Hydrolysis and large scale ultrafiltration study of alfalfa protein concentrate enzymatic hydrolysate

Batch enzymatic hydrolysis of insoluble Alfalfa Protein Concentrate by Delvolase was carried out at laboratory and at pilot-plant scale coupled to an ultrafiltration reactor with a mineral tubular membrane. Parametric studies were carried out on the batch system to determine the biochemical and hydrodynamical optimum conditions. The hydrolysis conditions selected were 40 degrees C, pH 9.5, initial substrate level 3 g protein/100 g and the enzyme substrate ratio 152 U/g protein. After 5 h of hydrolysis, 96% of the total amount of initial nitrogen was solubilized. The ultrafiltration conditions selected were a 10 000 Nominal Molecular Weight Cut-Off, a transmembrane pressure of 1.5 bar, a flux velocity of 0.8 m/s. Fifty percent of the initial nitrogen appeared in the permeate.     

双酶法水解辐照改性马铃薯淀粉动力学研究

为了解辐照改性马铃薯淀粉的酶解特性,用α-淀粉酶和糖化酶同时作用于马铃薯原淀粉和经400 kGy剂量辐照处理后淀粉,考察了pH值、酶解温度、α-淀粉酶用量、糖化酶用量对反应速率的影响.以米氏方程为基础,用Lineweaver-Burk法求解动力学参数.结果表明,辐照后马铃薯淀粉的酶解反应速率明显高于马铃薯原淀粉.在单一水解体系中,α-淀粉酶和糖化酶对辐照前后马铃薯淀粉的降解都遵循Michaelis-Menten方程,α-淀粉酶的Km分别为11.343 mg· mL-1和9.386 mg· mL-1,Vmax分别为0.406 mg（mL·min）-1和1.079 mg（mL·min）-1;糖化酶的Km分别为10.307 mg· mL-1和8.905 mg·mL-1,Vmax分别为0.338 mg（mL·min）-1和0.821mg（mL·min）-1;水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数Ki分别为1.298 mg·mL-1和0.934 mg·mL-1.研究结果表明辐照有效提高了马铃薯淀粉的酶解反应活性.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.     

Porcine pancreatic alpha-amylase hydrolysis of native starch granules as a function of granule surface area

Porcine pancreatic alpha-amylase activity on native starch granules is more accurately described as a function of surface area of the granules rather than of substrate concentration. The apparent K(m) of alpha-amylolysis of native starch from potato, maize, and rice expressed as a function of substrate concentration was largest for potato with a single value of V(max). However, the ratio of the slope of a Lineweaver-Burk plot to that of rice for enzymatic hydrolysis of native potato and maize starch were 7.78 and 2.58, respectively, which were very close to the ratio of surface area per mass of the two starch granules to that of rice. Therefore, the reciprocal of initial velocity was a linear function of the reciprocal of surface area for each starch granule. Surface area was calculated assuming the starch granules were spherical. The values obtained by this calculation were in good agreement with the value obtained by the photomicrographic method. By comparing enzymatic digestion of native maize granules to that of rice granules, it was concluded that the presence of pores in maize granules appeared to significantly affect overall rate of digestion after sufficient reaction time, but not at the very initial stage of hydrolysis.     

Purification of lysozyme using ultrafiltration

This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Continuous hydrolysis of goat whey in an ultrafiltration reactor: generation of alpha-lactorphin

Bovine whey hydrolysate has been developed and applied to areas such as nutrition, culture media, and isolation of bioactive peptides. In order to produce such a type of hydrolysate, it is possible to use goat whey, which constitutes also a food processing by-product. Enzymatic hydrolysis of goat whey by pepsin was carried out in a continuous ultrafiltration reactor. The permeate contained peptide hydrolysate that was resolved by RPHPLC. Second order derivative spectroscopy, amino acid analysis, and mass spectrometry revealed the presence of a biologically active peptide called alpha-lactorphin. This constitutes preliminary information about goat whey enzymatic degradation for future applications.     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.     

On the presence of starch bound phosphate in potato leaf starch

The presence of starch-bound phosphate in potato leaves collected late afternoon in the middle of July when the starch content is high (12.9% dry matter basis) was studied. Starch was extracted from the leaves with dimethylsulphoxide and fragments of starch were purified by ultrafiltration in two steps in combination with an -amylase hydrolysis. The fragments were analysed with ³¹P-NMR and no signals corresponding to phosphate monoesters linked to glucose at the C₃ and/or C₆ positions were detected. The results show that starch in potato leaves does not contain any detectable amounts of phosphate monoesters.     

Ultrafiltration of raw sewage using an immobilized enzyme membrane

Ultrafiltration of raw sewage was performed using multiple enzymes immobilized on non-cellulosic, ultrafiltration membranes. An increase of 12% in the permeate flux rate at quasi-steady state was observed due to the action of the immobilized enzymes. Enzymes were immobilized by physical sorption to minimize the loss of ultrafiltration capability of the membrane, due to the immobilization process. A mathematical model based on diffusive transport and enzymatic action is presented. A standard Marquardt algorithm and a fourth-order Runge-Kutta integration routine were used for estimation of the non-linear parameters in the model. A comparison of data presented here with the data reported earlier on the ultrafiltration of NFDM (non-fat dry milk), showed that the enzyme-membrane has a longer half-life in the case of NFDM than for raw sewage. Furthermore, the first-order enzyme decay rate is much faster in the multiple enzyme system used in raw sewage filtration than in the single enzyme system used in the ultrafiltration of NFDM.     

Structural analysis and reaction mechanism of the disproportionating enzyme (D‐enzyme) from potato

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D‐enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α‐1, 4‐glucan. D‐enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D‐enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26‐meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D‐enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D‐enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D‐enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.     

Study of performance of a tubular membrane reactor for an enzyme catalyzed reaction

A tubular membrane reactor offers many advantages over a solid wall reactor to carry out an enzyme catalyzed reaction. With proper membrane selectivity, the product, may be separated from the reacting stream and the enzyme recycled for continuous reuse. In most cases, enzyme reuse contributes to the economic feasibility of a continuous enzyme catalyzed process. Furthermore, the efficiency and performance of a membrane reactor is greater than that of a solid wall reactor. Continuous hydrolysis of starch by the enzyme β-amylase, carried out in a commercially available tubular membrane unit, is studied at different starch and enzyme concentrations for a given system pressure and inlet flow rate. Results show that the performance of the membrane reactor is in all cases greater than that of the solid wall reactor. A steady state in performance of permeation rate is, however, not reached by the membrane reactor, which shows a continuous decline within the periods examined in this study. This decline is caused in part by the aging of the starch solution, but mostly by the formation of a concentrated, or gel, layer at the membrane surface. This appears to be the main limiting factor for this process since the decline in reaction and permeation rate results in a severe decrease in the amount of maltose in the permeate.     

Purification of densonucleosis virus by tangential flow ultrafiltration

Purification at commercial scale of viruses and virus vectors for gene therapy applications and viral vaccines is a major separations challenge. Tangential flow ultrafiltration has been developed for protein purification. Here tangential flow ultrafiltration of parvoviruses has been investigated. Because these virus particles are small (18-26 nm), removal of host cell proteins will be challenging. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow some virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the 30, 50, and 100 kDa membranes, indicating possible entrapment of virus particle in the membrane pores. The permeate flux and level of protein rejection is strongly affected by the cell culture growth medium. The results indicate that when developing a new process, it is essential that the cell culture and purification operations be developed in parallel.     

Cereal-based biorefinery development: integrated enzyme production for cereal flour hydrolysis

Restructuring the traditional fermentation industry into viable biorefineries for the production of fuels, chemicals and plastics is essential in order to replace (petro)chemical processing. This work presents engineering aspects of Aspergillus awamori submerged fermentation for on-site production of an enzymatic consortium that contains glucoamylase, protease and phosphatase. The crude broth filtrate was used for the production of wheat flour hydrolysates. Improvements on traditional starch hydrolysis carried out in two stages (liquefaction and saccharification) were attempted through integration of unit operations and reduction of processing temperature and reaction duration. An initial increase of temperature to 68 degrees C and a subsequent decrease to 60 degrees C for the rest of the enzymatic hydrolysis resulted in a starch to glucose conversion yield of 94 and 92% when a wheat flour concentration and commercial starch concentration of 225 g L(-1) was used, respectively. The use of crude broth filtrates resulted in the simultaneous hydrolysis of wheat protein and phytic acid, as was indicated by the increase in free amino nitrogen and phosphorus concentration, respectively.     

Performance of a membrane reactor for cellobiose hydrolysis

A continuous enzymatic hollow fiber reactor (HFR), obtained by immobilizing cellobiose active cells into the shell side of hollow-fiber modules, was studied. The HFR yield was monitored by glucose analysis resulting from hydrolysis of cellobiose. The residence time of substrate in the bioreactor to obtain convenient hydrolysis yields was calculated from tests carried out by varying the reactor dilution rate in the range 0.001-0.004 L/min. The glucose yield was measured for 300 h (continuous substrate flux). The yield decreased from 40 to 15%. This decrease was due to the loss of specific activity in the operating conditions and to the pressure drop increase from 0.2 to 1.7 atm. The pressure drop increase is in turn dependent on the cell loading (0.2-2.1 g dry cell) and the substrate flux.     

Lysozyme separation by hollow-fibre ultrafiltration

This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.     

Pretreatment of starch raw materials and their enzymatic hydrolysis by immobilized glucoamylase

The pretreatment of starch raw materials such as sweet potato, potato and cassava has been carried out using various types of crusher, viz juice mixer, homogenizer and high-speed planetary mill. The effect of pretreatment of the materials on their enzymatic hydrolysis was studied. High-speed planetary mill treatment was the most effective and comparable with heat treatment (pasting). Various crushing times were used to examine the effect of crushing by mill treatment on the enzymatic hydrolysis. In the enzymatic hydrolysis of cassava, the use of both cellulase [1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and glucoamylase [1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] enhanced the d-glucose yield. The immobilization of glucoamylase was studied by radiation polymerization of hydrophilic monomers at low temperature, and it was found that enzymatic activity of the immobilized glucoamylase particles varied with monomer concentration and particle size. Starchy raw materials pretreated with the mill can be efficiently hydrolysed by immobilized glucoamylase.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

Enzymatic protein hydrolysis in a membrane reactor related to taste properties

Potato, Vicia faba and soybean proteins were hydrolysed enzymatically in a substrate feed membrane reactor system. Alkaline proteolytic enzymes and PM-10 membranes were used for the hydrolysis of potato protein. The taste of the ultrafiltrates, which was unpleasantly bitter and potato-like, was improved by application of gelatin. Also using PM-10 membranes, Vicia faba protein isolate was hydrolysed by alkaline and acid proteolytic enzymes. The bitterness of the ultrafiltrate decreased with the formation of an isoelectric precipitate, which was probably due to association of hydrophobic peptides. The reactor equipped with a cellulose acetate membrane delivered an acceptable enzymatic hydrolysate of Promine D during the first hours of ultrafiltration. This was not the case when similar processes were performed using non-cellulosic DM-5 membranes. The usefulness of ultrafiltration for obtaining bland protein hydrolysates seems to be limited to short-term processes with cellulose acetate membranes.     

Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

Use of Ultrafiltration membrane systems in stirred cell and in thin-Channel systems for immobilizing enzyme (sweet potato intrinsic and β-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. The immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato β-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%.