from MSW.     

Lemon juice clarification using fungal pectinolytic enzymes coupled to membrane ultrafiltration

Lemon juice was treated with Penicillium occitanis pectinase at various enzyme concentrations (0–1200 U/L), temperatures (25–50 °C) and times (0–90 min). The effect of these enzymatic treatments on the viscosity of the juice was evaluated. The optimum treatment conditions were: enzyme concentration 600 U/L, time 45 min and temperature 30 °C. Their application led to a 77% and 47% reduction of viscosity and turbidity, respectively. The enzymatic treatment was followed by ultrafiltration (cutoff value = 15 kDa). Analysis of the clarified juice indicated that enzyme depectinization permitted a higher permeate flux and a higher juice quality. The lemon juice obtained was clear, stable and characterized by viscosity = 0.7 mPa s, turbidity = 0.17 NTU, clarity (A_650nm) = 0.063 and color (A_420nm) = 0.232. Microbiological study showed that lemon juice was free from aerobes, molds, enterobacteriaceae and coliforms and was microbiologically stable during 3 months storage. Results suggested that enzymatic treatment coupled to ultrafiltration could be used for production of lemon juice with high commercial value.     

Optimization of enzymatic pretreatment for n-hexane extraction of oil from Silybum marianum seeds using response surface methodology

An investigation on enzymatic pretreatment for n-hexane extraction of oil from the Silybum marianum seeds was conducted. The optimum combination of extraction parameters was obtained with the response surface methodology (RSM) at a four-variable and five-level central composite design (CCD). The optimum parameters of enzymatic pretreatment were as follows: enzyme concentration of 2.0% (w/w), temperature of 42.8 °C, reaction time of 5.6 h, and pH of 4.8. After enzymatic pretreatment, the oil was extracted by n-hexane for 1.5 h, and the oil yield on a dry basis was 45.70%, which well matched with the predicted value (45.86%). The results of the effects of the enzymatic pretreatment for n-hexane extraction of oil from the aspects of oil yield, microstructure and the fatty acid compositions showed that the enzymatic pretreatment had not affected on the fatty acid compositions, but could cause structure breakage of the S. marianum seeds and accelerate releasing extra oil, which increased the oil yield by 10.46% compared with n-hexane extraction for 1.5 h without enzymatic pretreatment, and confirmed the efficacy of enzymatic pretreatment for n-hexane extraction of oil from the S. marianum seeds.     

Evaluation of radish (Raphanus sativus L.) peroxidase activity after high-pressure treatment with carbon dioxide

The objective of this work was to assess the influence of compressed CO₂ on the specific activity of peroxidase (POD) from radish (Raphanus sativus L.). The protein concentrate, in phosphate buffer, was obtained through the precipitation of proteins with (NH₄)₂SO₄. The peroxidase activity was determined using guaiacol as substrate. A semi-factorial experimental design was adopted to evaluate the effects of temperature (30–50 °C), exposure time (1–6 h), carbon dioxide density (ρ_R = 0.6–1.8, resulting in pressures from 70.5 to 254.4 bar), and depressurization rate (10–200 kg/m³/min) on the peroxidase activity. Samples of lyophilized proteic concentrate were also submitted to high pressure and the effect of water content on the enzymatic activity in compressed CO₂ was studied. The water content in lyophilized samples was determined by thermo-gravimetric analysis. The results showed that the treatment of the enzymatic concentrate with CO₂ at high pressure could be a promising route to increase the POD activity.     

Split addition of enzymes in enzymatic hydrolysis at high solids concentration to increase sugar concentration for bioethanol production

One challenge in making bioethanol production economical is to increase total solids in hydrolysis system while maintaining sugar conversion efficiency. Because the removal of excess water from hydrolysate requires enormous amounts of heat, large volume of reaction towers and high capital expenditure (CAPEX) for equipment, a lengthy operating time, and high operating costs. When solids loading in hydrolysis system increased from 5% to 20% with no mixing strategies, final sugar conversion decreased markedly. If cellulase is mixed with pulp at 5% solids and pressed to 20% solids, then 80% of the cellulase retained in the pulp thinned down the pulp mixture in 2 h. This thinning effect enabled additional cellulase, xylanase, and β-glucosidase to be mixed into the slurry. Sugar concentration was significantly improved; from 26 g/L to 121 g/L, while sugar conversion was remained as enzymatic hydrolysis with 5% total solids enzymatic hydrolysis. A US patent has been granted to NCSU for this concept and licenses have been granted to various companies.     

Fructooligosacharides production in aqueous medium with inulinase from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 immobilized and treated in pressurized CO2

This work investigated the influence of compressed CO₂ treatment on the enzymatic activity of immobilized inulinases, and the production of fructooligosacharides in aqueous medium using these enzymes. The effects of system pressure, exposure time and depressurization rate on the enzymatic activity were evaluated through central composite designs (CCD) 2³. Inulinase from Kluyveromyces marxianus NRRL Y-7571 presented an increase of 104% in the residual activity using CO₂ at 275 bar submitted to 6 h treatment, at a depressurization rate of 10 kg m^?3 min^?1. For Aspergillus niger commercial inulinase, a decrease in enzyme activity was observed (residual activity of 39%) using CO₂ treatment at 75 bar for 6 h exposure at the highest depressurization rate (200 kg m^?3 min^?1). Enzymatic activities changed significantly depending on the enzyme source and the experimental treatment conditions investigated. The values of FOS obtained using inulinases from A. niger were 30.64% of GF2; 13.90% of GF3 and 2.88% of GF4 in the medium containing inulin as substrate. Results demonstrate that the use of compressed CO₂ might be of technological importance as a preceding, preparation step, to improve enzyme activity, hence helping the development of new biotransformation processes.     

Comparison of red,brown and green seaweeds on enzymatic saccharification process

The production of bioethanol from seaweeds using acid hydrolysis and the enzymatic saccharification was studied. Red seaweed (Gelidium amansii), brown seaweed (Laminaria japonica), and green seaweed (Codium fragile) were selected, and the characteristics of their conversion to bioethanol were analyzed. The optimum conditions of the dilute acid hydrolysis preprocessing for bioethanol production from the seaweed were a reaction temperature of 150 °C, sulfuric acid content of 5.0 wt.%, and reaction time of 60 min. The seaweeds listed in order of bioethanol conversion performance are red seaweed > brown seaweed > green seaweed. The optimum dosage of enzyme was 2.0 mL per 10 g of seaweed. The optimal fermentation conditions for bioethanol production using seaweed included a commercial yeast dosage of 30 wt.% and a fermentation time of 3 days.     

A hybrid photocatalytic and enzymatic process using glucose oxidase immobilized on TiO2/polyurethane for removal of a dye

A rectangular recycling photo-bioreactor using glucose oxidase (GOx) immobilized on TiO₂/polyurethane (PU) was developed as a novel coupling of photodegradation and enzymatic process. This method was tested for removal of Acid Orange 7 (AO7), as a model pollutant. High efficiency of decolorization (>99%) was achieved after 22 min using the GOx/TiO₂/PU photo-biocatalyst. Roles of various processes including photodegradation (TiO₂/PU), enzymatic process (GOx/PU) and a coupling of photocatalytic–enzymatic (GOx/TiO₂/PU) process were investigated in the presence and absence of UV light. All the experiments were performed in a circulation photoreactor equipped with a 6 W UV lamp with rate of 5 mL/min.     

Kinetic enzymatic resolution in scCO2 – Design of continuous reactor based on batch experiments

We developed an efficient method for the lipase-catalyzed consecutive kinetic resolution of trans-1,2-cyclohexanediol in scCO₂ with vinyl-acetate as acetyl donor catalyzed by a commercial immobilized Candida antarctica lipase B (CAL-B). The reaction was optimized in scCO₂ in a batch reactor. The first acylation step is moderately enantioselective, preferring the formation of (1R,2R)-2-acetoxycyclohexane-1-ol, while the second acylation step is fully enantioselective. Michaelis–Menten type reaction constants and turnover number values were calculated. A combined extractor–enzymatic packed-bed reactor unit was designed with a residence time of a few seconds (the time requirement of a batch reaction was several hours). The reactions were performed at 10 MPa and 45 °C. The mean residence time in the enzymatic reactor was varied from 2 to 13 s by changing the flow rate of the CO₂. The implemented continuous reactor was optimized to achieve maximum productivity and enantiopure products. The optimum residence time in the reactor entirely confirmed the calculated operational parameters (calculated necessary residence time: 9.6 s, measured mean residence time for full conversion: 9 s). No loss of enzyme activity was observed after 28 h at continuous operation.     

Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution

Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of l-glutamate to form γ-aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L^− 1·min^− 1·(mg protein)^− 1, 120% higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.     

Electrochemical synthesis of a novel poly(2,5-dimethoxy aniline) nanorod for ultrasensitive glucose biosensor application

We report for the first time a rapid electrochemical synthesis of one-dimensional poly(2,5-dimethoxyaniline) nanorods (PDMA-NR) in the presence of surfactant. FE-SEM and TEM images confirm the PDMA-NR formation and the average diameter of single rod sizes in the range of ∼200–300 nm. An enzymatic glucose biosensor was fabricated through immobilizing glucose oxidase (GOx) into PDMA-NR matrix. The amperometric current response of PDMA-NR/GOx to glucose is linear in the concentration range between 1 and 10 μM with a detection limit of 0.5 μM (S/N = 3). The PDMA-NR/GOx electrode possesses high sensitivity (5.03 μA/μM), selectivity, stability, and reproducibility toward glucose.     

Effect of manganese(II) on the respiratory activity of biological sludge from wastewater treatment plant

The aim of this study was to determine the effect of manganese(II) on the respiratory activity and cellular metabolism of activated sludge from two different wastewater treatment plants (WWTP). The tested manganese concentrations were: 0.5, 1, 1.5, 2, 4 and 6 mg/L. The effect of this heavy metal on endogenous metabolism was studied by determining the specific endogenous respiration rate (SOUR_endo), chemical oxygen demand (COD) and measures of enzymatic activity (dehydrogenase activity), while the effect on the biodegradation capacity was determined through the terminal velocity of oxygen consumption (OUR_exo). Measurements were taken at 30 min, 3 h and 24 h of exposure of the sludge to the heavy metal. The results showed that concentrations below 1 ppm produce an increase in the respiratory activity of both sludges. Increasing the concentration of manganese (≥2 mg/L) resulted in substantial reductions in respiratory activity, being more significant for long exposure times. Finally, it was observed that activation of the endogenous respiration rate at low concentrations of manganese during the first hours of the test produces greater stabilization of the sludge, as identified by a lower terminal velocity of synthetic wastewater degradation and lower values of COD measured in the supernatant.     

Urease: A highly biocompatible catalyst for switchable Biginelli reaction and synthesis of 1,4-dihydropyridines from the in situ formed ammonia

Urease is a superior biocompatible catalyst for switching from the Biginelli reaction to urea-based synthesis of 1,4-dihydropyridines in water, where 100% switching occurs at 0.02 g/mL of enzyme. Hantzsch reaction with ammonium acetate (NH₄OAc) is inefficiently catalyzed by urease (70%, 4 h), and heavy metal ions inhibit the urease-catalyzed reactions with urea or NH₄OAc. Promotion of the urea-based Hantzsch reaction by urease and its inhibition with Hg^2 + supports specificity of urease for in situ generation of ammonia, whereas the role of urease in further transformations is not so specific. The features of this enzymatic method are reusability, mild reaction conditions, biocompatibility, generality, and high yield of products.     

Enzymatic conversion of corn oil into biodiesel in a batch supercritical carbon dioxide reactor and kinetic modeling

Synthesis of fatty acid methyl esters (FAME) as biodiesel from corn oil was studied in a batch supercritical carbon dioxide (SC-CO₂) bioreactor using immobilized lipase (Novozym 435) as catalyst. Effects of reaction conditions on the contents of FAME, monoacylglycerols (MAG), diacylglycerols (DAG), and triacyglycerols (TAG) were investigated at various enzyme loads (5–15%), temperatures (40–60 °C), substrate mole ratios (corn oil:methanol; 1:3–1:9), pressures (10–30 MPa), and times (1–8 h). The highest FAME content (81.3%) was obtained at 15% enzyme load, 60 °C, 1:6 substrate mole ratio, and 10 MPa in 4 h. A reaction kinetic model was used to describe the system, and the activation energy of the system was calculated as 72.9 kJ/mol. Elimination of the use of organic solvents, chemical catalysts and wastewater as well as reasonably high yields make the enzymatic synthesis of biodiesel in SC-CO₂ a promising green alternative to conventional biodiesel process.     

Effect of reaction parameters on conversion of krill (Euphausia superba) oil by immobilized lipase ethanolysis

Monoglyceride and diglyceride were produced by performing ethanolysis of krill oil with immobilized lipase and the influence of various parameters on the enzymatic ethanolysis was assessed. As an immobilized lipase, lipozyme TL-IM (thermonuces lanuginose) was used. Ethanolysis was done in non-pressurized and pressurized system to compare the reaction rate and yield. The optimal condition was found at 2.0 of ethanol mole ratio, temperature of 60 °C, lipases amount of 5 wt% in non-pressurized system. At pressurized system the optimal temperature and pressure was found at 50 °C and 10 MPa. However, at 50 °C monoglyceride was higher in pressurized system than in non-pressurized system.     

Production of concentrated natural beta-carotene from buriti (Mauritia vinifera) oil by enzymatic hydrolysis

The present work aimed at the extraction and concentration of β-carotene from crude and refined buriti oils using enzymatic hydrolysis as a process strategy. The performance of two commercial lipolytic preparations (Lipozyme TL IM and CALB L), as well as lipases from Yarrowia lipolytica, was evaluated. The parameters considered in the hydrolysis process were: temperature, enzyme loading and ratio buriti oil/water. Based on a previously conducted statistical design, the experimental conditions were set in order to maximize the free fatty acids (FFA) content in the oil, and simultaneously, minimize the loss of carotenoids. Lipozyme TL IM showed to be the most appropriate enzyme source for the hydrolysis of both oils. The optimized conditions determined for the crude buriti oil processing were 31 °C, 0.0047 g lipase mL^?1 (0.47 g lipase per 100 mL reactional mixture) and 2.33 (ratio oil/water), while for the refined oil, 45 °C, 0.0066 g lipase mL^?1 and 1.80 were the best conditions. At the optimized conditions, the maxima FFA release were 73.0% and 74.8% and the total carotenoids contents were 1578 and 793 mg kg^?1, respectively for the crude and the refined buriti oils, after 4 h of reaction agitated at 300 rpm. Following hydrolysis, oils were deacidified by winterization or phases partition with ethanol.     

Clarification of red raspberry juice using microfiltration with gas backwashing: A viable strategy to maximize permeate flux and minimize a loss of anthocyanins

Red raspberry (Rubus idaeus) juice was produced by maceration of raspberry pulp at 50 °C for 2 h using 400 mg kg^?1 Klerzyme^®150 enzymatic pectolitic preparation followed by raw juice clarification with gelatin and bentonite or cross-flow membrane filtration. A minimal loss of anthocyanins from 630 to 540 mg l^?1 was obtained when the juice was clarified using a ceramic multichannel microfilter (MF) with a pore size of 0.2 μm. A light transmission at 625 nm in MF permeate was above 85% and the residual pectin (900 mg l^?1) was completely removed. During ultrafiltration through ceramic or polysulfone membranes with a molecular weight cut-off of 30–300 kDa, the content of anthocyanins was reduced to 220–370 mg l^?1, but a light transmission at 625 nm was as high as 96%. The permeate flux in MF was maintained at high values above 170 l m^?2 h^?1 at 3 bar for more than 2 h by backwashing the membrane with a compressed air every 6 min for 1 min. The cake compression at high pressures was avoided by short filtration times between backwashing.     

Optimization of microwave-assisted enzymatic extraction of polyphenols from waste peanut shells and evaluation of its antioxidant and antibacterial activities in vitro

A microwave-assisted enzymatic extraction (MAEE) method was developed and optimized to enhance the polyphenols extraction yield from waste peanut shells. The optimum conditions were as follows: irradiation time 2.6 min, amount of cellulase 0.81 wt.%, a pH of 5.5, and incubation at 66 °C for 2.0 h. Under these conditions, the extraction yield of total polyphenols could reach 1.75 ± 0.06%, which was higher than other extraction methods including heat-refluxing extraction, ultrasonic-assisted extraction and enzyme-assisted extraction. The structural changes of the plant material after different extractions observed by scanning electron microscopy provided visual evidence of the disruption effect. Moreover, the crude extract was then purified by NKA-9 resin, the polyphenols content in the purified extract increased to 62.73%. The antioxidant activities of the crude and purified polyphenols extract were evaluated by DPPH and hydroxyl radicals, reducing power and β-carotene bleaching test. The antibacterial activities of purified extract were also tested using Oxford cup method. The results indicated that the MAEE method was efficient and environment-friendly, and the polyphenols have significant antioxidant and antibacterial activities, which can be used as a source of potential antioxidant and preservative.     

Aqueous enzymatic extraction of peanut oil and protein hydrolysates

The aqueous enzymatic process of simultaneously preparing oil and protein hydrolysates from peanut was investigated. The optimum parameters for hydrolysis using Alcalase 2.4L were established by the single-factor and orthogonal test. The optimal processing conditions were as follows: hydrolysis temperature 60 °C, pH 9.5, ratio of material to water 1:5 (w/w), alkaline extraction time 90 min, enzyme amount 1.5% (w/w) and hydrolysis time 5 h. Under these conditions, the free oil and protein hydrolysates yields were 79.32% and 71.38% respectively. In order to improve these yields, As1398 was chosen to hydrolyze the residue and emulsion. The total free oil and protein hydrolysates yields were increased to 91.98% and 88.21% respectively.