Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Industrial biosynthesis of ß-nicotinamide mononucleotide (ß-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L ß-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.

Modulating the performance of lipase-hydrogel microspheres in a "micro water environment".

In our previous work, we successfully stimulated lipase activity in an anhydrous reaction system using porous polyacrylamide hydrogel microsphere (PPAHM) as a carrier of lipase and free water. However, the effect of the existence state and content of water in lipase-porous polyacrylamide hydrogel microsphere (L-PPAHM) on the interfacial activation remained unclear. In this work, L-PPAHM with different water contents were obtained by water mist rehydration and were used to catalyze the synthesis of conjugated linoleic acid ethyl ester (CLA-EE). The results revealed that there were three existence states of water in L-PPAHM: bound water, semi-bound water and free water, and free water provided the "micro water environment" for the interfacial activation of lipase. The reusability of L-PPAHM with different water contents showed that the activity and stability of L-PPAHM could be achieved by varying the water content of L-PPAHM. The proportion of free water in L-PPAHM increased, and the activity of L-PPAHM increased, but the strength of hydrogen bond interaction between PPAHM and lipase weakened, resulting in the decrease of stability. L-PPAHM with 2/3 of water absorption could ensure sufficient immobilized lipase activity and stability, and its water absorption property could reduce the free water generated during esterification, thus increasing the yield of CLA-EE.

Hydrogen-bonded lipase-hydrogel microspheres for esterification application.

Lipase is the most widely used enzyme in industry. Due to its unique "lid" structure, lipase can only show high activity at the oil-water interface, which means that water is needed in the catalytic esterification process. However, the traditional lipase catalytic system cannot effectively control "micro-water" in the esterification environment, resulting in the high content of free water, which hinders the esterification reaction and reduces the yield. In this paper, a promising strategy of esterification catalyzed by polyacrylamide hydrogel immobilized lipase is reported. The porous polyacrylamide hydrogel microspheres (PHM) prepared by inverse emulsion polymerization are used as carrier to adsorb lipase by hydrogen bonding interaction. These hydrogel microspheres provide a "micro-water environment" for lipase in the anhydrous reaction system, and further provide an oil-water interface for "interface activation" of lipase. The obtained lipase-porous polyacrylamide hydrogel microspheres (L-PHMs) exhibit higher temperature and pH stability compared with free lipase, and the optimum enzymatic activity reach 1350 U/g (pH 6, 40 °C). L-PHMs can still remain about 49% of their original activity after 20 reuses. Furthermore, L-PHMs have been successfully applied to catalyze the synthesis of conjugated linoleic acid ethyl ester. The results suggest that this immobilization method opens up a new way for the application of lipase in ester synthesis.

A lipase/poly (ionic liquid)-styrene microspheres/PVA composite hydrogel for esterification application.

Enzymes are particularly attractive as biocatalysts for the green synthesis of chemicals and pharmaceuticals. However, the traditional enzyme purification and separation process is complex and inefficient, which limits the wide application of enzyme catalysis. In this paper, an efficient strategy for enzyme purification and immobilization in one step is proposed. A novel poly (ionic liquid)-styrene microsphere is prepared by molecular design and synthesis for adsorbing and purifying high activity lipase from fermentation broth directly. By optimizing the surface morphologies and charge of the microspheres, the enzyme loading is significantly improved. In order to further stabilize the catalytic environment of lipase, the resulting lipase/poly (ionic liquid)-styrene microspheres are immobilized in physical crosslinking hydrogel to obtain a complex lipase catalytic system, which can be prepared into various shapes according to the requirements of catalytic environment. In the actual catalytic reaction process, this complex lipase catalytic system exhibits excellent catalytic activity (6314.69 ± 21.27 U mg-1) and good harsh environment tolerance compared with the lipase fermentation broth (1672.87 ± 36.68 U mg-1). Under the condition of cyclic catalysis, the complex lipase catalytic system shows the outstanding reusability (After 8 cycles the enzymatic activity is still higher than that of the lipase fermentation broth) and is easily separated from the products.

Integration of Transcriptional and Post-transcriptional Analysis Revealed the Early Response Mechanism of Sugarcane to Cold Stress.

Cold stress causes major losses to sugarcane production, yet the precise molecular mechanisms that cause losses due to cold stress are not well-understood. To survey miRNAs and genes involved in cold tolerance, RNA-seq, miRNA-seq, and integration analyses were performed on Saccharum spontaneum. Results showed that a total of 118,015 genes and 6,034 of these differentially expressed genes (DEGs) were screened. Protein-protein interaction (PPI) analyses revealed that ABA signaling via protein phosphatase 2Cs was the most important signal transduction pathway and late embryogenesis abundant protein was the hub protein associated with adaptation to cold stress. Furthermore, a total of 856 miRNAs were identified in this study and 109 of them were differentially expressed in sugarcane responding to cold stress. Most importantly, the miRNA-gene regulatory networks suggested the complex post-transcriptional regulation in sugarcane under cold stress, including 10 miRNAs-42 genes, 16 miRNAs-70 genes, and three miRNAs-18 genes in CT vs. LT0.5, CT vs. LT1, and CT0.5 vs. LT1, respectively. Specifically, key regulators from 16 genes encoding laccase were targeted by novel-Chr4C_47059 and Novel-Chr4A_40498, while five LRR-RLK genes were targeted by Novel-Chr6B_65233 and Novel-Chr5D_60023, 19 PPR repeat proteins by Novel-Chr5C_57213 and Novel-Chr5D_58065. Our findings suggested that these miRNAs and cell wall-related genes played vital regulatory roles in the responses of sugarcane to cold stress. Overall, the results of this study provide insights into the transcriptional and post-transcriptional regulatory network underlying the responses of sugarcane to cold stress.