Enzyme Optimization for Lignocellulose Hydrolysis using Mechanistic Modeling

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) from Trichoderma reesei; β-glucosidase (BG), and β-xylosidase (BX) from Aspergillus niger. The model employs the synergistic action of these enzymes to predict optimum enzyme concentrations for hydrolysis of Avicel and ammonia fiber explosion (AFEX) pretreated corn stover. Glucan, glucan + xylan, glucose and glucose + xylose conversion predictions are given over a range of mass fractions of enzymes, and a range of enzyme loadings. Simulation results are compared with optimizations using statistically designed experiments. BG and BX are modeled in solution at later time points to predict the effect on glucose conversion and xylose conversion.

Optimization of xylanase biosynthesis by Aspergillus japonicus isolated from a Caatinga area in the Brazilian state of Bahia

The objective of this research was to investigate the potential of xylanase production by Aspergillus japonicus and to determine the effects of cultivation conditions in the process, aiming toward optimization of enzyme production. The best temperature, as well as the best carbon source, for biomass production was determined through an automated turbidimetric method (Bioscreen-C). The enzyme activity of this fungus was separately evaluated in two solid substrates (wheat and soybean bran) and in Vogel medium, adding other carbon sources. Temperature effects, cultivation time, and spore concentrations were also tested. The best temperature for enzyme and biomass production was 25°C; however, the best carbon source for growth (determined by the Bioscreen C) did not turn out to be a good inducer of xylanase production. Maximum xylanase activity was achieved when the fungus was cultivated in wheat bran (without the addition of any other carbon source) using a spore concentration of 1 × 107 spores/mL (25°C, pH 5.0, 120 h). A. japonicus is a good xylanase producer under the conditions presented in these assays. © 2006 Academic Journals.

Screening of culture condition for xylanase production by filamentous fungi

The objective of this research was to investigate xylanase production by filamentous fungi (Trichoderma viride) to determine the best cultivation conditions in the process, aiming toward optimization of enzyme production. The best temperature, as well as the best carbon source, for biomass production was determined through an automated turbidimetric method (Bioscreen-C). The enzyme activity of this fungus was separately evaluated in two solid substrates (wheat and soybean bran) and in Vogel medium, pure and by adding other carbon sources. Temperature effects, cultivation time, and spore concentrations were also tested. The best temperature and carbon source for enzyme and biomass production was 25 C and sorbitol, respectively. Maximum xylanase activity was achieved when the fungus was cultivated in wheat bran along with sorbitol (1%, w/v), using a spore concentration of 2 x 10(6) spores. mL(-1), pH 5.0, for 144 h cultivation. The study demonstrated not only the importance of the nature of the substrate in obtaining a system resistant to catabolic repression, but also the importance of the culture conditions for biosynthesis of this enzyme. T. viride showed a high potential for xylanase production under the conditions presented in these assays.

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg-1 protein and 7.03 U mg-1 protein for free and immobilized preparations, respectively). © 2013 Elsevier Ltd.

An evolutionary approach to the design of experiments for combinatorial optimization with an application to enzyme engineering

In a large number of problems the high dimensionality of the search space, the vast number of variables and the economical constrains limit the ability of classical techniques to reach the optimum of a function, known or unknown. In this thesis we investigate the possibility to combine approaches from advanced statistics and optimization algorithms in such a way to better explore the combinatorial search space and to increase the performance of the approaches. To this purpose we propose two methods: (i) Model Based Ant Colony Design and (ii) Naïve Bayes Ant Colony Optimization. We test the performance of the two proposed solutions on a simulation study and we apply the novel techniques on an appplication in the field of Enzyme Engineering and Design.

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer`s spent grain

Brewer`s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 degrees C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2(3) full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer`s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.

Extending the kinetic solution of the classic Michaelis-Menten model of enzyme action

The principal aim of studies of enzyme-mediated reactions has been to provide comparative and quantitative information on enzyme-catalyzed reactions under distinct conditions. The classic Michaelis-Menten model (Biochem Zeit 49:333, 1913) for enzyme kinetic has been widely used to determine important parameters involved in enzyme catalysis, particularly the Michaelis-Menten constant (K (M) ) and the maximum velocity of reaction (V (max) ). Subsequently, a detailed treatment of the mechanisms of enzyme catalysis was undertaken by Briggs-Haldane (Biochem J 19:338, 1925). These authors proposed the steady-state treatment, since its applicability was constrained to this condition. The present work describes an extending solution of the Michaelis-Menten model without the need for such a steady-state restriction. We provide the first analysis of all of the individual reaction constants calculated analytically. Using this approach, it is possible to accurately predict the results under new experimental conditions and to characterize and optimize industrial processes in the fields of chemical and food engineering, pharmaceuticals and biotechnology.

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

Optimization of the Tartrate-Resistant Acid Phosphatase Detection by Histochemical Method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.

Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA) for experimental plague

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

Background: Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results: We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA) representation. The optimization task is posed as a nonconvex nonlinear programming (NLP) problem that is solved by an outer- approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP) master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion: Our results show the utility of the suggested approach for investigating the evolution of adaptive responses to environmental changes. The proposed method can be used in other important applications such as the evaluation of parameter changes that are compatible with health and disease states.

Orthologous and heterologous expression of a protein from Saccharomyces cerevisiae: Optimization for a biotechnological application

In this thesis (TFG) the results of the comparison between different methods to obtain a recombinant protein, by orthologous and heterologous expression, are exposed. This study will help us to identify the best way to express and purify a recombinant protein that will be used for biotechnology applications. In the first part of the project the goal was to find the best expression and purification system to obtain the recombinant protein of interest. To achieve this objective, a system expression in bacteria and in yeast was designed. The DNA was cloned into two different expression vectors to create a fusion protein with two different tags, and the expression of the protein was induced by IPTG or glucose. Additionally, in yeast, two promoters where used to express the protein, the one corresponding to the same protein (orthologous expression), and the ENO2 promoter (heterologous expression). The protein of interest is a NAD-dependent enzyme so, in a second time, its specific activity was evaluated by coenzyme conversion. The results of the TFG suggest that, comparing the model organisms, bacteria are more efficient than yeast because the quantity of protein obtained is higher and better purified. Regarding yeast, comparing the two expression mechanisms that were designed, heterologous expression works much better than the orthologous expression, so in case that we want to use yeast as expression model for the protein of interest, ENO2 will be the best option. Finally, the enzymatic assays, done to compare the effectiveness of the different expression mechanisms respect to the protein activity, revealed that the protein purified in yeast had more activity in converting the NAD coenzyme.

Application of Doehlert experimental design in the optimization of experimental variables for the Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) cell lysis

This study aimed to verify the influence of pH and temperature on the lysis of yeast using experimental design. In this study, the enzymatic extract containing β-1,3-glucanase and chitinase, obtained from the micro-organism Moniliophthora perniciosa, was used. The experiment showed that the best conditions for lysis of Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) by lytic enzyme were pH 4.9 at 37 ºC and pH 3.9 at 26.7 ºC, respectively. The lytic enzyme may be used for obtaining various biotechnology products from yeast.