Alcohol Dehydrogenase Research Articles

Constructing a CdS QDs/silica gel composite with high photosensitivity and prolonged recyclable operability for enhanced visible-light-driven NADH regeneration

Visible-light-driven nicotinamide adenine dinucleotide (NADH) regeneration is one of the most effective measures, and cadmium sulfide (CdS) materials are typically used as low-cost photocatalysts. The CdS photocatalysts, however, still suffer from low regeneration efficiency and poor cycle stability. In this work, the CdS quantum dots (QDs) less than 10 nm embedded onto silica gel (CdS QDs/Silica gel) were constructed for visible-light-driven NADH regeneration by a successive ionic layer adsorption reaction and ball milling method. Results demonstrate that the photosensitivity of the CdS QDs/Silica gel composite was 31 times higher than that of the bulk CdS. Moreover, the conduction band (CB) edge of the CdS QDs/Silica gel composite is −1.34 eV, which is more negative 0.5 eV than that of the bulk CdS. The obtained CdS QDs/Silica gel composites showed the highest NADH regeneration yields of 68.8% under visible-light (LED, 420 nm) illumination and can be reused for over 40 cycles. Finally, the bioactivity of NADH toward enzyme catalysis is further confirmed by the hydrogenation of benzaldehyde to benzyl alcohol catalyzed with an alcohol dehydrogenase as enzyme catalysis.

Smart enzyme catalysts capable of self-separation by sensing the reaction extent

A smart biocatalyst should dissolve homogeneously for catalysis and recover spontaneously at the end of the reaction. In this study, we present a strategy for preparing self-precipitating enzyme catalysts by exploiting reaction-induced pH decreases, which connect the reaction extent to the catalyst aggregation state. Using poly(methacrylic acid)-functionalized gold nanoparticles as carriers, we construct smart catalysts with three model systems, including the glucose oxidase (GOx)-catalase (CAT) cascade, the alcohol dehydrogenase (ADH)-glucose dehydrogenase (GDH) cascade, and a combination of two lipases. All smart catalysts can self-separate with a nearly 100% recovery efficiency when a certain conversion threshold is reached. The threshold can be adjusted depending on the reaction demand and buffer capacity. By monitoring the optical signals caused by the dissolution/precipitation of smart catalysts, we propose a prototypic automation system that may enable unsupervised batch/fed-batch bioprocessing.

Identification of hepatoprotective peptides from porcine liver and its interaction with ethanol metabolizing enzymes in vitro

Alcoholic liver disease (ALD) is a worldwide disease caused by excessive ethanol consumption. In current study, hepatoprotective peptides were isolated from the porcine liver while their activities and interactions with ethanol metabolizing enzymes (alcohol dehydrogenase: ADH/acetaldehyde dehydrogenase: ALDH) were analyzed. The alcalase digested porcine liver hydrolysate (PLH) was confirmed to possess ADH/ALDH activation ability in a dose-dependent manner. The peptide sequences of PLH were identified by mass spectrometry. Molecular docking and in silico analysis were federatively used for screening. NTLPHPTAP had the lowest binding energy with ADH (−0.88 kcal/mol) and low toxicity. Fluorescence quenching data showed a dynamic fluorescence quenching constant (Kq) for NTLPHPTAP interacting with ADH/ALDH (ADH: 1.57 × 10−10 M−1 s−1; ALDH:1.961 × 10−10 M−1 s−1). Circular dichroism (CD) spectra and infrared spectrometer (FTIR) revealed that the enzyme secondary structures became flexible and disordered after interaction with NTLPHPTAP. Meanwhile, the ADH/ALDH was speculated to bind NTLPHPTAP via amido and carbonyl groups, respectively. These results indicated that the formation of ADH/ALDH-NTLPHPTAP complexes expanded the overall structure of the original enzymes and further increased the possibility of enzyme-substrate binding. In conclusion, the study proposed a method for the extraction of porcine liver hepatoprotective peptides and suggested further explanation for the interaction between NTLPHPTAP and ADH/ALDH in vitro.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.

Effects of Plant-Based Extract Mixture on Alcohol Metabolism and Hangover Improvement in Humans: A Randomized, Double-Blind, Paralleled, Placebo-Controlled Clinical Trial.

Hangovers are uncomfortable physiological symptoms after alcohol consumption caused by acetaldehyde, a toxic substance in which alcohol is metabolized by alcohol dehydrogenase (ADH). Rapid alcohol and acetaldehyde decomposition are essential to alleviate alcohol handling symptoms. This study investigated the effects of HY_IPA combined with Mesembryanthemum crystallinum, Pueraria lobata flower, and Artemisia indica on alleviating hangovers. A randomized, double-blind, parallel-group, placebo-controlled clinical study was conducted on 80 individuals with hangover symptoms. Alcohol intake was 0.9 g/bw with 40% whiskey, adjusted proportionately to body weight. The Acute Hangover Scale total score was 5.24 ± 5.78 and 18.54 ± 18.50 in the HY_ IPA and placebo groups, respectively (p < 0.0001). All nine indicators of the hangover symptom questionnaire were significantly improved in the HY_IPA group (p < 0.01). Blood alcohol and acetaldehyde concentrations rapidly decreased from 30 min in the HY_IPA group (p < 0.05). ADH and acetaldehyde dehydrogenase (ALDH) activities in the blood of the HY_IPA group were significantly higher than those in the placebo group at 0, 1, and 2 h after alcohol consumption (p < 0.01). The rapid hangover relief was due to increased ADH and ALDH. Therefore, HY_IPA effectively relieves hangover symptoms by decomposing alcohol and acetaldehyde when consumed before alcohol consumption.

Prevention of Swelling Phenomenon of Alginate Beads To Improve the Stability and Recyclability of Encapsulated Horse Liver Alcohol Dehydrogenase.

Horse Liver Alcohol Dehydrogenase (HLADH) has been immobilized on calcium-alginate beads and used for both oxidation and reduction reactions. To avoid swelling of the beads and their subsequent breakage, calcium ions were added to both reaction and storage solutions, allowing the beads to maintain the initial structural features. The techniques used for this purpose revealed that 2 mM Ca2+ is the optimal concentration, which does not significantly change the weight of the beads, the amount of water in them, and their external and internal structure. The optimized experimental procedure has been used to verify the properties of the enzyme in terms of reusability, storage, and thermal stability. The addition of calcium ions allows the enzyme to retain more than 80% of its initial activity for fourteen cycles and approximately 50% at the twentieth cycle. Moreover, when the biocatalyst has been stored in a buffer solution containing 2 mM Ca2+, the retention of enzyme activity after 30 days was 100%, compared to that measured before incubation. The encapsulated enzyme exhibits greater thermal stability than free HLADH up to at least 60 °C, preventing dimer dissociation into the two subunits.

Enhanced active-site electric field accelerates enzyme catalysis.

The design and improvement of enzymes based on physical principles remain challenging. Here we demonstrate that the principle of electrostatic catalysis can be leveraged to substantially improve a natural enzyme's activity. We enhanced the active-site electric field in horse liver alcohol dehydrogenase by replacing the serine hydrogen-bond donor with threonine and replacing the catalytic Zn2+ with Co2+. Based on the electric field enhancement, we make a quantitative prediction of rate acceleration-50-fold faster than the wild-type enzyme-which was in close agreement with experimental measurements. The effects of the hydrogen bonding and metal coordination, two distinct chemical forces, are described by a unified physical quantity-electric field, which is quantitative, and shown here to be additive and predictive. These results suggest a new design paradigm for both biological and non-biological catalysts.

Rate of dilution and redox ratio influence the refolding efficiency of recombinant fungal dehydrogenases

Dehydrogenases from fungi are attracting attention as industrial biocatalysts due to their high activity and chiral selectivity. However, these enzymes form insoluble aggregates when overexpressed in E. coli, limiting their industrial application. In the present study, we report the systematic development of a refolding process for selected, industrially relevant fungal dehydrogenases, viz., formate dehydrogenase from Candida boidinii (CbFDH) and formate and alcohol dehydrogenases from Geotrichum candium (GcFDH and GcADH, respectively). We first employed a screen to evaluate the effects of different variables on refolding including the buffer system, additives, and rate of dilution. The extent of refolding was determined by enzyme assays, circular dichroism, and tryptophan fluorescence. Our results showed that glycerol and reducing environment are essential for refolding of these dehydrogenases. Further, slow dilution of solubilized protein over 16 h dramatically improved the recovery of refolded enzymes compared to rapid dilution. The importance of slow dilution was further confirmed in a 10-fold scaled-up refolding trial. Overall, we demonstrate a robust method for refolding of fungal dehydrogenases, thus improving their availability for various biocatalytic applications.

Waterlogging Tolerance of Actinidia valvata Dunn Is Associated with High Activities of Pyruvate Decarboxylase, Alcohol Dehydrogenase and Antioxidant Enzymes.

Kiwifruit (Actinidia spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among Actinidia plants for improving the waterlogging tolerance of kiwifruit cultivars, the underlying mechanisms remain largely unknown. Here, a comparative study was undertaken using one wild germplasm, Maorenshen (A. valvata Dunn, MRS), and one cultivar, Miliang-1 (A. chinensis var. deliciosa (A.Chev.) A.Chev. cv. Miliang-1, ML). Under stress, the ML plantlets were seriously damaged with wilted chlorotic leaves and blackened rotten roots, whereas the symptoms of injury in the MRS plantlets were much fewer, along with higher photosynthetic rates, chlorophyll fluorescence characteristics and root activity under stress conditions. However, neither aerenchyma in the root nor adventitious roots appeared in both germplasms upon stress exposure. The activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), as well as their transcript levels, were constitutively higher in MRS than those in ML under both normal and stress conditions. Waterlogging stress significantly enhanced the PDC and ADH enzyme activities in both germplasms, which were 60.8% and 22.4% higher in the MRS roots than those in the ML roots under waterlogging stress, respectively. Moreover, MRS displayed higher activities of antioxidant enzymes, including SOD, CAT, and APX, as well as DPPH-radical scavenging ability, and decreased H2O2 and MDA accumulation under both normal and stress conditions. Our findings suggest that the waterlogging tolerance of the wild A. valvata germplasm was associated with high PDC and ADH, as well as antioxidant ability.

Biotransformation of Deoxynivalenol by a Dual-Member Bacterial Consortium Isolated from Tenebrio molitor Larval Feces.

In this study, a dual-member bacterial consortium with the ability to oxidize deoxynivalenol (DON) to 3-keto-DON, designated SD, was first screened from the feces of Tenebrio molitor larvae. This consortium consisted of Pseudomonas sp. SD17-1 and Devosia sp. SD17-2, as determined by 16S rRNA-based phylogenetic analysis. A temperature of 30 °C, a pH of 8.0-9.0, and an initial inoculum concentration ratio of Devosia to Pseudomonas of 0.1 were optimal single-factor parameters for the DON oxidation activity of the bacterial consortium SD. Genome-based bioinformatics analysis revealed the presence of an intact PQQ biosynthesis operon (pqqFABCDEG) and four putative pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) genes in the genomes of Pseudomonas strain SD17-1 and Devosia strain SD17-2, respectively. Biochemical analyses further confirmed the PQQ-producing phenotype of Pseudomonas and the DON-oxidizing enzymatic activities of two of four PQQ-dependent ADHs in Devosia. The addition of PQQ-containing a cell-free fermentation supernatant from Pseudomonas activated DON-oxidizing activity of Devosia. In summary, as members of the bacterial consortium SD, Pseudomonas and Devosia play indispensable and complementary roles in SD's oxidation of DON. Specifically, Pseudomonas is responsible for producing the necessary PQQ cofactor, whereas Devosia expresses the PQQ-dependent DON dehydrogenase, together facilitating the oxidation of DON.

An alcohol dehydrogenase 7 gene polymorphism associates with both acute and chronic pain in sickle cell disease.

Introduction: As the most distressing complication of sickle cell disease (SCD), pain is marked by considerable heterogenicity. In this study we explored the potential association of alcohol dehydrogenase 7 gene (ADH7) polymorphismrs971074 with sickle cell pain. Methods: We analyzed clinical phenotypes and thers971074 single-nucleotide polymorphismin ADH7 by MassARRAY-iPlex analysis in a cohort of SCD patients. Results: The synonymous rs971074 wassignificantly associated with both acute and chronic pain in SCD. Patients withthe minor T allele(s) recorded significantly more crisis episodes and severe chronic pain symptoms. Conclusion: Our study has identified the rs971074 minor T allele as a genetic biomarker potentially influencing acute and chronic pain. These findings may ultimately help inform strategies to develop precision pain therapies in SCD.

The Role of Intracellular Ca2+ and Mitochondrial ROS in Small Aβ1-42 Oligomer-Induced Microglial Death.

Alzheimer's disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aβ) accumulation, and the link between the Aβ1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aβ-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aβ species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aβ-caused neurotoxicity. In this study, we investigated whether different Aβ1-42 species-small oligomers, large oligomers, insoluble fibrils, and monomers-were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aβ1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca2+ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca2+ nor viability was significantly affected by larger Aβ1-42 species or monomers. In addition, the small Aβ1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aβ1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aβ-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Meyerozyma guilliermondii promoted the deposition of GSH type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers

Lignin is a crucial component in the wound tissue of tubers. The biocontrol yeast Meyerozyma guilliermondii increased the activities of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coenzyme coenzyme A ligase, and cinnamyl alcohol dehydrogenase, and elevated the levels of coniferyl, sinapyl, and p-coumaryl alcohol. The yeast also enhanced the activities of peroxidase and laccase, as well as the content of hydrogen peroxide. The lignin promoted by the yeast was identified as guaiacyl-syringyl-p-hydroxyphenyl type using Fourier transform infrared spectroscopy and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance. Furthermore, a larger signal area for G2, G5, G'6, S2, 6, and S'2, 6 units was observed in the treated tubers, and the G'2 and G6 units were only detected in the treated tuber. Taken together, M. guilliermondii could promote deposition of guaiacyl-syringyl-p-hydroxyphenyl type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers.

Class III Alcohol Dehydrogenase Plays a Key Role in the Onset of Alcohol-Related/-Associated Liver Disease as an S-Nitrosoglutathione Reductase in Mice.

Lipid accumulation in the liver due to chronic alcohol consumption (CAC) is crucial in the development of alcohol liver disease (ALD). It is promoted by the NADH/NAD ratio increase via alcohol dehydrogenase (ADH)-dependent alcohol metabolism and lipogenesis increase via peroxisome proliferator-activated receptor γ (PPARγ) in the liver. The transcriptional activity of PPARγ on lipogenic genes is inhibited by S-nitrosylation but activated by denitrosylation via S-nitrosoglutathione reductase (GSNOR), an enzyme identical to ADH3. Besides ADH1, ADH3 also participates in alcohol metabolism. Therefore, we investigated the specific contribution of ADH3 to ALD onset. ADH3-knockout (Adh3-/-) and wild-type (WT) mice were administered a 10% ethanol solution for 12 months. Adh3-/- exhibited no significant pathological changes in the liver, whereas WT exhibited marked hepatic lipid accumulation (p < 0.005) with increased serum transaminase levels. Adh3-/- exhibited no death during CAC, whereas WT exhibited a 40% death. Liver ADH3 mRNA levels were elevated by CAC in WT (p < 0.01). The alcohol elimination rate measured after injecting 4 g/kg ethanol was not significantly different between two strains, although the rate was increased in both strains by CAC. Thus, ADH3 plays a key role in the ALD onset, likely by acting as GSNOR.

Extended Scope and Understanding of Zinc-Dependent Alcohol Dehydrogenases for Reduction of Cyclic α-Diketones.

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring.

Physiological responses and Ethylene-Response AP2/ERF Factor expression in Indica rice seedlings subjected to submergence and osmotic stress

BackgroundThe increased frequency of heavy rains in recent years has led to submergence stress in rice paddies, severely affecting rice production. Submergence causes not only hypoxic stress from excess water in the surrounding environment but also osmotic stress in plant cells. We assessed physiological responses and Ethylene-Response AP2/ERF Factor regulation under submergence conditions alone and with ionic or nonionic osmotic stress in submergence-sensitive IR64 and submergence-tolerant IR64-Sub1 Indica rice cultivars.ResultsOur results indicate that both IR64 and IR64-Sub1 exhibited shorter plant heights and root lengths under submergence with nonionic osmotic stress than normal condition and submergence alone. IR64-Sub1 seedlings exhibited a significantly lower plant height under submergence conditions alone and with ionic or nonionic osmotic stress than IR64 cultivars. IR64-Sub1 seedlings also presented lower malondialdehyde (MDA) concentration and higher survival rates than did IR64 seedlings after submergence with ionic or nonionic osmotic stress treatment. Sub1A-1 affects reactive oxygen species (ROS) accumulation and antioxidant enzyme activity in rice. The results also show that hypoxia-inducible ethylene response factors (ERF)-VII group and alcohol dehydrogenase 1 (ADH1) and lactate dehydrogenase 1 (LDH1) genes exhibited different expression levels under nonionic or ionic osmotic stress during submergence on rice.ConclusionsTogether, these results demonstrate that complex regulatory mechanisms are involved in responses to the aforementioned forms of stress and offer new insights into the effects of submergence and osmotic stress on rice.

Effects of Cynanchum bungei Decne Addition on the Physicochemical Properties and Antioxidant Activity of Rice Wine

Cynanchum bungei Decne is an agricultural crop with a high starch content and contains bioactive compounds with anti-tumor, anti-depressant, anti-oxidant, and other activities. In this work, three concentrations of C. bungei Decne, namely, 5%, 15%, and 25%, were added to media to brew C. bungei Decne rice wine. The basic physical and chemical properties, antioxidant activities, sensory characteristics, and volatile components of C. bungei Decne rice wine were determined. Furthermore, the effects of C. bungei Decne extract on the cell viability, alcohol dehydrogenase activity, and glucose absorption capacity of Saccharomyces cerevisiae were analyzed. The results showed that the main active compound contents and antioxidant activity of the rice wine were increased with the increase in the C. bungei Decne added. However, the vitality of Saccharomyces cerevisiae was inhibited by C. bungei Decne to some extent. Combined with a sensory evaluation, 15% C. bungei Decne was found to be the optimal additive concentration with which to brew C. bungei Decne rice wine. These data provide a theoretical basis for the development of C. bungei Decne rice wine.

Enzymatic properties of alcohol dehydrogenase PedE_M.s. derived from Methylopila sp. M107 and its broad metal selectivity.

As an important metabolic enzyme in methylotrophs, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases play significant roles in the global carbon and nitrogen cycles. In this article, a calcium (Ca2+)-dependent alcohol dehydrogenase PedE_M.s., derived from the methylotroph Methylopila sp. M107 was inserted into the modified vector pCM80 and heterologously expressed in the host Methylorubrum extorquens AM1. Based on sequence analysis, PedE_M.s., a PQQ-dependent dehydrogenase belonging to a methanol/ethanol family, was successfully extracted and purified. Showing by biochemical results, its enzymatic activity was detected as 0.72 U/mg while the Km value was 0.028 mM while employing ethanol as optimal substrate. The activity of PedE_M.s. could be enhanced by the presence of potassium (K+) and calcium (Ca2+), while acetonitrile and certain common detergents have been found to decrease the activity of PedE_M.s.. In addition, its optimum temperature and pH were 30°C and pH 9.0, respectively. Chiefly, as a type of Ca2+-dependent alcohol dehydrogenase, PedE_M.s. maintained 60-80% activity in the presence of 10 mM lanthanides and displayed high affinity for ethanol compared to other PedE-type enzymes. The 3D structure of PedE_M.s. was predicted by AlphaFold, and it had an 8-bladed propeller-like super-barrel. Meanwhile, we could speculate that PedE_M.s. contained the conserved residues Glu213, Asn300, and Asp350 through multiple sequence alignment by Clustal and ESpript. The analysis of enzymatic properties of PedE_M.s. enriches our knowledge of the methanol/ethanol family PQQ-dependent dehydrogenase. This study provides new ideas to broaden the application of alcohol dehydrogenase in alcohol concentration calculation, biosensor preparation, and other industries.

Effect of inoculated Daqu on the spontaneous fermentation of Chinese liquor

Inoculated fermentation is widely used to improve the efficiency and quality of food production. However, it is still unclear how the inoculated multi-species starters influence food fermentation. Here we prepared two different types of Daqu, with/without the inoculation of Bacillus licheniformis in spontaneous Daqu fermentation, and studied their effects on liquor fermentation. These two types of Daqu were different in microbial community, and the inoculated Daqu had significantly higher relative abundance of Bacillus (69.2%) and lower relative abundance of Lactobacillus (3.2%) than those of Daqu without inoculation (Bacillus, 13.5%; Lactobacillus, 14.0%). After using with these two types of Daqu, metatranscriptomic analysis revealed that Kazachstania, Naumovozyma, Saccharomyces, Nakaseomyces and Lactobacillus were the transcriptional active genera during liquor fermentation. The transcription of Lactobacillus decreased on days 10 and 20 in liquor fermentation with the inoculated Daqu. The transcription of Kazachstania, Naumovozyma and Saccharomyces decreased on day 10 but increased on day 20 with the inoculated Daqu. Although lactate dehydrogenase decreased in Lactobacillus, alcohol dehydrogenase, aldehyde dehydrogenase and lactate dehydrogenase increased in Saccharomyces on day 20 in fermentation with inoculated Daqu, it indicated an extended succession of Saccharomyces in liquor fermentation. This would facilitate the increase of ethanol, acetic acid and lactic acid contents in liquor fermentation with inoculated Daqu. This work would be beneficial for improving Chinese liquor fermentation.

Phosphoproteomic analysis highlights phosphorylation of primary metabolism-related proteins in response to postharvest cold storage and subsequent shelf-life in tomato fruit

Tomato fruit are susceptible to postharvest chilling injuries when stored at low temperatures, resulting in quality deterioration and economic losses worldwide. Protein phosphorylation is an important post-translational modification that has been proven to be involved in plant cold tolerance. However, the changes of protein phosphorylation underlying cold stress responses in tomatoes remain poorly understood. In this study, microscopic analyses showed that endomembranes and intracellular organelles were broken down after cold storage, and the breakdown became more pronounced when the fruit was restored to room temperature. Primary metabolomic analysis revealed that large amounts of lipids, amino acids, sugars, and organic acids were altered following cold treatment. Additionally, we performed comparative phosphoproteomics and determined the overall phosphoprotein profile of tomato fruit after postharvest cold storage and subsequent shelf life. A total of 10260 phosphopeptides corresponding to 9640 phosphosites in 3740 phosphoproteins were identified. Differentially phosphorylated proteins were mainly involved in primary metabolism, such as glycerolipid metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid cycle. Correspondingly, the phosphorylation of large amounts of important primary metabolic enzymes, such as fatty acid desaturase, sucrose synthase, and alcohol dehydrogenase, was upregulated after cold treatment. The present study revealed the involvement of phosphorylation of primary metabolism-related proteins in the response to postharvest cold storage in tomato fruit.