Starch Liquefaction Research Articles

On-line FT-Raman spectroscopic monitoring of starch gelatinisation and enzyme catalysed starch hydrolysis

The hydrolysis of starch by α-amylase and amyloglucosidase and the kinetics of these technically important reactions were investigated by FT-Raman spectroscopy. During the technical starch hydrolysis process, three reactions proceed one after the other but partly in parallel: the gelatinisation, the hydrolysis of starch to dextrin (liquefaction) by α-amylase, and the hydrolysis of dextrin to glucose (saccharification) by glucoamylase. These three reactions were studied separately. Potato starch or dextrin in concentrations of 50 g/l were suspended or dissolved in water, the reaction chamber was placed in a heated water bath, and defined, varied amounts of enzymes in solution were added. The reactions were monitored on-line in a bypass loop. The greatest spectral changes were observed due to the swelling and gelatinisation of starch. The liquefaction of starch to dextrin was started with the addition of α-amylase at a temperature of 80°C and the spectral changes were monitored. In a similar way, a solution/suspension of dextrin was used to investigate the reaction of glucoamylase at 50°C. Both enzymatic reactions were performed at four different enzyme activities. All reactions showed distinctive spectral changes, which can, in principle, be evaluated for the determination of the degree of starch hydrolysis. As a Nd:YAG laser at 1064 nm was used, fluorescence excitation was not observed despite the use of crude, technical-grade enzymes. The findings demonstrate the potential use of Raman spectroscopy in monitoring and control of technical enzyme reactions.

糖質酵素の現状と将来

Being engaged in the enzyme industry, we should know the enzymes which are of interest to the sugar industry. The following three matters, which have been of interest for the past two decades, are described in this paper. 1) Starch liquefaction: Two-step liquefaction was applied in the past, and then a thermostable a-amylase was developed. This enzyme made it possible to perform more efficient liquefaction using a jet-cooker. Further improvement, such as a new liquefying a-amylase used under acidic coditions, is desired. 2) Starch saccharification: At first, a glucoamylase from Rhizopus was used for starch saccharification, and then an economical glucoamylase from Asper-gillus niger was developed and gained popularity in use. Finally, improvements such as the re-moval of transglucosidase and addition of pullulanase were developed and have contributed to high yields of glucose production. Further improvement is required. 3) Conversion of glucose into fruc-tose: At first, batch conversion was performed with intact cells. Development of an immobilized enzyme made it possible to perform continuous conversion. Further improvements, such as a new enzyme which converts to fructose over 50%, and works at acidic condition, are required.

Protein engineering of alpha-amylase for low pH performance.

Industrial-scale starch liquefaction is currently constrained to operating at pH 6.0 and above, as the enzyme used in the process, Bacillus licheniformis alpha-amylase, is unstable at lower pH under the conditions used. There is a need to develop an enzyme that can operate at lower pH. Recent progress has been made in engineering the B. licheniformis enzyme for improved industrial performance. The availability of crystal structures and subsequent analysis of improved variants, in a structural context, is revealing common factors and a rationale to make further improvements.

Development of Industrially Important .ALPHA.-Amylases.

A new calcium independent α-amylase has been developed for starch liquefaction. This amylase Termamyl LCTM gives the same performance in the absence of free calcium compared to TermamylTM, in the presence of 40 ppm free calcium. The improvement is a result of 5 single amino acid substitutions and one N-terminal alteration. Several new alkaline α-amylases were identified in Nature. We have isolated two amylases that display the desired activity profile but lack the overall stability necessary for a detergent a-amylase. A two amino acid deletion in the region from position 180-184 increased the overall stability both with respect to temperature and calcium sensitivity, to a satisfactory level comparable to that of TermamylTM.

Biotechnological Conversion of Sugar and Starchy Crops into Lactic Acid

Abstract Lactic acid can be used not only as a key substance in chemical synthesis, but also as a special agent in agriculture. For microbial production, original products of agriculture such as sweet sorghum stalks and rye grains can be used as raw material. In laboratory experiments, sweet sorghum stalks were milled, steam-treated and pressed. The sugar of the aqueous extract, which amounted to 89% of the total sugar of stalks, was completely converted into lactic acid by fermentation. The yield amounted to 94% (94 g of lactic acid/100 g of sugar consumed). Also in laboratory experiments, rye grains were milled, fractionated and hydrolyzed in a two-step process using commercial enzymes. The optimum temperature and pH value were 82·5°C and 5·8 for starch liquefaction and 51·6°C and 4·0 for saccharification of liquefied starch, respectively. Starch liquefaction was also influenced by particle size, the optimum value of which was 3 mm. For optimum starch saccharification, a process duration of 2 h was necessary. Of the solid starch, 99·6% was liquefied in the first hydrolysis step. In the second step, 97·9% of the liquefied starch was converted into glucose. Bioconversion of the glucose formed by starch hydrolysis into lactic acid also gave a maximum yield of 94%. Various possibilities of process improvement in order to make the whole operation less expensive are discussed, and a flow-sheet of an improved process for manufacturing lactic acid from cereals is presented.

Quantitative estimation of the action of α-amylase from Bacillus subtilis on native corn starch by HPLC and HPTLC

The optimal conditions of corn starch liquefaction and dextrinization using α-amylase from Bacillus subtilis to obtain maltodextrins with a dextrose equivalent of 12.9–23.5 were determined. The pattern of action of B. subtilis α-amylase on native corn starch was monitored using high-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC). It was possible to separate malto-oligosaccharides of up to 9 glucose units by HPLC and those of up to 12 glucose units by HPTLC, respectively. The results suggest that α-amylase from B. subtilis is an endo-specific enzyme which mainly produces two oligosaccharides, DP3 and DP6. It was shown that both methods can be successfully applied for the characterization of maltodextrins.

Effect of mixing on enzymatic liquefaction of sago starch

The effect of mixing as a function of agitation speed and impeller diameter on the rate and degree of enzymatic liquefaction of sago starch was carried out using a stirred tank reactor with a single Rushton turbine impeller. The performance of the reactor as a mixing device was first examined using different concentrations of carboxymethylcellulose, which exhibited pseudoplastic behaviour similar to that of the solution during the sago starch liquefaction process. A correlation between mixing time ( t m ) and Reynolds number (Re) in the form of t m = bRe c is presented; the constants for the correlation depended on viscosity of the fluid. For the two ratios of impeller diameter ( D i ) to tank diameter ( D t ) used, 0.407 and 0.542, agitation speed gave significant influence on both overall rate and degree of liquefaction of sago starch. Mixing time ( t m ) was independent of impeller diameter used, and correlated well with the overall rate of liquefaction ( P) (calculated as the reducing sugar produced divided by time of liquefaction) and expressed as P = 1.95 t m −0.362.

Enzymatic hydrolysis of sago starch in a twin-screw extruder

Simultaneous gelatinisation and liquefaction of sago starch with a thermostable α-amylase in a co-rotating twin-screw extruder was investigated. Maltodextrins with dextrose equivalent (DE) ranging from 0 to 10 were produced. Response surface methodology was used to study the influence of barrel temperature (70–130 °C), screw speed (70–160 rpm), enzyme concentration (0–1.0%) and feed moisture content (28.5–50.5%) on the extrudate properties. Specific mechanical energy (SME) consumption was calculated to be in the range of 21–91 Wh/kg for the various processing conditions. Structural changes were characterised by measuring DE, water solubility index (WSI), water absorption index (WAI), degree of degradation (DGR) and oligosaccharide content. Enzyme concentration, barrel temperature and feed moisture were the important variables affecting most of the measured physicochemical properties. Hydrolysis of starch granules was the fundamental reaction occurring during extrusion processing with both amylopectin and amylose degraded. Higher breakdown was achieved with lower SME consumption.

Pretreatment of Fermentation Feed for Lactic Acid Production; Liquefaction of Potato Starch in Lactic Acid Solution.

The liquefaction of potato starch, catalyzed by lactic acid produced in a fermentation process, was carried out as a means for pretreating lactic acid fermentation feed. Liquefaction yield and molecular weight distribution of liquefied starch fractions were largely dependent on pH values of solution. The feed was effectively liquefied in lactic acid solution, of which pH value was less than 3.5, under sterilization conditions of 388 K and 30 min. Starch in potato or its waste was also hydrolyzed into smaller molecular weight fractions, compared to soluble starch commercially available. The potato starch liquefaction proposed is considered to be a useful method for pretreatment of lactic acid fermentation feed.

A model study on Eudragit and polyethyleneimine as soluble carriers of α-amylase for repeated hydrolysis of starch

Eudragit and polyethyleneimine have been evaluated for their potential as soluble carriers of enzymes acting on macromolecular substrates. The present study is based on coupling of α-amylase (Termamyl) to these polymers for the degradation of starch. The enzyme was bound to both the polymers by carbodiimide coupling. Using soluble starch as substrate, about 96% of the enzyme activity was found to be retained on binding to Eudragit, while the binding to polyethyleneimine resulted in activation of the enzyme. Kinetic properties and optimal conditions for activity and stability of the soluble-immobilized enzyme preparations were compared with that of the free enzyme. The polymer-enzyme complexes were also used for the repeated hydrolysis of starch, the biocatalyst being recovered in between runs by precipitation. The enzyme in the immobilized form exhibited good stability during repeated runs of starch liquefaction.

The production of recombinant proteins in plants

AbstractFor recombinant proteins that are required in large quantities, plant agriculture probably represents the most cost‐effective means of production. However, the absence of efficient methods for the processing of harvested plant materials and for the cost‐effective purification of extracted proteins has so far limited the exploitation of plant expression systems. This problem, which is particularly acute for recombinant proteins required at high purity, is now being addressed. Thus, the application of oleosins as carrier proteins may provide a facile means of separating recombinant proteins from bulk seed proteins. Transgenic plants are being assessed for the production of a number of recombinant industrial enzymes. Significantly, the seeds from transgenic plants expressing genes encoding either phytase or α‐amylase can be more or less directly employed as enzyme formulations for poultry feed modification and for starch liquefaction, respectively. Furthermore, transgenic seeds provide a simple means of storing and transporting recombinant enzymes. The plant‐synthesised phytase and α‐amylase enzyme are active despite displaying non‐native patterns of glycosylation. Transgenic plants have been shown to be capable of synthesising a wide range of recombinant antibodies and antibody fragments that are typical of those used in industrial processes and in therapy. Plants may be particularly suited to the production of complex, multimeric immunoglobulins since genes encoding the different components of these complex molecules can be combined by simple sexual crossing of individual transgenic plants.

Liquefaction of starch by a single-screw extruder and post-extrusion static-mixer reactor

The production of ethanol has increased rapidly during the past few years, especially since enactment of the 1990 Clean Air Act. Production costs, however, are high, requiring improved conversion technologies. The gelatinization of starch with conventional jet cookers requires a minimum moisture content of 65% by weight. An alternative to jet cookers is extrusion, which requires significantly less moisture to gelatinize starch. Starch gelatinization and liquefaction was achieved using single-screw extrusion and a post-extrusion static-mixer reactor. The effects of moisture content, reactor temperature, reactor length, and enzyme concentration on liquefaction were evaluated using response surface methodology. The use of a static-mixer reactor tube improved mixing of the gelatinized slurry and increased residence times, resulting in increased starch degradation and reduced viscosity. Comparison of costs for extrusion and conventional technology indicates extrusion has potential as a cost-effective alternative.

Influence of extrusion variables on subsequent saccharification behaviour of sago starch

This study reports the effects of substrate concentrations (21.6–38.4 g/100ml), amyloglucosidase (AMG) concentrations (0.66-2.34 U/g) and hydrolysis time (14.8–65.2 h) on the saccharification of sago starch extrudates. Regression analysis established AMG concentration and hydrolysis time as the most significant factors; a generated regression equation adequately predicted the outcome of saccharification. Using 30 g/100 ml solid content, 65.2 h and 2.34 U/g of AMG, syrups of highest glucose content (87.9%) were attained. Effects of extrusion conditions, i.e. feed moisture content (21.6–38.4), enzyme (Termamyl 120L) concentrations (1.48–6.52%) and mass temperatures in the compression and die zones (70.5–97.5 .C) during the combined gelatinisation and liquefaction of sago starch, on the amount of glucose produced were evaluated. Enzyme concentrations and feed moisture contents had the most pronounced effects on saccharification, which ranged from 47.6 to 73.3% (DE = 85–94) and 54.5–93.8% (DE = 90–98) after 24 and 65 h, respectively. Response surface plots suggest that a higher percent saccharification can be achieved when sago starch is extruded at high Termamyl concentrations but low moisture contents.

Dynamic Modelling and Simulation of a Multipurpose Batch Pilot Plant

A detailed dynamic model if developed for a complex multi-purpose food processing batch pilot plant in which two tots of batch operations are performed: a Cleaning-In-Place (CP) operation and an enzymatic starch liquefaction process. A number of control, safety assessment and process improvement studies are then carried out using the dynamic model. Results show that the plant operating efficiency can be improved considerably. Some of the results were confirmed experimentally.

Synthesis of Branched Oligosaccharides from Starch by Two Amylases Cloned fromBacillus licheniformis

An E. coli cell extract containing thermostable α-amylase and maltogenic amylase expressed from the clone pTMA322 was used to produce an anomalously linked oligosaccharides (Alo) mixture from starch. The liquefaction and saccharification of starch were done by one-step procedure using the above cell extract. The resulted Alo mixture contained over 40% oligosaccharides having DP 3 or more including branched forms.

Enzymatic hydrolysis of sago starch

Various enzyme hydrolysis procedures were employed in this study. A kinetic model presents sago starch hydrolysis by Bacillus licheniformis α-amylase (Termamyl 120L), Bacillus subtilis β-glucanase (Cereflo 200L) and by Bacillus pullulanase (Promozyme 200L) at temperatures of 90°C and 60°C and substrate concentration of 15% solids. Quantitation of pH, temperature and enzyme dosage in different combinations was used in standard statistical techniques of experimental design and data analysis. Determination of the degree of hydrolysis was based on freezing point depression osmometry, high-performance size-exclusion chromatography (HPSEC), differential scanning calorimetry (DSC) and X-ray diffraction methods. Computer implementation of the model allows graphical evaluation and prediction in improvement of the conditions of enzyme hydrolysis of sago-resistant starch. Liquefaction and saccharification of sago starch were performed to establish the optimum conditions of enzymatic hydrolysis in comparison with other starches. Enzyme treatment resulted in a decrease in the degree of crystallinity of all hydrolysed starch samples. C-type crystals completely disappeared at 100°C.

Efficient production of active industrial enzymes in plants

An industrial bulk enzyme, Bacillus licheniformis α-amylase was produced in transgenic tobacco at a maximum level of about 0.3% of total soluble protein. The molecular weight of the enzyme produced in tobacco differed from the bacterial enzyme due to complex-type carbohydrate chains attached to the protein. Milled transgenic seeds containing α-amylase were successfully applied in the liquefaction of starch. The resulting hydrolysis products were virtually identical to those obtained from degradation with B. licheniformis α-amylase. No effect was observed of the presence of the enzyme on endogenous starch. Homogenization of transgenic leaves, followed by incubation at 95°C, resulted in degradation of the endogenous starch. Different approaches to the concept of production and application of industrial enzymes in plants are presented that show the efficacy of transgenic plants as a new source of active industrial enzymes.

A novel technique for determining half life of alpha amylase enzyme during liquefaction of starch

The half-life of alpha amylase has been determined in consideration with the stabilizing effect of starch (30% w/v). Initially, enzyme of known activity was taken. During starch liquefaction, the residual activity of enzyme was estimated interms of its’ reducing power.

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

Thermoanaerobacter sp. CGTase: Its Properties and Application.

A novel CGTase (Cyclomaltodextrin glucanotransferase) has been isolated from a strain of Thermoanaerobacter, a thermophilic anaerobe. The enzyme is extremely heat stable and has a temperature optimum of 90-95°C at pH 6.0. It is active over a broad pH range, and exhibits more than 80% activity from pH 5.0-6.7. In the presence of starch, the enzyme is stable at temperatures in excess of 100°C. In addition to producing cyclodextrins from starch, Thermoanaerobacter sp. CGTase has excellent starch liquefying properties. It is possible to liquefy a 35% DS starch slurry at pH 4. 5, in the absence of calcium, using the "jet cooker" process developed for B. licheniformis α-amylase (105°C for 5 min, 90-95°C for 90 min). Saccharification of the liquefied starch with A. niger glucoamylase can therefore be carried out without further pH adjustment. Some of the properties of Thermoanaerobacter sp. CGTase are described, and examples of the use of the enzyme for cyclodextrin production, intermolecular transglycosylation and starch liquefaction are given. The gene coding for Thermoanaerobacter sp. CGTase has been transfered to a Bacillus host, making large-scale production of the enzyme in commercially acceptable yields possible.