Mathematical expressions describing enzyme velocity and inhibition at high enzyme concentration

ABSTRACTEnzyme behaviour is typically characterised in the laboratory using very diluted solutions of enzyme. However, in vivo processes usually occur at [ST] ≈ [ET] ≈ Km. Furthermore, the study of enzyme action usually involves analysis and characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived to apply to the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and small molecule (inhibitor or activator) kinetic interactions. These expressions are simple and can be understood as an extension of the classical Michaelis-Menten equations. A first analysis of these expressions permits to deduce some differences at high vs low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised following classical approaches, these equations also show some differences that need to be taken into account. The most important one may be the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for the translation in vivo of in vitro experimental data or for modelling in vivo and biotechnological processes.

Machine-learning-guided library design cycle for directed evolution of enzymes: the effects of training data composition on sequence space exploration

Machine learning (ML) is becoming an attractive tool in mutagenesis-based protein engineering because of its ability to design a variant library containing proteins with a desired function. However, it remains unclear how ML guides directed evolution in sequence space depending on the composition of training data. Here, we present a ML-guided directed evolution study of an enzyme to investigate the effects of a known "highly positive" variant (i.e., variant known to have high enzyme activity) in training data. We performed two separate series of ML-guided directed evolution of Sortase A with and without a known highly positive variant called 5M in training data. In each series, two rounds of ML were conducted: variants predicted by the first round were experimentally evaluated, and used as additional training data for the second-round prediction. The improvements in enzyme activity were comparable between the two series, both achieving enzyme activity 2.2-2.5 times higher than 5M. Intriguingly, the sequences of the improved variants were largely different between the two series, indicating that ML guided the directed evolution to the distinct regions of sequence space depending on the presence/absence of the highly positive variant in the training data. This suggests that the sequence diversity of improved variants can be expanded not only by conventional ML using the whole training data, but also by ML using a subset of the training data even when it lacks highly positive variants. In summary, this study demonstrates the importance of regulating the composition of training data in ML-guided directed evolution.

A Modified Brewing Procedure Informed by the Enzymatic Profiles of Gluten-Free Malts Significantly Improves Fermentable Sugar Generation in Gluten-Free Brewing

The mashing step underpins the brewing process, during which the endogenous amylolytic enzymes in the malt, chiefly β-amylase, α-amylase, and limit dextrinase, act concurrently to rapidly hydrolyze malt starch to fermentable sugars. With barley malts, the mashing step is relatively straightforward, due in part to malted barley’s high enzyme activity, enzyme thermostabilities, and gelatinization properties. However, barley beers also contain gluten and individuals with celiac disease or other gluten intolerances should avoid consuming these beers. Producing gluten-free beer from gluten-free malts is difficult, generally because gluten-free malts have lower enzyme activities. Strategies to produce gluten-free beers commonly rely on exogenous enzymes to perform the hydrolysis. In this study, it was determined that the pH optima of the enzymes from gluten-free malts correspond to regions already typically targeted for barley mashes, but that a lower mashing temperature was required as the enzymes exhibited low thermostability at common mashing temperatures. The ExGM decoction mashing procedure was developed to retain enzyme activity, but ensure starch gelatinization, and demonstrates a modified brewing procedure using gluten-free malts, or a combination of malts with sub-optimal enzyme profiles, that produces high fermentable sugar concentrations. This study demonstrates that gluten-free malts can produce high fermentable sugar concentrations without requiring enzyme supplementation.

Process Improvisation Strategies for the Enhancement of Lipase Activity

In this present study, lipase-producing bacteria were isolated and screened from an indigenous soil sample and were used for lipase production with high enzyme activity. In the production medium, different production media were screened and lipase production was induced by olive oil, 14 mL/L. It was observed from Luedeking and Piret model that the lipase production was mixed growth associated with maximum activity at 37°C and at pH 7. Statistical optimization using Response Surface Methodology was performed to understand the interaction of different parameters and the standardized conditions obtained were as follows: Peptone 10 g/L, yeast extract 7.5 g/L and olive oil 14 mL/L. The predicted data were validated and the model predicted was significant with a maximum specific activity of 1.1 µmole/min/mg proteins. The lipasespecific activity was enhanced by 10% and 23% after a single parameter and statistical optimization.

A model of digestive tooth corrosion in lizards: experimental tests and taphonomic implications

Corrosion patterns induced by gastric fluids on the skeleton of prey animals may depend on the nature of the corrosive agents (acid, enzymes) as well as on the composition of the hard parts and the soft tissues that surround them. We propose a framework for predicting and interpreting corrosion patterns on lizard teeth, our model system, drawing on the different digestive pathways of avian and non-avian vertebrate predators. We propose that high-acid, low-enzyme systems (embodied by mammalian carnivores) will lead to corrosion of the tooth crowns, whereas low-acid, high-enzyme systems (embodied by owls) will lead to corrosion of the tooth shafts. We test our model experimentally using artificial gastric fluids (with HCl and pepsin) and feeding experiments, and phenomenologically using wild-collected owl pellets with lizard remains. Finding an association between the predictions and the experimental results, we then examine corrosion patterns on nearly 900 fossil lizard jaws. Given an appropriate phylogenetic background, our focus on physiological rather than taxonomic classes of predators allows the extension of the approach into Deep Time.

Optimization of a fluorogenic assay to determine caspase 3/7 activity in meat extracts

Usefulness of general-purpose fluorogenic assay kits to determine caspase 3/7 activity of biological extracts is highly compromised in meat-based samples due to their scarce enzyme concentration. In the present work, a straightforward protocol is presented with two main purposes: 1) to enhance sensitivity of the fluorogenic approach addressing caspase 3/7 activity in tissues showing scarce enzyme concentration such as skeletal muscle, and 2) to reduce/economize the volume of employed reagents. The enzyme extraction procedure, peptide substrate, dithiothreitol concentration and detection settings were appropriately optimized for use in microtiter-plate fluorometers. As a result, low to high enzyme activity extracts (from 10,000 to 260,000 relative fluorescence units) can be measured under developed sampling and experimental conditions. The fact that enzyme reactions took place in 96-microtiter well plates reduces the consumption of chemical compounds when analysing a high number of samples, thus contributing to environment sustainability.

Progress in Enzymatic Biodiesel Production and Commercialization

Enzymatic biodiesel production has attracted tremendous interest due to its well-recognized advantages. However, high enzyme costs limit the application of enzymatic processes in industrial production. In the past decade, great improvements have been achieved in the lab and the industrial scale, and the production cost of the enzymatic process has been reduced significantly, which has led to it being economically competitive compared to the chemical process. This paper summarizes the progress achieved in enzymatic biodiesel research and commercialization, including reducing enzyme cost, expanding low-quality raw materials, and novel reactor designs. The advantages and disadvantages of different enzymatic processes are also compared.

Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose

Background Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose. Results We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR. Conclusion Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

Preparation of enzymatically highly active pegylated-D-amino acid oxidase and its application to antitumor therapy

Background: D-amino acid oxidase (DAO) is an H2O2-generating enzyme, and tumor growth suppression by selective delivery of porcine DAO in tumors via the cytotoxic action of H2O2 has been reported. DAO isolated from Fusarium spp. (fDAO) shows much higher enzyme activity than porcine DAO, although the application of fDAO for antitumor treatment has not yet been determined. Objective: The purpose of this study was to prepare enzymatically highly active pegylated-fDAO, and to determine whether it accumulates in tumors and exerts a potent antitumor effect in tumor-bearing mice. Methods: Polyethylene glycol (PEG; Mw. 2000) was conjugated to fDAO to form PEGylated fDAO (PEG-fDAO). PEGfDAO was intravenously administered into S180 tumor-bearing mice, and the body distribution and antitumor activity of PEG-fDAO was determined. Results: The enzyme activity of PEG-fDAO was 26.1 U/mg, which was comparable to that of fDAO. Intravenously administered PEG-fDAO accumulated in tumors with less distribution in normal tissue except in the plasma. Enzyme activity in the tumor was 60–120 mU/g-tissue over 7–20 h after i.v. injection of 0.1 mg of PEG-fDAO. To generate the H2O2 in the tumor tissue, PEG-fDAO was intravenously administered, and then, D-phenylalanine was intraperitoneally administered after a lag time. No remarkable tumor suppression effect was observed under conditions used in this study, compared to the non-treated group. Conclusion: The results suggest that PEG-fDAO maintained high enzymatic activity after pegylation. Treatment with PEGfDAO conferred high enzyme activity on tumor tissue; 3–6 fold higher than that of previously reported pDAO; however, high enzyme activity in the plasma limited repeated treatment owing to lethal toxicity, which seemingly led to poor therapeutic outcome. Overall, the use of PEG-fDAO is promising for antitumor therapy, although the suppression of DAO activity in the plasma would also be required rather than only the increase in DAO activity in the tumor for an antitumor effect.