A process for converting starch or partially hydrolyzed starch into a syrup containing dextrose includes the steps of saccharifying starch hydrolyzate in the presence of a saccharifying starch hydrolyzate in the presence of a mutated glucoamylase or related enzyme and increasing the selectivity of the enzyme for α-(1→4)- glucosidic bonds by the glucoamylase or related enzyme by including at least one mutation, the mutation substituting an amino acid of the enzyme with at least one amino acid chosen by comparison with structurally related regions of other enzymes that selectively hydrolyze only α-(1→4) glucosidic bonds. Enzymes made in accordance with the present invention are also disclosed.
In conclusion, an integrated bio-process of trehalose production from kudzu root starch through two-stage enzymatichydrolysis into malt syrup was established with the help of the intelligent visualization method in this study. The high-yield of 94.6 % maltose was realized by α -amylase, β -amylase and pullulanase pretreatment under the optimum DE of 19 and sacchari- ficaiton condition of 32.5 U β -amylase/g starch and 1.25 U pullulanase/g starch. Furthermore, the optimum con- ditions of trehalose production were obtained with the maximum trehalose conversion rate of 70.6 %, which was considered as a good candidate for the large-scale pro- duction of trehalose in the near future. Thus, application of the visualization method for optimization of param- eters of enzyme catalytic reaction conditions was proved successful, and its further application in biocatalysis technology was expected.
of xylose to glucose concentration. The Student’s t- distribution and the corresponding P-value, along with the parameters was shown in Table-3. The P-values are used as a tool to check the significance of each coefficient, which will help to explain the pattern of mutual interactions between the best variables. The parameter coefficient and the corresponding P-value suggested that, pH do has significant effect on all three responses (P- values<0.05). Whereas only for xylose concentration response analysis, temperature significantly influenced these response. The sample 3D response surfaces plots were employed to illustrate the interaction of temperature and pH and their effects onenzymatic hydrolysis result (Figure-1).The estimated optimum condition for enzymatichydrolysis for each objective function were presented in Figure-1.
bilized lignin. The previous works have characterized the glass transition behavior of lignin, where it trans- forms from a hard or glass-like state into a rubbery or viscous state upon heating (Ko et al. 2015). Hot-water pretreatments reaching temperatures above the range for lignin phase transition cause lignin to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls upon cooling (Donohoe et al. 2008). During cool- ing after pretreatment, coalesced lignin could harden and either became trapped within the cell wall layers or settle out of the bulk liquid phase, potentially deposit- ing back onto the biomass surface. Typically, the solid fraction is separated by filtration. The solid fraction allows solubilized lignin to condense and precipitate out on the cellulosic residue interfering with the enzy- matic hydrolysis of cellulose to glucose (Selig et al. 2007). Flow-through hot-water pretreatment, where the sol- ids residence time is longer than that of the liquid, has been shown to effectively dissolve more lignin compared with pretreatments, in which liquid and solids have the same residence time (Mosier et al. 2005). This method also generates solids that are more reactive on enzymatichydrolysis, because of removing the solubilized lignin. However, continuous flow-through operation is thought to use an excessive amount of water and energy (Liu and Wyman 2005).
Cellulases firstly adsorb onto solid substrate to cleave cellulose chain and then release soluble cellobiose, which can be further hydrolyzed to glucose by β-glucosidase. For cellulase cocktails that were designed to have a synergistic effect, only some portion of loaded enzymes adsorbs on the substrates as bound enzymes. Cellobiohydrolases (CBH) possess a catalytic core and one or more carbohydrate-binding module connected by a linker. Carbohydrate-binding module is believed to have a high affinity for crystalline cellulose and surface hydrogen bonds are considered to disrupt by a linker. These combined actions can facilitate anchoring the cellulose microfibril into catalytic core (Ding & Xu, 2004). The typical catalytic cycle involves binding at the end of cellulose chain, cleavage of a β-1,4 glycosidic bond to yield cellobiose, and finally translocation along the chain for the next cycle. On the other hand, the other portion of enzyme in the liquid phase, such as β- glucosidases and desorbed CBH, are free enzymes that have flexible mobility and potentially available for recycling. Enzymatichydrolysis is a dynamic equilibrium process, in which the concentration of bound enzyme and free enzyme is always changing due to the amount of available substrate and its surface characteristics.
β -glucosidase is an enzyme which catalyses the hydrol- ysis of compounds with β -d-glucosidic linkages (Shewale 1982). It has been established that cellulase enzyme com- plex is made up of three different enzymes (exo β -1,4- glucanase, endo- β -1,4-glucanase and β -glucosidase) which work together to hydrolyse crystalline cellulose. Endoglucanases starts cellulose hydrolysisprocess, dis- rupting internal β -1,4-glucosidic bonds along the cellu- lose chain, increasing the number of ends available for exoglucanases (Shewale 1982). These hydrolysis reac- tions occur in the amorphous regions of cellulose. Exo- glucanases may then cleave off two units of cellobiose from each end of these shorter cellulose chains (Ogeda et al. 2012). Cellobiose and higher cellodextrins are pro- duce when endo-glucanase is used for a long time. The cellobiose and cellodextrins are finally hydrolysed by β -glucosidase to glucose. β -glucosidase plays a major role in cellulose hydrolysis by removing cellobiose which inhibits the action of exo and endo glucanases (Shewale 1982).
The performance of genetic algorithm (GA) in nonlinear kinetic parameter estimation of tapioca starchhydrolysis was studied and compared with the Gauss-Newton method. Both methods were employed for determining the model parameters of the modified version of Gonzalez-Tello model. To estimate and validate the model parameters, experimental works involving hydrolysing tapioca starch were conducted. The model was then used to predict glucose concentration profile for a given initial condition of the tapioca hydrolysisprocess. In terms of error index values, both methods produced good results. This study showed that the impact of user defined parameters of the GA was insignificant as compared with the influence of initial parameters of the Gauss-Newton method on the predictive performance. Furthermore, the GA approach requires no guessing of the initial values and is able to produce reasonable solutions for the estimated parameters.
ANNs can handle incomplete data and deal with nonlinear problems. It can also perform prediction and generalization immediately after the training process . Artificial NNs seem to be a feasible alternative in several instances, and their application for biotechnological processes is continuously growing . With respect to biotechnological processes in particular, several studies can be found in literature, such as the description of the α-amilase inactivation, the prediction of the final concentration of ethanol in a batch fermentation process and as a soft-sensor [7-9]. ANN models are generally used for prediction, function approximation, classification, and clustering . However, few papers were reported about ANN-basedmodel for enzymatichydrolysis. The aim of the present study is to check the validity of ANN to predict the glucose production under various enzymatichydrolysis conditions with available experimental data and compare ANN results with kinetic model results.
The first reported model for hydrolysis of insoluble polysaccharides using stochastic modeling approach was developed by Fenske et al. . This was a limited SMM model as it did not capture the actual structural proper- ties of cellulose (e.g. crystallinity, DP, fibril structure) and multi enzyme dynamics. Hydrolysis was performed on a two dimensional matrix representing a single sur- face of cellulose with short chain length of 20. It was a theoretical study and results were not validated with experimental data. More recently, similar approach was used by Asztalos et al.  to model cellulose hydrolysis which had reasonable accuracy in predicting the hy- drolysis trends for endoglucanse and CBH enzymes. Dy- namic enzyme-substrate interactions were captured to some extent in the model. However the model did not include some important structural features of cellulose and had a limited usability. It was a two dimensional model in which all glucose chains were accessible to all enzymes, which in not the case in actual process. Degree of crystallinity was not considered in the cellulose struc- ture, and consequently, activity difference of enzymes in amorphous and crystalline regions found to be signifi- cant by many researchers was not incorporated into the model [1,20,27]. Inhibition by cellobiose or glucose was not accounted into the model, which is very important parameter during cellulose hydrolysis. Therefore, despite these relatively recent advances, there is no SMM model to date that considers complex enzyme-substrate inter- actions. The objective of this study was to develop a de- tailed SMM model that can predict hydrolysis profiles of cellulose with high accuracy by capturing the complex- ities of cellulose structure and hydrolysis mechanism. The aim was to develop a general hydrolysis model that can be used for various conditions (different substrates, enzymes, hydrolysis conditions) considering structural properties of feedstock (crystallinity, degree of polymerization, accessi- bility), enzyme properties (mode of actions, synergism and inhibition) and most importantly dynamic changes in these properties during hydrolysis.
Finding of optimal hydrolysis conditions is important for increasing the yield of saccharides. The higher yield of saccharides is usable for increase of the following fermentation effectivity. In this study optimal conditions (pH and temperature) for amylolytic enzymes were searched. As raw material was used waste bread. Two analytical methods for analysis were used. Efficiency and process of hydrolysis was analysed spectrophotometrically by Somogyi-Nelson method. Final yields of glucose were analysed by HPLC.
Cassava pulp, a low cost solid byproduct of cassava starch industry, has been proposed as a high potential ethanolic fermentation substrate due to its high residual starch level, low ash content and small particle size of the lignocellulosic fibers. As the economic feasibility depends on complete degradation of the polysaccharides to fermentable glucose, the comparative hydrolytic potential of cassava pulp by six commercial enzymes were studied. Raw cassava pulp (12% w/v, particle size <320 µm) hydrolyzed by both commercial pectinolytic (1) and amylolytic (2) enzymes cocktail, yielded 70.06% DE. Hydrothermal treatment of cassava pulp enhanced its susceptibility to enzymatic cleavageas compared to non-hydrothermal treatment raw cassava pulp. Hydrothermal pretreatment has shown that a glucoamylase (3) was the most effective enzyme for hydrolysisprocess of cassava pulp at temperature 65 °C or 95 °C for 10 min and yielded approximately 86.22% and 90.18% DE, respectively. Enzymatic pretreatment increased cassava pulp vulnerability to cellulase attacks. The optimum conditions for enzymatic pretreatment of 30% (w/v) cassava pulp by a potent cellulolytic/ hemicellulolytic enzyme (4) was achieves at 50 °C for 3, meanwhile for liquefaction and saccharification by a thermo-stable α-amylase (5) was achieved at 95 °C for 1 and a glucoamylase (3) at 50 °C for 24 hours, respectively, yielded a reducing sugar level up to 94,1% DE. The high yield of glucose indicates the potential use of enzymatic-hydrothermally treated cassava pulp as a cheap substrate for ethanol production.
According to results from hydrothermal treatment, the percentage of cellulose content is as shown in Fig. 1. The Figure shows time versus cellulose percent of the hardwood by using hydrothermal treatment process. At reaction time (180min), Sample No. (HS-5) gave the higher percentage of cellulose for optimum condition. The more reaction time, the higher percentage of cellulose. So, hardwood sample (HS-5) is the best conditions. Then, sawdusts from softwood treated by preheating to boil 80±5˚C followed by adding sodium hydroxide solution (5% by wt of sawdust) for 60min to 240min.So, sawdusts from softwood (SS-5) treated by preheating to boil 80±5˚C followed by adding sodium hydroxide solution (5% wt of sawdust) for 180min was best condition compared to that other conditions .
The use of biodegradable polymers is one of the strategies used to minimize environmental problems caused by the inappropriate disposal of plastic articles. Thus, biodegradation is an extremely important phenomenon and needs to be clearly elucidated because the rate of biodegradation of the same sample may vary from one method to another, due to the different environmental conditions and the degrading factor to which the same is exposed. Such factors may be biotic or abiotic. In addition, characteristics such as crystallinity, morphology and the surface profile of the materials may influence its degradation. In this context, the aim of this work is to understand the enzymatichydrolysisprocess for two biodegradable polymers with adverse behavior, thermoplastic starch (TPS), poly (lactic acid) (PLA) and their blends. The materials were characterized by contact angle (CA) and scanning electron microscopy (SEM). The samples were submitted to the enzymatichydrolysis assay in the presence of the α-amylase and proteinase K enzymes. The results obtained allow us to conclude that the incorporation of TPS in the matrix of the PLA confers greater roughness to the mixtures, making them more susceptible to hydrolysis and consequent biodegradation. With respect to the enzymatichydrolysis, it was possible to conclude that the samples showed higher affinity with the proteinase-K enzyme.
To investigate the role of product inhibition in high-solids enzymatichydrolysis, various amounts of sugar were added to a hydrolysis of filter paper. An example of such an experiment (at large laboratory scale) is seen in Figure 3. With 50 g/l glucose added, the rate of hydrolysis during the first few hours was significantly reduced compared with the reference, in particular for the 5% solids hydrol- ysis where the initial phase of fast conversion was com- pletely absent. As there is a constant enzyme dosage per gram of solids in the experiments, the ratio between glu- cose and enzyme is much higher at 5% than 20% solids (for the hydrolyses with 50 g/l glucose added) and the stronger inhibition is thus not surprising. Although 4 h makes up a small part of the whole hydrolysis time, the fast rate of hydrolysis in the first phase is responsible for conversion of a major part of the substrate. Interestingly, after approximately 4 h, the rate of hydrolysis at 20% is nearly identical despite the difference in glucose level. This indicates that one of two things is happening. Either there are other and stronger factors inhibiting the hydrol- ysis after the first phase, thereby 'masking' the product inhibition, or there is a certain glucose level threshold, above which the enzymes are inhibited to a similar extent and thus resulting in a similar hydrolysis rate.
Ozone can be used to degrade lignin and hemicellulose in many lignocellulosic materials such as wheat straw (Ben-Ghedalia and Miron, 1981), bagasse, green hay, peanut, pine (Neely, 1984), cotton straw (Ben-Ghedalia and Shefet, 1983), and poplar sawdust (Vidal and Molinier, 1988). The degradation was essentially limited to lignin and hemicellulose was slightly attacked, but cellulose was hardly affected. The rate of enzymatichydrolysis increased by a factor of 5 following 60% removal of the lignin from wheat straw in ozone pretreatment (Vidal and Molinier, 1988). Enzymatichydrolysis yield increased from 0 to 57% as the percentage of lignin decreased from 29% to 8% after ozonolysis pretreatment of poplar sawdust (Vidal and Molinier, 1988). Ozonolysis pretreatment has the following advantages: (1) it effectively removes lignin; (2) it does not produce toxic residues for the downstream processes; and (3) the reactions are carried out at room temperature and pressure (Vidal and Molinier, 1988). However, a large amount of ozone is required, making the process expensive.
The development of biorefinery industry requires inex- pensive sugar stream for downstream biological and/ or chemical conversion [1, 2]. Biomass pretreatment followed by enzymatichydrolysis has been viewed as a viable way to obtain sugars from biomass due to its fractionation effect and the high selectivity of enzymes for the hydrolysis of polysaccharides (cellulose and hemicellulose) to sugars (e.g., glucose and xylose) . This pathway has been intensively studied for years but two aspects are still in need of improvements to fur- ther improve the economics of sugar-based biorefinery processes. First, an environment-friendly pretreatment method is needed. Various pretreatment methods, such as dilute-acid pretreatment , alkali pretreatment , organosolv pretreatment [e.g. ethanol, tetrahydrofuran, and γ-valerolactone (GVL)] [6, 7] and SPORL (sulfite pretreatment to overcome recalcitrance of lignocellu- lose) , have been successfully developed to produce cellulase-digestible substrates. However, post-treatments are required to remove and/or recover the chemicals or solvents for environmental and economic considerations [8–10]. These additional treatments increase the com- plexity and the cost of the processing. Second, reducing the use of costly cellulolytic enzymes is still necessary. Klein-Marcuschamer and Liu et al. [11, 12] conducted a techno-economic analysis of bioethanol production from typical lignocelluloses (such as poplar) and demonstrated that the cost of the enzymes would be as high as US $1.47/gal ethanol. Such a high enzyme cost will undoubt- edly limit the development of cellulosic biofuel industry.
Various researchers have shown that removal of the CBM component of individual cellulases reduces the hydrolytic activity of the catalytic module on insoluble, crystalline substrates such as microcrystalline cellulose (Avicel), cotton, and filter paper, whereas their activity on soluble or amorphous cellulose remains largely unaf- fected [5,22,23]. In addition, CBMs isolated from both bacteria and fungi have been suggested to facilitate cel- lulose hydrolysis by physically disrupting the structure of the fibrous cellulosic network and releasing small par- ticles, without showing any detectable hydrolytic activity, which is normally quantified by the release of reducing sugars (the so-called disruptive function) [7,8]. In recent studies investigating the morphological and structural changes of cotton fibers after treatment with purified CBM from fungal CBH1, it was found that CBM could promote non-hydrolytic disruption of crystalline cellu- lose by weakening and splitting the hydrogen bonds (as observed by infra-red spectroscopy and X-ray diffrac- tion), thereby freeing cellulose chains [24,25]. Molecular dynamic simulations also provided a nanoscopic view of the mechanism, showing that strong and medium hydrogen bonds decreased dramatically when CBM was bound to the cellulose surface of cotton fibers .
It is apparent from the steps in hydrolysis that the properties of the substrate, the multiple nature of the cellulase complex, and the mass transfer effects inﬂuence cellulose hydrolysis. A prerequisite for hydrolysis to occur is direct physical contact between the enzyme molecules and the surface of cellulose (Lee and Fan, 1982). Cellulase enzymes have a speciﬁc three-dimensional shape and their catalytic power depends on adsorption onto the surface of a substrate in lock-and-key fashion (Etters, 1998). A higher surface area enhances the accessibility of the enzyme molecules to the surface (Lee and Fan, 1982). Since cellulases are highly substrate speciﬁc in their action, any changes in the structure and ac- cessibility of the substrate has a profound inﬂuence on the kinetics of the hydrolysis reaction. Yarn type and fabric construction inﬂuence the hydrolysis rate (Karmakar, 1998). Key parameters for the cellulose substrate are accessible surface area, crystallinity, and pore dimensions. Changes in any of these fac- tors, such as structural changes brought about by pre-treatments, inﬂuence the hydrolysis reaction. It has been reported that mercerized and raised fabrics are more accessible to enzymatic attack, because they have a more accessible structure (Cavaco-Paulo and Almeida, 1996a). Crystallinity is another structural feature regarded as important. The cellulolytic en- zyme acts to a greater degree on the more accessible amorphous regions, so as crystallinity increases, cellulose becomes resistant to further hydrolysis (Lee and Fan, 1982). Milling of cellulose can in-
Appearance and morphological structure of poplar catkin fibers are shown in Fig. 1. Abundant white catkin appears on poplar branches in every spring, as shown in Fig. 1a- c. Poplar catkin fibers showed cylindrical hollow structure (Fig. 1d) with smooth surface (Fig. 1e). Similar hollow structure was also observed with other natural fibers such as kapok and milkweed fibers [18, 19]. The average outer diameter of poplar catkin fibers was 8 µm which a range from 4 to 14 µm, more than a half size less compared with kapok fiber at 20 µm . Because of its fine fiber struc- ture feature, poplar catkin fiber can be used directly without high economic and energetic costs of grinding process com- pared with lignocellulosic materials (such as wood), which accounting for 33% of the power requirement of the entire process . In addition, its hollow structure provided a large and effective surface area, for enzyme adsorption and catalytic reactions during enzymatichydrolysis.
regenerated from these solutions by precipitation was essentially amorphous and porous, which made the sub- sequent enzymatichydrolysis more efficient [8-11]. ILs are non-volatile with a low vapor pressure, and can be easily separated by distillation or condensation . Pre- treatment with ILs is considered an environmentally friendly alternative to conventional pretreatment meth- ods . Although pretreatment with ILs is a viable method, it faces three major challenges [14,15]: 1) the slow rate of dissolution within these liquids  means that it takes a long time for complete dissolution of the biomass; 2) the high viscosity of the solutions  causes agglomeration of cellulose and a resulting high consumption of energy for stirring; and 3) the high cost of ILs  makes them uneconomic for commercial use. To overcome these drawbacks, Sui et al. prepared a homogeneous cellulose solution by adding N,N- dimethylformamide (DMF) into a 1-allyl-3-methylimida- zolium chloride;([AMIM]Cl). Additional DMF compo- nent reduced the viscosity of the whole solvent at room temperature . Luo et al. reported that mixtures com- posed of dipolar aprotic intercrystalline swelling agents (for example, acetone, dioxane, pyridine, N-oxide, N- methyl pyridine, and hexamethylphosphoramide), and ILs can also dissolve wood pulp . Rinaldi developed a series of solvent systems called ‘organic electrolyte solutions’ (OESs), which contained a polar aprotic sol- vent (for example, DMF, N,N-dimethylacetamide, 1, 3- dimethyl-2-imidazolidinone, and dimethyl sulfoxide (DMSO)) and only a small molar fraction of ILs; these solutions had a strong ability to dissolve cellulose quickly . Because of their potential novel properties, OESs might be useful, environmentally friendly, and cost-saving solvents for pretreatment. However, no study has yet been performed to characterize and hydro- lyze OES-pretreated cellulose.