Graphene microcrystal (GMC) is a type of glassy carbon fabricated from lignin, in which the microcrystals of graphene are chemically bonded by sp³ carbon atoms, forming a glass-like microcrystal structure. The lignin is refined from sugarcane bagasse using an ethanol-based organosolv technique which is used for the fabrication of GMC by two technical schemes: The pyrolysis reaction of lignin in a tubular furnace at atmospheric pressure; and the hydrothermal carbonization (HTC) of lignin at lower temperature, followed by pyrolysis at higher temperature. The existence of graphene nanofragments in GMC is proven by Raman spectra and XRD patterns; the ratio of sp² carbon atoms to sp³ carbon atoms is demonstrated by XPS spectra; and the microcrystal structure is observed in the high-resolution transmission electron microscope (HRTEM) images. Temperature and pressure have an important impact on the quality of GMC samples. With the elevation of temperature, the fraction of carbon increases, while the fraction of oxygen decreases, and the ratio of sp² to sp³ carbon atoms increases. In contrast to the pyrolysis techniques, the HTC technique needs lower temperatures because of the high vapor pressure of water. In general, with the help of biorefinery, the biomass material, lignin, is found to be qualified and sustainable material for the manufacture of GMC. Lignin acts as a renewable substitute for the traditional raw materials of glassy carbon, copolymer resins of phenol formaldehyde, and furfuryl alcohol-phenol.

In order to discover a broad-specificity and high stability chitinase, a marine fungus, Aspergillus fumigatus df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene (AfChi28) from A. fumigatus df347 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme AfChi28 was purified and characterized. AfChi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc)₂, (GlcNAc)₃ and (GlcNAc)₄. Na⁺, Mg²⁺, K⁺, Ca²⁺ and Tris at a concentration of 50 mM had a strong stimulatory effect on AfChi28. The crude enzyme and pure enzyme exhibited the highest specific activity of 0.737 mU/mg and 52.414 mU/mg towards colloidal chitin. The DxDxE motif at the end of strand β5 and with Glu154 as the catalytic residue was verified by the AlphaFold2 prediction and sequence alignment of homologous proteins. Moreover, the results of molecular docking showed that molecular modeling of chitohexaose was shown to bind to AfChi28 in subsites −4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that AfChi28 is a promising chitinase for the preparation of desirable chitin oligosaccharides, and provides a foundation for elucidating the catalytic mechanism of chitinases from marine fungi.

Sugarcane bagasse (SCB) is an abundant agricultural waste, rich in cellulose and hemicellulose, that could be used as an ideal raw material for succinic acid (SA) production. A two-step chemical pretreatment, involving alkali extraction and alkaline hydrogen peroxide treatment, was utilized to treat SCB, followed by multi-enzyme hydrolysis to obtain a reducing sugar hydrolysate mainly composed of glucose and xylose. Optimization of the multi-enzyme hydrolysis of pretreated SCB resulted in a final reducing sugar concentration of 78.34 g/L. In order to enhance the bioconversion of SCB to SA and to reduce the production costs, the initial reducing sugar concentration, nitrogen source, and MgCO₃ content were further optimized. The results demonstrated that the inexpensive corn steep liquor powder (CSLP) could be utilized as an alternative nitrogen source to yeast extract for the production of SA; and the optimal concentrations of initial reducing sugar, CSLP, and MgCO₃ were 70 g/L, 18 g/L, and 60 g/L, respectively. When fed-batch fermentation was conducted in a 2 L stirred bioreactor, approximately 72.9 g/L of SA was produced, with a yield of 83.2% and a productivity of 1.40 g/L/h. The high SA concentration, yield, and productivity achieved in this study demonstrate the potential of SCB, an agricultural waste, as a viable alternative substrate for Actinobacillus succinogenes GXAS137 to produce SA. This lays a solid foundation for the resource utilization of agricultural waste and cost-effective industrial-scale production of SA in the future.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a K_m of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

This study aimed to acquire a low-cost medium to produce high-yields of lipases from Burkholderia sp. Bps1 by using a statistic method so as to optimize the compositions of medium in the following two steps: 1) a single factor experiment (SFE) was conducted to screen the most significant factors affecting lipase activity to determine the optimal carbon source (molasses), nitrogen source (peptone) and inducer (palm oil); 2) a central composite design (CCD) and response surface methodology (RSM) were used to further optimize the previously mentioned factors to acquire an optimal combination consisting of 9.7 g/L molasses, 13.7 g/L peptone and 0.60% (v/v) palm oil. The optimized lipases activity reached 153.537 U/mL which was 30 times greater than the activity of the primary enzyme. This research used a statistical method to improve the production of lipases from submerged fermentation and formed a theoretical foundation for the industrial production of lipases using low cost raw materials.

Lipases are important industrial enzymes with a wide range of applications across various sectors. Cold-active lipases are particularly well suited for industrial processes that operate at low temperatures (such as food processing and environmental remediation) due to their high catalytic efficiency and energy-saving benefits. In this study, a novel lipase—LipU (GenBank accession: PV094892)—was heterologously expressed from Bacillus cereus U2 and characterized for its low-temperature adaptability and alkaline resistance. LipU belongs to the lipase Subfamily I.5 and shares the highest amino acid sequence identity (53.32%) with known homologs. Enzymatic assays revealed that LipU exhibits optimal activity at 20 °C and pH 11. It retained 95% of its initial activity after 24 h of incubation at 4 °C and pH 11.0. Furthermore, the activity of LipU was enhanced by Ca²⁺, Na⁺, Tween 20, and Tween 80, whereas it was inhibited by Cu²⁺, Zn²⁺, Mn²⁺, and sodium dodecyl sulfate (SDS). LipU demonstrated tolerance to various organic solvents of differing polarity; after 1 h of exposure to 15% (v/v) ethanol, n-butanol, isoamyl alcohol, dimethyl sulfoxide, or glycerol, it retained over 78.6% of its activity. These properties make LipU a promising candidate for industrial applications, including for leather degreasing, alkaline wastewater treatment, and low-temperature biocatalysis.