Mulberry (Morus alba L.) leaf powder modified the processing of meat alternatives: Principal component analysis from apparent properties to chemical bonds.

For texture control in plant-meat alternatives, the interrelationship between apparent characteristics and chemical bonds in high-fiber formulations remains unclear. The influence of mulberry leaf powder on apparent characteristics and chemical bonds of raw materials, block and strip products at addition amounts of 0.5-25% was analyzed. The results showed that 8% addition significantly increased the chewiness of the block by 98.12%. The strips' texture shows a downward trend, and the processing produced more redness and color difference. Additives promoted the formation of voids, lamellar and filamentous structures, and the strip produced more striped structures. Disulfide bonds significantly increased in the block, and the ß-turn in the secondary structure enhanced by 12.20%. The ß-turn transformed into a ß-sheet in strips. Principal component analysis revealed that the texture improvement was associated with producing disulfide bonds and ß-turn, providing a basis for high-fiber components to improve products' apparent characteristics by chemical bonds.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Molecular modification and biotechnological applications of microbial aspartic proteases.

The growing preference for incorporating microbial aspartic proteases in industries is due to their high catalytic function and high degree of substrate selectivity. These properties, however, are attributable to molecular alterations in their structure and a variety of other characteristics. Molecular tools, functional genomics, and genome editing technologies coupled with other biotechnological approaches have aided in improving the potential of industrially important microbial proteases by addressing some of their major limitations, such as: low catalytic efficiency, low conversion rates, low thermostability, and less enzyme yield. However, the native folding within their full domain is dependent on a surrounding structure which challenges their functionality in substrate conversion, mainly due to their mutual interactions in the context of complex systems. Hence, manipulating their structure and controlling their expression systems could potentially produce enzymes with high selectivity and catalytic functions. The proteins produced by microbial aspartic proteases are industrially capable and far-reaching in regulating certain harmful distinctive industrial processes and the benefits of being eco-friendly. This review provides: an update on current trends and gaps in microbial protease biotechnology, exploring the relevant recombinant strategies and molecular technologies widely used in expression platforms for engineering microbial aspartic proteases, as well as their potential industrial and biotechnological applications.

Advances in the One-Step Approach of Polymeric Materials Using Enzymatic Techniques.

The formulation in which biochemical enzymes are administered in polymer science plays a key role in retaining their catalytic activity. The one-step synthesis of polymers with highly sequence-controlled enzymes is a strategy employed to provide enzymes with higher catalytic activity and thermostability in material sustainability. Enzyme-catalyzed chain growth polymerization reactions using activated monomers, protein-polymer complexation techniques, covalent and non-covalent interaction, and electrostatic interactions can provide means to develop formulations that maintain the stability of the enzyme during complex material processes. Multifarious applications of catalytic enzymes are usually attributed to their efficiency, pH, and temperature, thus, progressing with a critical structure-controlled synthesis of polymer materials. Due to the obvious economics of manufacturing and environmental sustainability, the green synthesis of enzyme-catalyzed materials has attracted significant interest. Several enzymes from microorganisms and plants via enzyme-mediated material synthesis have provided a viable alternative for the appropriate synthesis of polymers, effectively utilizing the one-step approach. This review analyzes more and deeper strategies and material technologies widely used in multi-enzyme cascade platforms for engineering polymer materials, as well as their potential industrial applications, to provide an update on current trends and gaps in the one-step synthesis of materials using catalytic enzymes.

More efficient barley malting under catalyst: Thermostability improvement of a ß-1,3-1,4-glucanase through surface charge engineering with higher activity.

ß-1,3-1,4-Glucanase is an indispensable biocatalyst in barley brewing industry for its crucial effect in reducing the viscosity of mash. However, the unsatisfactory thermostability greatly limited its application performance. In this study, structure-based surface charge engineering was conducted aiming at thermostability improvement of BisGlu16B, a highly active ß-1,3-1,4-glucanase from Bispora sp. MEY-1. By applying the enzyme thermal stability system (ETSS), residues D47, D213, and D253 were inferred to be critical sites for thermal properties. Single (D47A, D213A, and D253A) and combination (D47A/D213A/D253A) mutants were generated and compared with BisGlu16B. Among all improved mutants, D47A/D213A/D253A outstanded in thermostability. In comparison with BisGlu16B, its T50 and Tm were respectively increased by 7.0 °C and 4.3 °C, while the t1/2 at 70 °C was 8.1 times that of the wild type. Furthermore, the catalytic activity of D47A/D213A/D253A also increased by 42.5%, compared with BisGlu16B (42,900 ± 300 U/mg vs. 30,100 ± 800 U/mg). Comparing with BisGlu16B and commercial enzyme treatment groups, under simulated malting conditions, efficiency improvement was observed in decreasement of viscosity (35.5% and 90.7%) and filtration time (30.9% and 34.6%) for D47A/D213A/D253A treatment group. Molecular dynamics simulation showed that mutation sites A47, A213, and A253 increased the protein rigidity by lowering the overall root mean square deviation (RMSD). This study may bring optimization of technology and improvement of producing efficiency to the present brewing industry.

Long noncoding RNA transcriptome analysis reveals novel lncRNAs in Morus alba 'Yu-711' response to drought stress.

Drought stress has been a key environmental factor affecting plant growth and development. The plant genome is capable of producing long noncoding RNAs (lncRNAs). To better understand white mulberry (Morus alba L.) drought response mechanism, we conducted a comparative transcriptome study comparing two treatments: drought-stressed (EG) and well-watered (CK) plants. A total of 674 differentially expressed lncRNAs (DElncRNAs) were identified. In addition, 782 differentially expressed messenger RNAs (DEmRNAs) were identified. We conducted Gene Ontology (GO) and KEGG enrichment analyses focusing on the differential lncRNAs cis-target genes. The target genes of the DElncRNAs were most significantly involved in the biosynthesis of secondary metabolites. Gene regulatory networks of the target genes involving DElncRNAs-mRNAs-DEmRNAs and DElncRNA-miRNA-DEmRNA were constructed. In the DElncRNAs-DEmRNAs network, 30 DEmRNAs involved in the biosynthesis of secondary metabolites are collocated with 46 DElncRNAs. The interaction between DElncRNAs and candidate genes was identified using LncTar. In summary, quantitative real-time polymerase chain reaction (qRT-PCR) validated nine candidate genes and seven target lncRNAs including those identified by LncTar. We predicted that the DElncRNAs-DEmRNAs might recruit microRNAs (miRNAs) to interact with gene regulatory networks under the drought stress response in mulberry. The findings will contribute to our understanding of the regulatory functions of lncRNAs under drought stress and will shed new light on the mulberry-drought stress interactions.

Loop engineering of a thermostable GH10 xylanase to improve low-temperature catalytic performance for better synergistic biomass-degrading abilities.

Lignocellulosic biorefining for producing biofuels poses technical challenges. It is usually conducted over a long time using heat, making it energy intensive. In this study, we lowered the energy consumption of this process through an optimized enzyme and pretreatment strategy. First, the dominant mutant M137E/N269G of Bispora sp. MEY-1XYL10C_ΔN was obtained by directed evolution with highcatalytic efficiency (970 mL/s∙mg)and specific activity (2090 U/mg)at 37 °C, and thermostability was improved (T50 increased by5 °C). After pretreatment with seawater immersionfollowing steam explosion,bagasse was co-treated with cellulase and M137E/N269G under mild conditions (37 °C), the resulting highest yield of fermentable sugars reached 219 µmol/g of bagasse,46% higher than that of the non-seawater treatment group, with the highest degree of synergy of 2.0. Pretreatment with seawater following steam explosion and synergistic hydrolysis through high activity xylanase and cellulase helped to achieve low energy degradation of lignocellulosic biomass.

Dual promoter strategy enhances co-expression of α-L-rhamnosidase and enhanced fluorescent protein for whole-cell catalysis and bioresource valorization.

Developing circular economy is the only way to improve the efficiency of resource utilization. Whole-cell catalysis is an effective method to recycle enzymes, improve catalytic efficiency, and reduce production costs. The enzyme, α-L-rhamnosidase has considerable application prospects in the field of biocatalysis as it can hydrolyze a variety of α-L rhamnoses. In the present study, the genes for α-L-rhamnosidase (rhaB1) and enhanced fluorescent protein (EGFP) were co-expressed using a bi-promoter expression vector pRSFDuet1 and their enzymatic properties were evaluated. To our knowledge, this study has established an effective rhamnosidase-fluorescent indicator and whole-cell catalytic system for the first time. Moreover, we analyzed the change in the activity of the crude rhaB1-EGFP as well as its whole-cell during the biocatalysis process using fluorescence intensity. Recombinant rhaB1-EGFP as a product which contains rhaB1 and EGFP showed higher thermal stability, pH stability, and conversion efficiency than rhaB1, and its optimum temperature for rutin catalysis was ideal for industrial applications. Moreover, under the optimal conditions of a rutin concentration of 0.05 g/L, pH of 6.0, temperature of 40 °C, a yield of 92.5% was obtained. Furthermore, we demonstrated the relationship between the fluorescence intensity and enzyme activity. This study established a highly efficient whole-cell catalytic system whose activity can be evaluated by fluorescence intensity, providing a reference for enzyme recycling.

Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase.

BACKGROUND: Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. RESULTS: Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T 50 (3-4.7 °C) and T m (1.1-4.7 °C), and extended t 1/2 (by 1.8-2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. CONCLUSIONS: This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers.

Microfluidic tools for lipid production and modification: a review.

Microfluidics has great potential as an efficient tool for a large range of applications in industry. The ability of such devices to deal with an extremely small amount of fluid has additional benefits, including superlatively fast and efficient mass and heat transfer. These characteristics of microfluidics have attracted an enormous amount of interest in their use as a novel tool for lipid production and modification. In addition, lipid resources have a close relationship with energy resources, and lipids are an alternative renewable energy source. Here, recent advances in the application of microfluidics for lipid production and modification, especially in the discovery, culturing, harvesting, separating, and monitoring of lipid-producing microorganisms, will be reviewed. Other applications of microfluidics, such as the modification of lipids from microorganisms, will also be discussed. The novel microfluidic tools in this review will be useful in applications to improve lipid production and modification in the future.