New advances in protein engineering for industrial applications: Key takeaways.

Recent advancements in protein/enzyme engineering have enabled the production of a diverse array of high-value compounds in microbial systems with the potential for industrial applications. The goal of this review is to articulate some of the most recent protein engineering advances in bacteria, yeast, and other microbial systems to produce valuable substances. These high-value substances include α-farnesene, vitamin B12, fumaric acid, linalool, glucaric acid, carminic acid, mycosporine-like amino acids, patchoulol, orcinol glucoside, d-lactic acid, keratinase, α-glucanotransferases, ß-glucosidase, seleno-methylselenocysteine, fatty acids, high-efficiency ß-glucosidase enzymes, cellulase, ß-carotene, physcion, and glucoamylase. Additionally, recent advances in enzyme engineering for enhancing thermostability will be discussed. These findings have the potential to revolutionize various industries, including biotechnology, food, pharmaceuticals, and biofuels.

Current status and emerging frontiers in enzyme engineering: An industrial perspective.

Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the introduction of new-to-nature chemical reactivities are also possible through the same mechanism. Discovering new protocols that enhance the catalytic active protein that possesses novelty in terms of being stable, active, and, stereoselectivity with functions could be identified as essential areas in terms of concurrent bioorganic chemistry (synergistic relationship between organic chemistry and biochemistry in the context of enzyme engineering). However, with our current level of knowledge about protein folding and its correlation with protein conformation and activities, it is almost impossible to design proteins with specific biological and physical properties. Hence, contemporary protein engineering typically involves reprogramming existing enzymes by mutagenesis to generate new phenotypes with desired properties. These processes ensure that limitations of naturally occurring enzymes are not encountered. For example, researchers have engineered cellulases and hemicellulases to withstand harsh conditions encountered during biomass pretreatment, such as high temperatures and acidic environments. By enhancing the activity and robustness of these enzymes, biofuel production becomes more economically viable and environmentally sustainable. Recent trends in enzyme engineering have enabled the development of tailored biocatalysts for pharmaceutical applications. For instance, researchers have engineered enzymes such as cytochrome P450s and amine oxidases to catalyze challenging reactions involved in drug synthesis. In addition to conventional methods, there has been an increasing application of machine learning techniques to identify patterns in data. These patterns are then used to predict protein structures, enhance enzyme solubility, stability, and function, forecast substrate specificity, and assist in rational protein design. In this review, we discussed recent trends in enzyme engineering to optimize the biochemical properties of various biocatalysts. Using examples relevant to biotechnology in engineering enzymes, we try to expatiate the significance of enzyme engineering with how these methods could be applied to optimize the biochemical properties of a naturally occurring enzyme.

Detergent-stable amylase production by Paenibacillus lactis strain OPSA3 isolated from soil; optimization by response surface methodology.

This study aimed to isolate thermostable, alkaliphilic, and detergent-tolerant amylase-producing bacteria. Pure isolates from environmental samples were screened on a starch-based medium (pH 11), and selected isolates were identified using cultural and molecular techniques. Product optimization studies were conducted, and secreted amylase was partially purified using 40% (w/v) saturation ammonium sulfate at 4 °C. The wash performance of concentrated amylase was analyzed. A novel isolate, Paenibacillus lactis OPSA3, was selected for further studies. The isolate produced amylase optimally when grown on banana peels and soybean extracts, which are agro-wastes. Optimization by Response surface Methodology resulted in a 2.1-fold increase in alkaliphilic amylase production. A 2.46-fold purification was achieved, with an enzyme activity yield of 79.53% and specific activity of 26.19 Umg-1. Wash performance analysis using the amylase supplemented with boiled commercial detergent (kiln®) showed good cleaning efficiency. The amylase has the potential for application as a component of green detergent.

Probiotic therapy, African fermented foods and food-derived bioactive peptides in the management of SARS-CoV-2 cases and other viral infections.

The current paper focuses on the impact of probiotics, African fermented foods and bioactive peptides on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection severity and related viral infections. Using probiotics or bioactive peptides as therapeutic adjuncts appears superior to standard care alone. Probiotics play critical roles in innate and adaptive immune modulation by balancing the gut microbiota to combat viral infections, secondary bacterial infections and microbial dysbiosis. African fermented foods contain abundant potential probiotic microorganisms such as the lactic acid bacteria (LAB), Saccharomyces, and Bacillus. More so, fermented food-derived bioactive peptides play vital roles in preventing cardiovascular diseases, hypertension, lung injury, diabetes, and other COVID-19 comorbidities. Regularly incorporating potential probiotics and bioactive peptides into diets should enable a build-up of the benefits in the body system that may result in a better prognosis, especially in COVID-19 patients with underlying complexities. Despite the reported therapeutic potentials of probiotics and fermented foods, numerous setbacks exist regarding their application in disease management. These shortfalls underscore an evident need for more studies to evaluate the specific potentials of probiotics and traditional fermented foods in ameliorating SARS-CoV-2 and other viral infections.

Enhancement of the Anti-inflammatory Effect of Bromelain by Its Immobilization on Probiotic Spore of Bacillus cereus.

The therapeutic application of bromelain is limited due to its sensitivity to operating conditions such as high acidity, gastric proteases in the stomach juice, chemicals, organic solvents and elevated temperature. We hypothesized that bromelain immobilized on probiotic bacterial spores would show enhanced therapeutic activity through possible synergistic or additive effects. In this study, the oedema inhibition potential of bromelain immobilized on probiotic Bacillus spores was compared to the free enzyme using the carrageenan paw oedema model with Wistar rats. In batch A rats (carrageenan-induced inflammation 30 min after receiving oral treatments), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed the highest oedema inhibition, 89.20 ± 15.30%, while group 4 treated with a lower dose of free bromelain had oedema inhibition of 60.25 ± 13.00%. For batch B rats (carrageenan-induced inflammation after receiving oral treatment for three days), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed higher inhibition percentage (81.94 ± 8.86) than group 4 treated with a lower dose of free bromelain (78.45 ± 4.46) after 24 h. Our results showed that used alone, the enzyme and the spores produced oedema inhibition and improved the motility of the rats. The spore-immobilized bromelain formulation performed approximately 0.9-fold better than the free bromelain and the free spores at the lower evaluated dose.

Stabilizing enzymes by immobilization on bacterial spores: A review of literature.

The ever-increasing applications of enzymes are limited by the relatively poor performance in harsh processing conditions. As a result, there are constant innovations in immobilization protocols for improving biocatalyst activity and stability. Bacterial spores are cheap to generate and highly resistant to environmental stress. The spore core is sheathed by an inner membrane, the germ cell wall, the cortex, outer membrane, spore coat and in some species the exosporium. The spore surface is anion-rich, hydrophobic and contains several reactive groups capable of interacting and stabilizing enzyme molecules through electrostatic forces, hydrophobic interactions and covalent bonding. The probiotic nature of spores obtained from non-toxic bacterial species makes them suitable carriers for the enzyme immobilization, especially food-grade enzymes or those intended for therapeutic use. Immobilization on spores is by direct adsorption, covalent attachment or surface display during the sporulation phase. Hindrances to the immobilization on spore matrix include the production rates, operational instability, and reduced catalytic properties due to conformational changes in enzyme. This paper reviews bacterial spore as a heterofunctional support matrix gives reasons why probiotic bacillus spores are better options and the diverse technologies adopted for spore-enzyme immobilization. It further suggests directions for future use and discusses the commercialization prospects.

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius.

Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius. The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol-1) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.

Stabilizing bromelain for therapeutic applications by adsorption immobilization on spores of probiotic Bacillus.

Bromelain, a protease from pineapple plant can be applied as oral drug for the treatment of inflammation and certain diseases. Unlike most conventional supports, immobilization on edible support will make the enzyme suitable for therapeutic use. In this study, spores of probiotic Bacillus sp was used for the adsorption of bromelain. Effect of pH, temperature and enzyme concentration on bromelain immobilization was studied, followed by characterization of the enzymes. Maximum bromelain coupling (%) (50.607 ±â€¯4.194) was obtained when immobilization was carried out at pH 6.0, 24 °C for 150 min. The immobilized enzyme showed optimum activity at pH 8 and 80 °C, while the free enzyme had 6 and 60 °C as its optimum pH and temperature, respectively. Bromelain Vmax increased after immobilization while Km decreased. Activation energy, Ea was 26.513 kJ/mol and 20.942 kJ/mol for the free and immobilized enzymes, respectively. The immobilized bromelain also showed significantly greater storage and thermal stability than the free bromelain. At 80 °C, the free bromelain lost all its activity after 50 min while the immobilized enzyme lost only 46.89% activity. Bromelain was successfully immobilized on Bacillus spores with improved catalytic and non-catalytic properties and this holds great potential for its growing therapeutic applications.

Chemical modification with phthalic anhydride and chitosan: Viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius.

The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time compared to regular amylases. Chemical modification is a simple and rapid method toward their stabilization for a wider application. RSDA from Aspergillus carbonarius was modified with either phthalic anhydride (PA) or chitosan. Activity retention was 87.3% for PA-modified and 80.9% for chitosan-modified RSDA. Optimum pH shifted from 5 to 7 after PA-modification. Optimum temperature changed from 30°C (native) to 30-40°C and 60°C for PA-modified and chitosan-modified, respectively. Activation energy (kJmol-1) for hydrolysis was 13.5, 12.7, and 10.2 while the activation energy for thermal denaturation was 32.8, 80.3, 81.9 for free, PA-modified and chitosan-modified, respectively. The specificity constants (Vmax/Km) were 73.2 for PA-modified, 63.1 for chitosan-modified and 77.1 for native RSDA. The half-life (h) of the RSDA at 80°C was increased from 6.1 to 25.7 for the PA-modified and 138.6 for the chitosan derivative. Modification also led to increase in D value, activation enthalpy and Gibbs free energy of enzyme deactivation. Fluorescence spectra showed that center of spectral mass decreased for the PA-modified RSDA but increased for chitosan modified RSDA.

Extracellular amylase production of a thermotolerant Fusarium sp: isolated from Eastern Nigerian soil

In this work, an α- amylase producing Fusarium sp. was isolated from the soil at 50 0C. Growth and enzyme production occurred at 30, 45 and 55 ºC. Soybean meal at 1 percent concentration, supplemented with 0.2 percent NH4Cl and 2.5 percent corn starch elicited the highest amylase yield. Optimum pH for the enzyme was pH 6.5 which retained over 60 percent of its activity after 24 h incubation at the pH range of 4.5-7.0. The enzyme showed high activity from 40-70 0C with optimal activity at 50 ºC and 78 percent activity was retained after incubation at 70 ºC for 30min. Catalytic function of the crude amylase was stimulated by Mg (136 percent), Ca (118 percent) and Zn (118 percent) at 2mM concentrations. The enzyme hydrolyzed cassava, potato and yam starches effectively.