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Describes an inquiry-based biochemistry activity on the industrial applications of cellulase, which is known as biostoning, and used to soften denim jeans. (YDS)

Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure, expanded activities, data analysis, and discussion. (PVD)

A biotechnologist-turned-teacher introduces a series of laboratory modules incorporating concepts from microbiology, cellular biology, molecular biology, biochemistry, and evolution. The Amylase Project aims to distill the biotechnology process into a few short steps using amylase, the easiest enzyme to detect of those commonly produced by microorganisms. Description of modules includes enrichment possibilities. (PVD)

Reviews new developments in biotechnology--designer enzymes, biomining, gene cloning. Includes student outcomes, quiz, glossary, and a design brief on genetics. (SK)

Presents results of a study that examines the development and use of computer simulations for high school science instruction and for integrated laboratory and computerized tests that are part of the biology matriculation examination in Israel. Eleven implications for teaching are presented. (MDH)

The idea that errors and misconceptions in biology textbooks are often slow to be discovered and corrected is discussed. Selected errors, misconceptions, and topics of confusion about cell metabolism are described. Fermentation, respiration, Krebs cycle, pentose phosphate pathway, uniformity of catabolism, and metabolic pathways as models are included. (KR)

Presented is a teaching progression that enhances learning through low-cost, manipulative transparencies. Discussed is instruction about restriction enzymes, plasmids, cutting plasmids, plasmid maps, recording data, and mapping restriction sites. Mapping wheels for student use is included. (CW)

Uses a commercially available meat tenderizer for the source of an enzyme that can degrade meat protein and asks students to test the effects of incubating human hair in water with or without this enzyme. Teaches scientific methodology and the importance of experimental controls. (Author/YDS)

Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)

A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are included. (KR)

Diabetic foot ulcers (DFU) are chronic, refractory wounds caused by diabetic neuropathy, vascular disease, and bacterial infection, and have become one of the most serious and persistent complications of diabetes mellitus because of their high incidence and difficulty in healing. Its malignancy results from a complex microenvironment that includes a series of unfriendly physiological states secondary to hyperglycemia, such as recurrent infections, excessive oxidative stress, persistent inflammation, and ischemia and hypoxia. However, current common clinical treatments, such as antibiotic therapy, insulin therapy, surgical debridement, and conventional wound dressings all have drawbacks, and suboptimal outcomes exacerbate the financial and physical burdens of diabetic patients. Therefore, development of new, effective and affordable treatments for DFU represents a top priority to improve the quality of life of diabetic patients. In recent years, nanozymes-based diabetic wound therapy systems have been attracting extensive interest by integrating the unique advantages of nanomaterials and natural enzymes. Compared with natural enzymes, nanozymes possess more stable catalytic activity, lower production cost and greater maneuverability. Remarkably, many nanozymes possess multienzyme activities that can cascade multiple enzyme-catalyzed reactions simultaneously throughout the recovery process of DFU. Additionally, their favorable photothermal-acoustic properties can be exploited for further enhancement of the therapeutic effects. In this review we first describe the characteristic pathological microenvironment of DFU, then discuss the therapeutic mechanisms and applications of nanozymes in DFU healing, and finally, highlight the challenges and perspectives of nanozyme development for DFU treatment.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Introduction: Africa faces immense food and health insecurity challenges, a problem partly attributed to food loss and waste during postharvest handling and distribution. In the context of research to meet the sustainable development goals, this project specifically addressed the postharvest loss of the ripe indigenous eggplant (Solanum anguivi lam) fruit called Igba Yinrin by Yoruba in South-West Nigeria, which is usually discarded in farms. The study was carried out on ripe and unripe fruits to better understand their value by comparing their effects in diabetes treatment. Methods: The study sought to assess the effects of a diet including ripe or unripe mature eggplant fruits in the sucrose-induced diabetic-like fruit fly. Bioactive compounds were identified and quantified with HPLC-UV, while the antioxidant vitamin (A, C, E), carotenoid, and mineral (Na, K, Ca, Mg, Fe, P, and Zn) content was analyzed in the fruits. Extracts were used to investigate their in vitro anti-inflammatory properties on cyclooxygenases (COX 1 and 2), 5-lipoxygenase (5-LOX), and anti-diabetes enzymes [α-amylase and α-glucosidase], while extract-supplemented diets (0.25-1% concentration) were fed to the fruit flies for 14 days. Results: Interestingly, the results showed that the ripe fruits had a significantly (p < 0.05) higher total phenol and flavonoid content, as well as a higher content of vitamins, carotenoids, and minerals, than the unripe fruits. The in vivo activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione transferase (GST)] and the total thiol level increased, while the blood glucose, reactive oxygen species (ROS), and malondialdehyde (MDA) levels decreased in Drosophila melanogaster (fruit fly). An in silico docking analysis showed strong binding affinity of the above-mentioned enzymes under investigation with the ligands hesperidin, naringin, and myricetin, which are bioactive compounds contained in the examined extracts. Conclusions: There was no significant difference in the biological effects of the ripe and unripe fruit extracts on inflammatory and anti-diabetes enzyme activities, which means that the ripe fruit, usually discarded, could serve as a sustainable alternative source of food nutrients.

The effective design of combinatorial libraries to balance fitness and diversity facilitates the engineering of useful enzyme functions, particularly those that are poorly characterized or unknown in biology. We introduce MODIFY, a machine learning (ML) algorithm that learns from natural protein sequences to infer evolutionarily plausible mutations and predict enzyme fitness. MODIFY co-optimizes predicted fitness and sequence diversity of starting libraries, prioritizing high-fitness variants while ensuring broad sequence coverage. In silico evaluation shows that MODIFY outperforms state-of-the-art unsupervised methods in zero-shot fitness prediction and enables ML-guided directed evolution with enhanced efficiency. Using MODIFY, we engineer generalist biocatalysts derived from a thermostable cytochrome c to achieve enantioselective C-B and C-Si bond formation via a new-to-nature carbene transfer mechanism, leading to biocatalysts six mutations away from previously developed enzymes while exhibiting superior or comparable activities. These results demonstrate MODIFY's potential in solving challenging enzyme engineering problems beyond the reach of classic directed evolution.

Levulinic acid, which can be obtained from biomass, has sparked great interest as a biologically-based chemical building block with wide versatility and potential. Its esterification with alcohols of different chain lengths is a promising valorization process for obtaining esters with various applications in the areas of biofuels/biolubricants, food and cosmetics, among others. In this work, the enzymatic esterification of levulinic acid and 1-octanol using a biocatalyst derived from Araujia sericifera latex was studied in systems with and without solvent. The influence of the molar ratio between alcohol and acid (ranging from 2:1–1:9), the biocatalyst loading (between 7.5 % and 17.5 % relative to the acid), the volume of n -heptane used as reaction solvent (from 0 to 4 ml), and the reaction time (6 hours) were investigated. The activity and stability of the biocatalyst in successive uses were also analyzed. A conversion of 49 % was achieved when the reaction was carried out in a solvent-free system, using an alcohol/acid molar ratio of 1:7 and after 5 h of reaction. On the other hand, the conversion was 65.1 % when the reaction was conducted in a system containing 1 ml of n -heptane as solvent, an alcohol/acid molar ratio of 1:8, and 5 h of reaction. In both cases, a temperature as low as 30 °C and an agitation speed of 300 RPM were used. • Enzymatic route for levulinic acid esterification using biocatalyst. • Araujia sericifera latex-derived biocatalyst demonstrates high activity. • Achieved 65.1 % conversion of levulinic acid in the synthesis of octyl levulinate. • Solvent-free and n-heptane solvent systems investigated. • Studied reaction parameters for optimal conversion. [ABSTRACT FROM AUTHOR]

The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)). While coimmobilization of ficin with BSA presented no effect on the activity versus any of the assayed substrates, coimmobilization with hemoglobin permitted to improve the immobilized ficin specificity for casein versus hemoglobin, but still significant activity versus hemoglobin remained. Second, aldehyde-dextran has been employed to modify the immobilized ficin, trying to generate steric hindrances to avoid the entry of large proteins (hemoglobin) while enabling the entry of small ones (casein). This also increased the size specificity of ficin, but still did not suppress the activity versus hemoglobin. The combination of both strategies and the use of 37ºC during the proteolysis enabled to almost fully nullify the hydrolytic activity versus hemoglobin while preserving a high percentage of the activity versus casein. The modifications improved enzyme stability and the biocatalyst could be reused for 5 cycles without alteration of its properties. • Ficin extract immobilized on glyoxyl was able to hydrolyze casein and hemoglobin. • Ficin coimmobilization with large hemoglobin increases its specificity by casein. • Ficin modification with aldehyde dextran increases its specificity by casein. • Both strategies together almost fully nullify the activity versus hemoglobin. [ABSTRACT FROM AUTHOR]

Biocatalytic membranes have great potential in various industrial sectors, with the immobilization of enzymes being a crucial stage. Immobilizing enzymes through covalent bonds is a complex and time-consuming process for large-scale applications. Polydopamine (PDA) offers a more sustainable and eco-friendly alternative for enzyme immobilization. Therefore, surface modification with polydopamine as mussel-inspired antifouling coatings has increased resistance to fouling. In this study, α-amylase enzyme was covalently bound to a bioactive PDA-coated polyethersulfone (PES) membrane surface using cyanuric chloride as a linker. The optimal activity of α-amylase enzyme immobilized on PES/PDA membrane was obtained at temperature and pH of 55°C and 6.5, respectively. The immobilized enzyme can be reused up to five reaction cycles with 55 % retention of initial activity. Besides, it maintained 60 % of its activity after being stored for five weeks at 4°C. Additionally, the immobilized enzyme demonstrated increased Michaelis constant and maximum velocity values during starch hydrolysis. The results of the biofouling experiment of various membranes in a dead-end cell demonstrated that the PES membrane's water flux increased from 6722.7 Lmh to 7560.2 Lmh after PDA modification. Although α-amylase immobilization reduced the flux to 7458.5 Lmh due to enhanced hydrophilicity, compared to unmodified membrane. The findings of this study demonstrated that the membrane produced through co-deposition exhibited superior hydrophilicity, enhanced coating stability, and strong antifouling properties, positioning it as a promising candidate for industrial applications. [Display omitted] • α-Amylase was covalently immobilized onto polydopamine-functionalized polyethersulfone membrane. • Surface modification with polydopamine generated a mussel-inspired coating to inhibit fouling. • The immobilization technique effectively preserved the activity of α-amylase enzyme. • The biocatalyst membrane exhibited superior stability and strong antifouling properties. [ABSTRACT FROM AUTHOR]

Carrot juice color changes from orange to brown immediately after production. Blanching is a suitable way to preserve and commercialize this product. In this study, an ohmic device was used as a heating source, and polyphenol oxidase enzyme was selected as an indicator of enzyme inactivation sufficiency to color change of carrot juice. Fresh carrot juice was subjected to thermal processing under 3 temperature levels of 70, 80, and 90 degrees of Celsius and 4 time levels of 0, 20, 40 and 60 seconds with a constant voltage of 100 volts and the enzyme inactivation, Brix, pH and color indexes L*, a* and b* were checked. Constant values of reaction rate, activation energy, D-value and Q10 were investigated. The kinetics of inactivation of polyphenol oxidase of carrot juice samples were calculated at 70, 80 and 90 degrees of Celsius. The changes of enzyme inactivation, Brix, pH and a* parameter with increasing temperature and time were significant at 1% probability level by completely random design. The level of enzyme inactivation was also measured by the conventional water bath method and compared with the ohmic method, and the results indicated the greater efficiency of the ohmic method. As the temperature increased, the Brix level increased and the color of carrot juice changed from orange-yellow to orange-red (p≤0.05) [ABSTRACT FROM AUTHOR]

Purpose: To investigate the relationship between the changes of lactate dehydrogenase (LDH), neuron‐specific enolase (NSE), keratin‐19 fragment antigen 21‐1 (cyfra21‐1), and liver metastases of lung cancer. Methods: Eighty patients who had lung cancer that had spread to their liver diagnosed in our hospital from October 2021 to October 2023 (Group A), 80 individuals with advanced lung cancer who have metastasized to other sites (Group B), and 80 individuals with lung cancer who have not spread (control group) were selected as the study objects. LDH, NSE, and serum cyfra21‐1 levels of patients in the three groups were detected, and pathological results were used as the diagnostic gold standard. ROC curves were drawn to examine the clinical value of NSE, cyfra21‐1, and LDH levels in the distinct types of lung cancer identification of liver metastases. Results: There was no remarkable variation in pathological types among the three groups (p > 0.05), but there were remarkable variations in TNM stage and lymph node metastasis among the three groups (p < 0.05). The levels of cyfra21‐1, NSE, and LDH in Group A and Group B were greater compared to those in the control group (p < 0.05), and the levels of cyfra21‐1, NSE, and LDH in Group A were greater compared to those in Group B (p < 0.05). The critical value, sensitivity, specificity, and area under the curve (AUC) of serum cyfra21‐1 in the distinct identification of liver metastasis of lung cancer were 8.22 ng/mL, 37.42%, 65.18%, and 0.508, respectively. The critical value, sensitivity, specificity, and AUC of NSE for distinct types of lung cancer identification of liver metastases were 23.96 ng/mL, 64.56%, 81.23%, and 0.723, respectively. The critical value, sensitivity, specificity, and AUC of LDH for differential diagnosis of liver metastasis of lung cancer were 304.78 U/L, 75.65%, 85.73%, and 0.821. Conclusion: The serum levels of NSE, cyfra21‐1, and LDH in patients with liver metastasis of lung cancer were remarkably greater compared to patients without liver metastasis, which can be useful as a clinical auxiliary in determining lung cancer metastasis. [ABSTRACT FROM AUTHOR]