Comparison of Biochemical Characteristics, Action Models, and Enzymatic Mechanisms of a Novel Exolytic and Two Endolytic Lyases with Mannuronate Preference

Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the β-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H195NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.

Fungal Laccases: The Forefront of Enzymes for Sustainability

Enzymatic catalysis is one of the main pillars of sustainability for industrial production. Enzyme application allows minimization of the use of toxic solvents and to valorize the agro-industrial residues through reuse. In addition, they are safe and energy efficient. Nonetheless, their use in biotechnological processes is still hindered by the cost, stability, and low rate of recycling and reuse. Among the many industrial enzymes, fungal laccases (LCs) are perfect candidates to serve as a biotechnological tool as they are outstanding, versatile catalytic oxidants, only requiring molecular oxygen to function. LCs are able to degrade phenolic components of lignin, allowing them to efficiently reuse the lignocellulosic biomass for the production of enzymes, bioactive compounds, or clean energy, while minimizing the use of chemicals. Therefore, this review aims to give an overview of fungal LC, a promising green and sustainable enzyme, its mechanism of action, advantages, disadvantages, and solutions for its use as a tool to reduce the environmental and economic impact of industrial processes with a particular insight on the reuse of agro-wastes.

Application of crude pectolytic enzymes from Saccaharomyces cerevisiae (ATCC 52,712) to starch extraction in Ghana: effects of enzyme technology on pasting characteristics in different cassava varieties

Previous work on enzyme application to starch extraction enhanced yield and starch recovery rates as well as modified some physicochemical properties of starches for potential alternative application to industry. The response of the technology, however, showed some sensitivity to variety. The knowledge gap therefore was to establish whether such physicochemical responses (by the technology) to variety affects the pasting parameters of the starches extracted. The pasting parameters of starches extracted from four different cassava varieties (‘Nkabom’, ‘Afisiafi’, ‘Bankyehemaa’ and ‘Esambankye’), with the aid of crude pectolytic enzymes from the Saccharomyces cerevisiae (ATCC 52,712), were investigated. Although a general response pattern was observed for most of the pasting parameters measured, which includes general enhancements (P < 0.05) in starch gelatinization viscosity, with improvements in gelatinization time and temperature and peak viscosities in most of the varieties, there were significant differences (P < 0.05) in their respective peak time and temperature requirements for the attainment of peak viscosity. Values for the breakdown viscosity were also generally increased (P < 0.05). The technology also increased values for setback viscosity in both the ‘Nkabom’ and ‘Bankyehemaa’ varieties but reduced setback values in the ‘Afisiafi’ and ‘Esambankye’ varieties. As pasting properties are one of the most important characteristics of starch that determine its overall utility, knowledge from this study should inform how adoption of the technology would help diversify the various cassava varieties for appropriate domestic and industrial applications while harnessing its benefits of improved starch yield.

Productivity of Turi (Sesbania grandiflora) as a multi purposes plant by eco enzyme application

Turi (Sesbania grandiflora) is a multi-purpose plant, including leaves for animal feed, flowers for human food and wood for wood pellets. Eco enzyme is produced from the fermentation of fruits containing enzymes and organic acids. The application of Eco enzyme, among others as biocatalist to improve plant productivity. This paper aims to study dilution of Eco Enzym which was applied to Turi plants to increase its productivity This study used a completely randomized design, namely: T1: Eco enzyme dilution 1: 100; T2: 1: 200; T3: 1: 300 whereas there were 5 replications. Parameters were plant height, stem diameter, number of branches, leaf width and number of flowers. The results of this research were that the parameters of plant height, leaf of width, number of flowers had significant effect by the 1:100 dilution treatment (p<0,05). However, there was no significant effect on stem diameter and and the number of branches. The best response to eco enzyme was 1: 100.

Protein Corona Hinders N-CQDs Oxidative Potential and Favors Their Application as Nanobiocatalytic System

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.

Effect of different cell disruption methods on lipid yield of Schizochytrium sp.

In this study, it was investigated to increase the lipid yield of the microalgae Schizochytrium sp., by applying different cell disruption methods. Therefore, acid treatment with HCl, osmotic shock, enzyme applications and ultrasonic homogenizer were tried in this algae species combined with the Bligh and Dyer and Soxhlet methods as an alternative to classical lipid extraction methods. As a result of the study, the highest lipid value (21.72 ± 0.74%) was obtained in enzyme application with Bligh and Dyer method (BDE). The cell disruption processes increased the lipid yield compared to the control groups. The highest PUFA DHA was found in the range of 4.58 ± 2.44-19.25 ± 0.09%, and the highest value was observed in the BDE group. Highest SFA was palmitic acid. Effective results were observed in the Bligh and Dyer applied groups in terms of both total lipid and total fatty acids. In cell disruption methods, particularly in enzyme and HCl extraction, good results were obtained in terms of fatty acids. The highest total fatty acids and the highest lipid content were detected in the Bligh and Dyer enzyme (BDE). Enzyme applications are also advantageous because of being environmentally friendly. Lipid health indices such as n-6/n-3, PUFA/SFA, Atherogenicity index (AI), Thrombogenicity index (TI) and hypocholesterolemic/hypercholesterolemic ratios (HH) were almost favorable. With this study, an appropriate lipid extraction methods were determined to provide an economical and environmental friendly suggestion for future studies to be used in areas such as food, feed and cosmetics.

Gut Microbiota from Lower Groups of Animals: An Upcoming Source for Cellulolytic Enzymes with Industrial Potentials

Cellulosic plant materials are a reliable source of renewable energy. Cellulose-based plant materials are now being used for bioenergy production as alternatives to fossil fuels. The traditional way of converting lignocellulosic materials to ethanol and other bioenergy is an expensive and environmentally unsafe process. Several research works have been conducted to find outsource of low-cost cellulolytic enzymes. Initially, fungal species were considered as sources of cellulolytic enzymes. Later on, several studies showed that bacterial species are a more potent source of cellulose-degrading enzymes. Phytophagous lower invertebrates are a good source of cellulolytic gut bacteria. They utilize a wide variety of plant materials as their food source. In this review, thorough literature studies have been made to explore the invertebrate groups that are novel sources of cellulolytic gut bacteria with high efficacy for enzyme production. This study also encompasses a brief description of cellulose, the activity, and cellulase enzyme application in industrial aspects.

Biodegradation of cassava root sieviate with enzymes extracted from isolated fungi

This study was carried out to investigate changes in proximate, sugars and cell wall components of cassava root sieviates (CRS) treated with extracted enzymes obtained from Aspergillus niger (An), Trichoderma viridae (Tv), Rhizopus stolonifer (Rs) and Mucor mucedo (Mm) applied on autoclaved CRS at 250ml/kg. Another treatment that had a commercial enzyme Roxazyme G2G (RG2G) as the degrading agent was prepared and the enzyme was applied at 150g/tonne as recommended by the manufacturer. At the end of the7 thday after enzyme application, the chemical analysis showed a higher bioavailability of nutrients in the degraded samples than the undegraded sample. Non starch polysaccharides (NSPs) in the CRS was negatively related to crude protein, ash and metabolisable energy (P<0.05) but positively related to crude fibre, pectin, neutraldetergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), hemicellulose and cellulose content . The highest value of metabolisable energy (ME), Ash, crude protein and phosphorus were : 2807.81kcal/kg, 17.32,18.32 and 0.91 g/100gdry matter respectively; obtained when A.niger, T.viridae, A. niger and R. stolonifer enzymes were added respectively .The lowest values of crude fibre, pectin, NDF,ADF, ADL, hemicellulose and cellulose were: 4.82,3.52,30.74, 17.12, 3.74,13.71 and 14.46g/100g dry matter respectively obtained when enzymes extracted from A.niger , R. stolonifer, T. viride ,A. niger, R. stolonifer, M. mucedo and T. viride were applied . Results of levels of sugars (µg/ml) in undegraded CRS showed that glucose level increased by 72.40% (257.34 to 932.22), 67.18%(257.34 to 784.20), 53.75%(257.34 to 556.40), 50.13%(257.32 to 516.06) and 32.53%(257.34 to 381.43) when enzyme preparations from A. niger, T.viridae , R.stolonifer, M.mucedo, and Roxazyme G2G were applied on CRS for degradation respectively . Other sugars that were significantly (P<0.05) increased after biodegradation were galactose, fructose and sucrose. Significant (P<0.05) differences were also expressed in the mineral analysis. After biodegradation, the degraded CRS had better mineral bioavailability as there were improvements in the minerals quantity. The results revealed that the use of enzymes from the above named fungi and the commercial enzymes defiberised the CRS and hydrolyzed the cell walls and hence promoted better availability of energy, crude proteins and other nutrients which were hitherto unavailable.