Duobin Mao

Microbial enzymes can be exploited to improve the sensory quality and reduce the harmful effects of tobacco-originated compounds in tobacco-derived products. In this study, a novel oligogalacturonide lyase (OGL) gene from Klebsiella variicola GB3 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The recombinant enzyme was purified to a protein concentration of 18.63 mg/mL. OGL exhibited the highest activity toward tetragalacturonic acid (GalA ₄ ), with kinetic parameters of K _m = 3.89 ± 0.18 mM and V _max = 36.41 ± 1.20 μmol/min/mg, and optimal catalytic activity at 45 °C and pH 6.0. The enzyme remained stable over a moderate temperature range (30–40 °C) and a broad pH range (6.0–8.0), while Ca ²⁺ significantly enhanced its catalytic activity. The treatment with 1% OGL degraded approximately 32.18% of tobacco pectin, accompanied by significant increases in galacturonic acid (39.86%) and oligogalacturonides (403.85%). Sensory evaluation indicated improved aroma quality, increased smoothness, and reduced irritation in OGL-treated tobacco strands. GC-MS analysis confirmed that OGL treatment significantly enhanced the production of key aroma compounds, including norisoprenoids, furans, and terpenoids, while reducing undesirable off-flavor compounds. Collectively, these results indicate that OGL efficiently degrades tobacco pectin, promotes the release of aroma precursors, and the formation of key flavor compounds. This study highlights the potential of OGL as a promising biocatalyst for improving aroma quality and enabling the value-added bioconversion of tobacco resources.

1. As an annually grown herbaceous plant of the Solanaceae family, Nicotiana tabacum represents one of the most commercially valuable agricultural crops worldwide and is usually used as the raw mat...

Cembranoids compounds are mainly distributed in higher plants of the Pinaceae and Solanaceae families, as well as soft corals, and serve as important aroma precursors. Their degradation products are widely used in various aspects of life, such as farnesal, which finds extensive applications in the pharmaceutical industry. In this study, a strain (Stenotrophomonas maltophilia H3-1) was isolated from soil, which can efficiently degrade α-cedriene-4,6-diol.Based on GC-MS detection of α-cembratriene-4,6-diol degradation efficiency of the H3-1 strain, it was determined that the optimal culture conditions for H3-1 are 40°C, pH 8, maltose concentration of 2 g/L, and ammonium sulfate concentration of 2 g/L. Under the optimum culture conditions, the degradation rate of 1 mg/mL α-cembratriene-4,6-diol by H3-1 strain within 36 h was as high as 85.86%. This indicates the strong biodegradation ability of the H3-1 strain towards α-cembratriene-4,6-diol. Through GC-MS analysis, we identified the biodegradation products of α-cembratriene-4,6-diol, including farnesal. This provides a theoretical basis and microbial resources for further study on the degradation products and applications of α-cembratriene-4,6-diol.

To improve the sensory quality of aged flue-cured tobacco (FCT), Bacillus subtilis subsp, H11 was inoculated on aged FCT leaves named Pingdingshan DCFB. The metagenome and thecharacteristic aroma substances of aged FCT with different fermentation times (0 h, 12 h, 24 h, and 36 h) were systematically analyzed. The results showed that the content of aroma components and sensory quality of aged FCT were significantly improved when the strain was treated at 35 °C with 25% moisture for 24 h. The inoculation of H11 had a strong influence on the microbial composition and metabolism of the aged FCT leaf surface. Five microorganisms Pantoea (35.04%, 20.12–56.95%), Enterobacter (22.16, 13.60–39.82%), Pseudomonas (12.12, 3.13–26.17%), Terribacillus (8.00%, 4.65–13.01%) and Bacillus (6.54%, 0.67–16.96%) accounted for the largest proportion during the process of fermentation. The content of most neutral flavor components such as ketones and aldehydes in FCT after fermentation was higher than that priorto fermentation. After 24 h fermentation, 3-furfural, 5-methylfurfural, dihydrokiwi lactone and megalotrienone increased by 71.42%, 49.19%, 21.09%, and 10.56%, respectively. Correlation analysis between groups showed that Pseudomonas was significantly correlated with (E, E)-2, 4-heptadienal (P < 0.05), Franconibacter was correlated with damascus ketone (P < 0.05), and Terribacillus was related to the production of β-citral (P < 0.05). GH9 may be involved in the formation of damasone (P < 0.05), and 4-cyclopentene-1, 3-dione was significantly correlated with glycoside hydrolase family 5 (GH5) (P < 0.05). The correlation between 4-oxyisophorone and GH31, GH103, GH73, and GH3 was significant (P < 0.05). Microorganisms and GHs may play important roles in FCT fermentation.

Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by various countries due to its toxic effect. In this study, a strain of HSM-C2 that can biodegrade coumarin with high efficiency was isolated from soil and identified as Pseudomonas putida through performing 16S rDNA sequence analysis. The HSM-C2 catalyzed the biodegradation up to 99.83% of 1 mg/mL coumarin within 24 h under optimal culture conditions, such as 30 °C and pH 7, which highlights the strong coumarin biodegrading potential of this strain. The product, such as dihydrocoumarin, generated after the biodegradation of coumarin was identified by performing GC-MS analysis. The present study provides a theoretical basis and microbial resource for further research on coumarin biodegradation.

3-Hydroxy-β-ionone, a flavor and fragrance compound with fruity violet-like characteristics, is widely applied in foodstuff and beverages, and is currently produced using synthetic chemistry. In this study, a novel lutein cleavage enzyme (EhLCD) was purified and characterized from Enterobacter hormaechei YT-3 to convert lutein to 3-hydroxy-β-ionone. Enzyme EhLCD was purified to homogeneity by ammonium sulfate precipitation, Q-Sepharose, phenyl-Sepharose, and Superdex 200 chromatography. The molecular mass of purified EhLCD, obtained by SDS-PAGE, was approximately 50 kDa. The enzyme exhibited the highest activity toward lutein, followed by zeaxanthin, β-cryptoxanthin, and β-carotene, suggesting that EhLCD exhibited higher catalytic efficiency for carotenoid substrates bearing 3-hydroxy-ionone rings. Isotope-labeling experiments showed that EhLCD incorporated oxygen from O₂ into 3-hydroxy-β-ionone and followed a dioxygenase reaction mechanism for different carotenoid substrates. These results indicated that EhLCD is the first characterized bacterial lutein cleavage dioxygenase. Active EhLCD was also confirmed to be a Fe²⁺-dependent protein with 1 molar equivalent of non-haem Fe²⁺. The purified enzyme displayed optimal activity at 45 °C and pH 8.0. The optimum concentrations of the substrate, enzyme, and Tween 40 for 3-hydroxy-β-ionone production were 60 μM lutein/L, 1.5 U/mL, and 2% (w/v), respectively. Under optimum conditions, EhLCD produced 3-hydroxy-β-ionone (637.2 mg/L) in 60 min with a conversion of 87.0% (w/w), indicating that this enzyme is a potential candidate for the enzymatic synthesis of 3-hydroxy-β-ionone in biotechnological applications.

In this report, the use of immobilized nicotine hydroxylase from Pseudomonas sp. ZZ-5 (HSPH_ZZ) for the production of 2,5-dihydroxypyridine (2,5-DHP) from 6-hydroxy-3-succinoylpyridine (HSP) in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) is described. HSPH_ZZ was covalently immobilized on Immobead 150 (ImmHSPH_ZZ). ImmHSPH_ZZ (obtained with 5–30 mg of protein per gram of support) catalyzed the hydrolysis of HSP to 2,5-DHP. At a protein loading of 15 mg g⁻¹, ImmHSPH_ZZ converted 93.6% of HSP to 2,5-DHP in 6 h. The activity of ImmHSPH_ZZ was compared with that of free HSPH_ZZ under various conditions, including pH, temperature, enzyme concentration, substrate concentration and stability over time, and kinetic parameters were measured. The results showed that ImmHSPH_ZZ performed better over wider ranges of pH and temperature when compared with that of HSPH_ZZ. The optimal concentrations of ImmHSPH_ZZ and substrate were 30 mg L⁻¹ and 0.75 mM, respectively. Under optimal conditions, 94.5 mg L⁻¹ of 2,5-DHP was produced after 30 min with 85.4% conversion. After 8 reaction cycles and 6 days of storage, 51.3% and 75.0% of the initial enzyme activity remained, respectively. The results provide a framework for development of commercially suitable immobilized enzymes that produce 2,5-DHP.

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 degrees C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 degrees C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.