Highly elastic, fatigue-resistant, antibacterial, conductive, and nanocellulose-enhanced hydrogels with selenium nanoparticles loading as strain sensors.

The fabrication of highly elastic, fatigue-resistant and conductive hydrogels with antibacterial properties is highly desirable in the field of wearable devices. However, it remains challenging to simultaneously realize the above properties within one hydrogel without compromising excellent sensing ability. Herein, we fabricated a highly elastic, fatigue-resistant, conductive, antibacterial and cellulose nanocrystal (CNC) enhanced hydrogel as a sensitive strain sensor by the synergistic effect of biosynthesized selenium nanoparticles (BioSeNPs), MXene and nanocellulose. The structure and potential mechanism to generate biologically synthesized SeNPs (BioSeNPs) were systematically investigated, and the role of protease A (PrA) in enhancing the adsorption between proteins and SeNPs was demonstrated. Additionally, owing to the incorporation of BioSeNPs, CNC and MXene, the synthesized hydrogels showed high elasticity, excellent fatigue resistance and antibacterial properties. More importantly, the sensitivity of hydrogels determined by the gauge factor was as high as 6.24 when a high strain was applied (400-700 %). This study provides a new horizon to synthesize high-performance antibacterial and conductive hydrogels for soft electronics applications.

Superoxide Dismutase Catalyzed Size-Adjustable Selenium Nanoparticles in Saccharomyces boulardii.

Selenium nanoparticles (SeNPs) are important and safe food and feed additives that can be used for dietary supplementation. In this study, a mutagenic strain of Saccharomyces boulardii was employed to obtain biologically synthesized SeNPs (BioSeNPs) with the desired particle size by controlling the dosage and duration of sodium selenite addition, and the average particle size achieved was 55.8 nm with protease A encapsulation. Transcriptomic analysis revealed that increased expression of superoxide dismutase 1 (SOD1) in the mutant strain effectively promoted the synthesis of BioSeNPs and the formation of smaller nanoparticles. Under sodium selenite stress, the mutant strain exhibited significantly increased expression of glutathione peroxidase 2 (GPx2), which was significantly greater in the mutant strain than in the wild type, facilitating the synthesis of glutathione selenol and providing abundant substrates for the production of BioSeNPs. Furthermore, based on the experimental results and transcriptomic analysis of relevant genes such as sod1, gpx2, the thioredoxin reductase 1 gene (trr1) and the thioredoxin reductase 2 gene (trr2), a yeast model for the size-controlled synthesis of BioSeNPs was constructed. This study provides an important theoretical and practical foundation for the green synthesis of controllable-sized BioSeNPs or other metal nanoparticles with potential applications in the fields of food, feed, and biomedicine.

Preparation of Yeast Extract from Brewer's Yeast Waste and Its Potential Application as a Medium Constituent.

Yeast extract serves as a source of nutritional components essential for human dietary requirements, feed formulations, and the vital growth factors and nutrients necessary for microorganisms. However, the production cost of yeast extract using cultivated active dry yeast is relatively high. This study aims to utilize the autolysis of discarded yeast post beer brewing to produce yeast extract. The concentration, temperature, pH, and time conditions are systematically optimized. It reveals that the yield of amino nitrogen and solids in the extract was increased by 3.3% and 20.9% under the optimized conditions (1.2% wall-breaking enzyme, 1% yeast extract enzyme, and a hydrolysis time of 24 h) than that of the documented 4.03% and 69.05%. Additionally, a comparative analysis with commercially available yeast powder demonstrates that the yeast extract derived from this study adequately fulfills the nutritional requirements for microbial growth. Hence, the utilization of discarded beer yeast presents an opportunity for the valuable reclamation of waste yeast, showcasing promising potential applications.

Facile green synthesis of wasted hop-based zinc oxide nanozymes as peroxidase-like catalysts for colorimetric analysis.

Hops are a common ingredient in beer production, and a considerable quantity of hops is usually discarded as a waste material once the brewing process is completed. Transforming this waste material into valuable nanomaterials offers a sustainable approach that has the potential to significantly mitigate environmental impact. Herein, a facile and green protocol for the production of zinc oxide nanozymes (ZnO NZs) using wasted hop extract (WHE) as a natural precursor was demonstrated. The process involved a hydrothermal synthesis method followed by a calcination step to form the final ZnO NZs. The results revealed that lupulon, the main ß-acid in hops, particularly the phenolic hydroxy group, is primarily responsible for the biosynthesis of ZnO NZs. The WHE-ZnO NZs exhibited exceptional peroxidase-like (POD-like) activity and served as effective catalysts for the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). Analysis of the catalytic mechanism revealed that the POD-like activity of these WHE-ZnO NZs originated from their ability to expedite the transfer of electrons between TMB and H2O2, resulting in the enzymatic kinetics following the standard Michaelis-Menten mechanism. Furthermore, we developed a straightforward and user-friendly colorimetric technique for detecting both H2O2 and glucose. By utilizing the WHE-ZnO NZs as POD-like catalysts, we achieved a linear detection range of 1-1000 µM and a limit of detection of 0.24 µM (S/N = 3) for H2O2 detection and a linear range of 0-100 mM and a detection limit of 16.73 µM (S/N = 3) for glucose detection. These results highlighted the potential applications of our waste-to-resource approach for nanozyme synthesis in the field of analytical chemistry.

Glycoside hydrolases in the biodegradation of lignocellulosic biomass.

Lignocellulose is a plentiful and intricate biomass substance made up of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are polysaccharides characterized by different compositions and degrees of polymerization. As renewable resources, their applications are eco-friendly and can help reduce reliance on petrochemical resources. This review aims to illustrate cellulose, hemicellulose, and their structures and hydrolytic enzymes. To obtain desirable enzyme sources for the high hydrolysis of lignocellulose, highly stable, efficient and thermophilic enzyme sources, and new technologies, such as rational design and machine learning, have been introduced in detail. Generally, the efficient biodegradation of abundant natural biomass into fermentable sugars or other intermediates has great potential in practical applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03819-1.

Artificial Intelligence Aided Lipase Production and Engineering for Enzymatic Performance Improvement.

With the development of artificial intelligence (AI), tailoring methods for enzyme engineering have been widely expanded. Additional protocols based on optimized network models have been used to predict and optimize lipase production as well as properties, namely, catalytic activity, stability, and substrate specificity. Here, different network models and algorithms for the prediction and reforming of lipase, focusing on its modification methods and cases based on AI, are reviewed in terms of both their advantages and disadvantages. Different neural networks coupled with various algorithms are usually applied to predict the maximum yield of lipase by optimizing the external cultivations for lipase production, while one part is used to predict the molecule variations affecting the properties of lipase. However, few studies have directly utilized AI to engineer lipase by affecting the structure of the enzyme, and a set of research gaps needs to be explored. Additionally, future perspectives of AI application in enzymes, including lipase engineering, are deduced to help the redesign of enzymes and the reform of new functional biocatalysts. This review provides a new horizon for developing effective and innovative AI tools for lipase production and engineering and facilitating lipase applications in the food industry and biomass conversion.

Designing multifunctional biocatalytic cascade system by multi-enzyme co-immobilization on biopolymers and nanostructured materials.

In recent decades, enzyme-based biocatalytic systems have garnered increasing interest in industrial and applied research for catalysis and organic chemistry. Many enzymatic reactions have been applied to sustainable and environmentally friendly production processes, particularly in the pharmaceutical, fine chemicals, and flavor/fragrance industries. However, only a fraction of the enzymes available has been stepped up towards industrial-scale manufacturing due to low enzyme stability and challenging separation, recovery, and reusability. In this context, immobilization and co-immobilization in robust support materials have emerged as valuable strategies to overcome these inadequacies by facilitating repeated or continuous batch operations and downstream processes. To further reduce separations, it can be advantageous to use multiple enzymes at once in one pot. Enzyme co-immobilization enables biocatalytic synergism and reusability, boosting process efficiency and cost-effectiveness. Several studies on multi-enzyme immobilization and co-localization propose kinetic advantages of the enhanced turnover number for multiple enzymes. This review spotlights recent progress in developing versatile biocatalytic cascade systems by multi-enzyme co-immobilization on environmentally friendly biopolymers and nanostructured materials and their application scope in the chemical and biotechnological industries. After a succinct overview of carrier-based and carrier-free immobilization/co-immobilizations, co-immobilization of enzymes on a range of biopolymer and nanomaterials-based supports is thoroughly compiled with contemporary and state-of-the-art examples. This study provides a new horizon in developing effective and innovative multi-enzymatic systems with new possibilities to fully harness the adventure of biocatalytic systems.

Expression and Characterization of a GH38 α-Mannosidase from the Hyperthermophile Pseudothermotoga thermarum.

This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0-6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co2+ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu2+, Zn2+, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins.

An acetate-independent pathway for isopropanol production via HMG-CoA in Escherichia coli.

Isopropanol has a good potential as a new fuel substitution. In the model biosynthesis pathway of isopropanol synthesis, acetoacetyl-CoA is converted to acetoacetate by acetoacetyl-CoA transferases, which requires an acetate molecule as a substrate. Herein, a novel isopropanol synthesis pathway based on mammalian ketone metabolic pathway was developed. In this pathway, acetoacetyl-CoA is condensed with acetyl-CoA to generate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, and then catalyzed by HMG-CoA lyase to generate acetoacetate. This process is acetate-independent. Under the same experimental system using glycerol as carbon source, the E. coli strain MG::ISOP1 containing the novel pathway produced 11.7 times more isopropanol than the strain MG::ISOP0 containing the model pathway. The pta-ackA knockout mutant strain MG∆pta-ackA::ISOP1, which reduced the conversion of acetyl-CoA to acetate, further increased the production from 76 mg/L to 360 mg/L. In another strategy, knocking out atoDA to block the acetoacetate degradation pathway in strain MG∆atoDA::ISOP1 increased the production to 680 mg/L. By knocking out both of pta-ackA and atoDA, strain MGΔpta-ackAΔatoDA::ISOP1 produced 964 mg/L of isopropanol, which was 12.7 times that of MG::ISOP1. This study indicated that the novel pathway is competent for isopropanol synthesis, and provides a new perspective for biosynthesis of isopropanol.

Digging and identification of novel microorganisms from the soil environments with high methanol-tolerant lipase production for biodiesel preparation.

Converting renewable biomass into carbon-neutral biofuels is one of the most effective strategies to achieve zero carbon emissions and contribute to environmental protection. Microorganisms from the soil were primarily screened on the rhodamine B-plate for highly-active lipase producing strains and re-screened on a tributyrin-methanol plate using crude lipases produced from the initially screened-out strains. The lipase-producing strains with higher methanol-tolerant lipase were identified based on morphological characteristics and 16S rDNA sequencing. The crude lipases with much higher methanol-tolerance from screened top-4 strains, Stenotrophomonas maltophilia D18, Lysinibacillus fusiformis B23, Acinetobacter junii C69, and A. pittii C95 showed temperature optima of 25 °C, 35 °C, 30 °C, and 30 °C at pH 7.0, respectively, while their pH optima were 8.0, 7.0, 7.5, and 7.5 at each optimum temperature, respectively. After 24-h incubation, they retained more than 85% of their original activities in 25%, 15%, 20%, and 20% of methanol, respectively. They catalyzed the conversion of soybean oil into biodiesel by yields of 63.1%, 35.4%, 74.6%, and 78.5% after 24-h reactions, respectively. In conclusion, the as-isolated microorganisms producing high methanol-tolerant lipase are considered promising to provide robust biocatalyst for efficient biodiesel preparation and other industrial applications.

Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation.

Methanol tolerance of lipase is one of the important factors affecting its esterification ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, eight sites (Val139, Ala146, Leu147, Pro218, Val286, Ala287, Val306, and Gly307) with high B value indicating more flexibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsified tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over wild-type CalB (CalB-WT), respectively. By modeling, a newly formed hydrogen bond in CalB-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance. CalB-P218W and -V306N transesterified the soybean oil into biodiesel at 30 °C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24 h reactions. These results may provide a basis for molecular engineering of CalB and expand its applications in fuel industries. The as-developed semi-rational method could be utilized to enhance the stabilities of many other industrial enzymes.

Expanding the Biocatalytic Scope of Enzyme-Loaded Polymeric Hydrogels.

In recent years, polymeric hydrogels have appeared promising matrices for enzyme immobilization to design, signify and expand bio-catalysis engineering. Therefore, the development and deployment of polymeric supports in the form of hydrogels and other robust geometries are continuously growing to green the twenty-first-century bio-catalysis. Furthermore, adequately fabricated polymeric hydrogel materials offer numerous advantages that shield pristine enzymes from denaturation under harsh reaction environments. For instance, cross-linking modulation of hydrogels, distinct rheological behavior, tunable surface entities along with elasticity and mesh size, larger surface-volume area, and hydrogels' mechanical cushioning attributes are of supreme interest makes them the ideal candidate for enzyme immobilization. Furthermore, suitable coordination of polymeric hydrogels with requisite enzyme fraction enables pronounced loading, elevated biocatalytic activity, and exceptional stability. Additionally, the unique catalytic harmony of enzyme-loaded polymeric hydrogels offers numerous applications, such as hydrogels as immobilization matrix, bio-catalysis, sensing, detection and monitoring, tissue engineering, wound healing, and drug delivery applications. In this review, we spotlight the applied perspective of enzyme-loaded polymeric hydrogels with recent and relevant examples. The work also signifies the combined use of multienzyme systems and the future directions that should be attempted in this field.

Characterization of a Novel Alginate Lyase from Marine Bacterium Vibrio furnissii H1.

Alginate lyases show great potential for industrial and medicinal applications, especially as an attractive biocatalyst for the production of oligosaccharides with special bioactivities. A novel alginate lyase, AlyH1, from the marine bacterium Vibrio furnissii H1, which has been newly isolated from rotten seaweed, was purified and characterized. The purified enzyme showed the specific activity of 2.40 U/mg. Its molecular mass was 35.8 kDa. The optimal temperature and pH were 40 °C and pH 7.5, respectively. AlyH1 maintained stability at neutral pH (7.0-8.0) and temperatures below 30 °C. Metal ions Na⁺, Mg2+, and K⁺ increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax values of AlyH1 were 2.28 mg/mL and 2.81 U/mg, respectively. AlyH1 exhibited activities towards both polyguluronate and polymannuronate, and preferentially degraded polyguluronate. Products prepared from sodium alginate by AlyH1 were displayed to be di-, tri-, and tetra-alginate oligosaccharides. A partial amino acid sequence (190 aa) of AlyH1 analysis suggested that AlyH1 was an alginate lyase of polysaccharide lyase family 7. The sequence showed less than 77% identity to the reported alginate lyases. These data demonstrated that AlyH1 could be as a novel and potential candidate in application of alginate oligosaccharides production with low polymerization degrees.

A unique mono- and diacylglycerol lipase from Penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity.

A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5-9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries.

Improving the thermostability of a mesophilic family 10 xylanase, AuXyn10A, from Aspergillus usamii by in silico design.

To improve the thermostability of a mesophilic GH family 10 xylanase, AuXyn10A, from Aspergillus usamii E001, its modification was performed by in silico design. Based on the comparison of B-factor values, a mutant xylanase ATXyn10 was predicted by substituting a segment YP from Tyr(25) to Pro(34) of AuXyn10A with the corresponding one from Asn(24) to Ala(32) of TaXyn10, a thermophilic GH family 10 xylanase from Thermoascus aurantiacus. Analysis of a TaXyn10 crystal structure indicated that there is a close interaction between segments YP and FP. For that reason, another mutant xylanase ATXyn10(M) was designed by mutating Ser(286) and His(288) of ATXyn10 into the corresponding Gly(285) and Phe(287) in the FP of TaXyn10. Then, two ATXyn10- and ATXyn10(M)-encoding genes, ATxyn10 and ATxyn10 (M), were expressed in Pichia pas toris GS115. The temperature optimum of recombinant (re) ATXyn10(M) was 60 °C, 10 °C higher than that of reAuXyn10A. Its thermal inactivation half-life (t(1/2)) at 55 °C was 10.4-fold longer than that of reAuXyn10A. As compared with reAuXyn10A, reATXyn10(M) displayed a slight decrease in K(m) value and a significant increase in V(max) value from 6,267 to 8,870 U/mg.

Enhancing the thermostability of a cold-active lipase from Penicillium cyclopium by in silico design of a disulfide bridge.

Cysteine mutants of a cold-active lipase (PcLipI) from Penicillium cyclopium were designed by the software Disulfide by Design Ver. 1.20 in an effort to improve enzyme thermostability by addition of a disulfide bridge. Those mutants predicted by molecular dynamics simulation to have better thermostability than the wild type were first expressed in Escherichia coli BL21(DE3) and then, for further investigation, in Pichia pastoris GS115. By replacing Val248 and Thr251 with cysteines to create a disulfide bridge, the recombinant lipases reE-PcLipV248C-T251C (expressed in E. coli) and reP-PcLipV248C-T251C (expressed in P. pastoris) were obtained. Both had enhanced thermostability with half-lives at 35 °C about 4.5- and 12.8-fold longer than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. The temperature optima of reE-PcLipV248C-T251C and reP-PcLipV248C-T251C were 35 and 30 °C, which were each 5 °C higher than those of the parent PcLipI expressed in E. coli and P. pastoris. The K ms of reE-PcLipV248C-T251C and reP-PcLipV248C-T251C toward tributyrin were 53.2 and 39.5 mM, while their V maxs were 1,460 and 3,800 U/mg, respectively. PcLipV248C-T251C had better thermostability and catalytic efficiency than the other mutants and the parent PcLipI.

Exploration of disulfide bridge and N-glycosylation contributing to high thermostability of a hybrid xylanase.

A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase AEx11A revealed that a disulfide bridge (Cys(5)-Cys(32)) and an N-glycosylation site (Asn(42)) were imported into AEx11A by N-terminal substitution of AoXyn11A with EvXyn11(TS). Two mutant genes AEx11A(C5T) and AEx11A(N42Q) were constructed by mutating Cys(5)- and Asn(42)-encoding codons of AEx11A into Thr(5)- and Gln(42)-encoding ones, and heterologously expressed in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11A(C5T) (reAEx11A(C5T)) was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3 and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A and reAEx11A(C5T) in pH characteristics and kinetic parameters. Furthermore, both reAEx11A(N42Q) and reAEx11A displayed no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the N-glycosylation had no effect on.