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Industrial biosynthesis of β-nicotinamide mononucleotide (β-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 β-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.

Probiotics can regulate gut microbiota and protect against acute alcohol-induced liver injury through the gut–liver axis. However, efficacy is strain-dependent, and their mechanism remains unclear. This study investigated the effect of lactic acid bacteria (LAB), including Lacticaseibacillus paracaseiE10 (E10), Lactiplantibacillus plantarumM (M), Lacticaseibacillus rhamnosusLGG (LGG), Lacticaseibacillus paracaseiJN-1 (JN-1), and Lacticaseibacillus paracaseiJN-8 (JN-8), on the prevention of acute alcoholic liver injury in mice. We found that LAB pretreatment reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) and reduced hepatic total cholesterol (TC) and triglyceride (TG). JN-8 pretreatment exhibited superior efficacy in improving hepatic antioxidation. LGG and JN-8 pretreatment significantly attenuated hepatic and colonic inflammation by decreasing the expression of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) and increasing the expression of interleukin 10 (IL-10). JN-1 and JN-8 pretreatments have better preventive effects than other LAB pretreatment on intestinal barrier dysfunction. In addition, the LAB pretreatment improved gut microbial dysbiosis and bile acid (BA) metabolic abnormality. All of the strains were confirmed to have bile salt deconjugation capacities in vitro, where M and JN-8 displayed higher activities. This study provides new insights into the prevention and mechanism of LAB strains in preventing acute alcoholic liver injury.

W18O49nanowires (W18O49NWs) with unique one-dimension structures and excellent electron/ions transport properties have attracted increasing attention in academia and industry because of their potential applications in many energy-related devices. In the past decades, many research articles related to W18O49have been published, but there are insufficient review articles focusing on W18O49NWs. In this review, we present the crystal structure of W18O49and briefly introduce the synthesis methods and growth mechanism of W18O49NWs. Moreover, their applications in energy conversion and storage devices are summarized. Finally, the current challenges and opportunities for applying W18O49NWs are provided. We hope this review can promote the development of W18O49NWs in energy conversion, storage, and other promising applications.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorataremain unknown. Herein, we first observed that the methanol extract of C. kanehiraetrunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphoratatriterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorataand demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Phospholipase D (PLD) is an essential enzyme for the enzymatic phosphatidylserine synthesis and Bacillus subtilisis a potential microbial cell factory for the PLD production, but its widespread application in the food industry is limited by low expression levels and the use of antibiotics. In this study, a multi-copy recombinant strain BS-PLD312 was constructed through screening genome location and the number of PLD expression cassettes. The enzyme activity of BS-PLD312 can reached 392.88 U/mL in the absence of antibiotics and inducers. Furthermore, a food grade reaction system within egg yolk was established and the phosphatidylserine content reached 35.13 g/L in egg yolk. This reaction system is applicable for synthesis of other PLs and regulating the composition of various phospholipids in egg yolk. The protocols established in this study shows great potential for application in industrial PLD production and expanded the practical application of PLD in food industry.

ABSTRACTDiet is a key player in gut–liver axis. However, the effect of different dietary patterns on gut microbiota and liver functions remains unclear. Here, we used rodent standard chow and purified diet to mimic two common human dietary patterns: grain and plant-based diet and refined-food-based diet, respectively and explored their impacts on gut microbiota and liver. Gut microbiota experienced a great shift with notable increase in Desulfovibrio, gut bile acid (BA) levels elevated significantly, and liver inflammation was observed in mice fed with the purified diet. Liver inflammation and elevated gut BA levels also occurred in mice fed with the chow diet after receiving Desulfovibrio desulfuricansATCC 29,577 (DSV). Restriction of sulfur-containing amino acids (SAAs) prevented liver injury mainly through higher hepatic antioxidant and detoxifying ability and reversed the elevated BA levels due to excess Desulfovibrio. Ex vivofermentation of human fecal microbiota with primary BAs demonstrated that DSV enhanced production of secondary BAs. Higher concentration of both primary and secondary BAs were found in the gut of germ-free mice after receiving DSV. In conclusion, Restriction of SAAs in diet may become an effective dietary intervention to prevent liver injury associated with excess Desulfovibrioin the gut.

The microbial community and product flavor of fermented foods are subject to seasonal environmental fluctuations due to open production environment. However, research on the impact of external environmental changes caused by seasonal shifts on brewing is limited. Here, we studied changes in physicochemical indicators and flavor compounds of fermented grains during Baijiu fermentation in four seasons, as well as the succession and assembly of microbial communities. The temperature throughout fermentation was highest in summer, followed by spring and autumn, and lowest in winter. Winter had slower rates of moisture growth, total acidity accumulation, and sugar consumption than the other seasons. At the end of fermentation, the highest total organic acid content was identified in summer, and the lowest level of volatile compounds was found in winter. We found significant differences in microbial community structure across different seasons. Meanwhile, compared to fungal community, the succession of bacterial community was more susceptible to brewing environment, with reducing sugar being the main driving factor in the early stage and total acidity, organic acids, and moisture being the main driving factors in the late stage. The microbial co-occurrence networks in autumn and winter were more complex than those in spring and summer. Furthermore, brewing in the spring and summer promoted the accumulation of acids in Baijiu, esters in summer, ethyl esters in autumn, and aldehydes and ketones in winter. This study offers a fresh perspective on the microbial brewing mechanism of Baijiu by exploring the seasonal variations of microbiota and metabolome.

Trehalose is a non-reducing disaccharide connected by α-1,1-glycosidic bonds; it is widely distributed in bacteria, fungi, yeast, insects, and plant tissues and plays various roles. It can be hydrolyzed by trehalase into two glucose molecules. Trehalases from different sources have been expressed in Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, baculovirus-silkworm, and other expression systems; however, it is most common in E. coli. The structural characteristics of different glycoside hydrolase (GH) family trehalases and the sources of trehalase have been analyzed. The catalytic mechanism of GH37 trehalase has also been elucidated in detail. Moreover, the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity. We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance. We suggest that the level of recombinant production is far from meeting industrial requirements, and the catalytic performance of trehalase needs to be improved urgently. Finally, we discuss developmental prospects and future trends.

β-Nicotinamide mononucleotide (NMN) is a direct precursor of the important coenzyme NAD+in the human body and has broad application prospects in healthcare. This study engineered an Escherichia coliBL21 (DE3) strain to efficiently biosynthesize NMN from nicotinamide (NAM) and xylose. Firstly, the nicotinamide phosphoribosyltransferase (Nampt) from Chitinophaga pinensiswas selected and heterologous expressed to construct the NAD+ remediation pathway for NMN synthesis from nicotinamide (NAM) and in vivoprecursor phosphoribosyl pyrophosphate (PRPP). Then, the supply of PRPP was enhanced by constructing the metabolic flux from d-xylose. To facilitate NMN accumulation, the branch pathways, including PRPP competitive pathway, carbon flow competitive pathway, and NMN downstream metabolic decomposition pathway were fine-tuned using CRISPR/Cas9 editing tools. Combined with process optimization of whole-cell biocatalytic reaction, a NMN titre of 497.5 mg·L−1was obtained. Finally, the scale-up culture was carried out on a 5 L fermenter, and the yield reached 760.2 mg·L−1. This work achieved green biosynthesis of NMN using xylose as substrate and enhanced the productivity by systematic engineering strategies, presenting more possibilities for the synthesis of NMN from a wide range of monosaccharides.

Multispecies solid-state fermentation is a traditional processing technique for the traditional Chinese food, such as cereal vinegar, Baijiu,etc. Generally, few abundant and many rare microbes were involved in such processes, and the necessity and roles of the latter are less studied. Here the co-occurrence patterns of abundant and rare bacterial community and abiotic factors influencing their community assembly were investigated in acetic acid fermentation following starter inoculation, using Zhenjiang aromatic vinegar as a model system. Abundant taxa that contribute to the function of accumulating acid exhibited a ubiquitous distribution while the distribution of rare taxa along the fermentation process unraveled. The species composition of the rare taxa significantly altered, but abundant taxa were maintained after inoculation. Moreover, the diversity of rare taxa changed more significantly than that of abundant taxa. Both abundant and rare sub-communities, which were contributed more with species turnover than species richness, were demonstrated to be driven by pH, acetic acid, ammonium nitrogen, and ethanol. Stochastic processes regulated the assembly of both sub-communities, but more prominent on rare sub-communities. Co-occurrence network was more governed by rare sub-communities, and the co-variations between microbial communities were predominantly positive, implying that rare taxa played more important role in the fermentation stability and network robustness. Furthermore, seven network connectors were identified, and three of them belonged to rare taxa. These microbes of different modules were enriched at particular phases of fermentation. These results demonstrate the ecological significance of rare bacteria and provide new insights into understanding the abiotic factors influence microbial structure in traditional fermented foods.

Using the previous batch of fermented product to initiate fermentation (i.e., community-level bioaugmentation) is a common production technique for fermented foods. As the saccharification, fermentation, and aroma-producing agent in strong-flavor Baijiu brewing, medium-temperature Daqu is usually produced by spontaneous fermentation without any exogenous microorganisms inoculated. Here, we selected Daqu from day 12 (FerDq) and day 30 (MatDq) of the previous batch as starters to perform bioaugmentation. Bioaugmentation enhanced total acidity, enzymatic activities, and volatiles, particularly alcohols and pyrazines. Metagenomics revealed that bioaugmentation with 5% or 10% (w/w) FerDq enhanced the relative abundances of Staphylococcusand Bacillus, whereas bioaugmentation with 5% or 10% (w/w) MatDq enriched Bacillus, Aspergillus, Talaromyces, and Rasamsonia. The increase in liquefying activity in MatDq groups was due to the increase in alpha-amylase and glycogen phosphorylase abundances, mainly caused by the enrichment of Aspergillus, Talaromyces, and Bacillus. Bioaugmentation with FerDq significantly increased the gene abundance of carboxyesterase and esterifying activity of Daqu via Staphylococcusand Bacillus. The enriched Bacillus, Staphylococcus, and Paenibacillusin FerDq groups facilitated the production of pyrazines. The metabolic pathway of phenylethyl alcohol biosynthesis varied among different groups. In the control group, it was mainly produced by aryl-alcohol dehydrogenase, which originated from Lactobacillales, while in the bioaugmented groups, it was mainly produced through alcohol dehydrogenase and formaldehyde dehydrogenase, which were derived from Rhizopusand Bacillusin the FerDq groups and from Rasamsonia, Talaromyces, Rhizopus, and Bacillusin the MatDq groups. This study revealed the feasibility of producing enzymatic activity- and aroma-enhanced Daqu through community-level bioaugmentation.

Cicada flowers, which are edible and medicinal mushrooms, are the fruiting bodies of Isaria cicadae, a fungus that is parasitic on the larvae of cicada pupae. We hypothesize that host factors might possess stimulatory activity on metabolite synthesis in Isaria cicadae. Here, we first compared the microbial community structures of different wild cicada flowers across geographical regions, compartments, and growth stages via high-throughput sequencing. Isaria cicadaeTZC-3, an isolate of the most abundant operational taxonomic unit (OTU6782) in all the fungal communities, was isolated from wild cicada flowers. Furthermore, the effects of cicada pupae on metabolite synthesis in Isaria cicadaeTZC-3 were studied in submerged culture. The contents of intercellular polysaccharides, adenosine, N6-(2-hydroxyethyl)-adenosine, free amino acids, and hydrolyzed monosaccharides in the mycelia cultured with cicada pupa powder (4%) were significantly increased as compared with the contents in the control group. This indicates that a cicada pupa can act as an elicitor for metabolite synthesis in Isaria cicadae.

Clostridium tyrobutyricumis a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. However, the limited genetic traits of C. tyrobutyricumresulted in its constrained applicability in the food industry due to the absence of publicly available data. We performed a comparative genomic analysis of 13 C. tyrobutyricumstrains isolated from different habitats to investigate the adaptation mechanisms and metabolic potentials of the C. tyrobutyricumstrains isolated from fermentation environments (FE). The results showed that the genetic diversity of FE- associated C. tyrobutyricumstrains was higher than that of strains isolated from non-fermentation environments (NFE). Based on evaluating safety-related genes, FE-associated strains were likely non-pathogenic to humans or animals. Many environmental adaptation-related genes involved in energy production and conversion, phosphotransferase system (PTS), etc., were significantly enriched in FE-associated strains, which was conducive to their survival in the corresponding habitats. The analysis of carbohydrate-active enzymes revealed that the CAZyme categories of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) were found to be significantly enriched in FE-associated strains, which was beneficial for carbohydrate utilization (including starch, cellulose, etc.). Fermentation experiments revealed that strains isolated from the Chinese liquor fermentation environment were capable of decomposing carbohydrates and producing organic acids. Collectively, this study provided insights into the genomic features of C. tyrobutyricumand the theoretical basis for further use, research, and development of these strains.

A keratinase gene kerBvwas mined from soil metagenomes. The open reading frame consisted of 1149 bp and potentially encoded a protein of 382 amino acid residues. It shared the same active site with several reported typical keratinases via analysis of the amino acid sequence. The keratinase was successfully expressed in B. subtilisWB600 with pMA5 expression vector. The maximum activity of 164.8 U/mL in the fermentation supernatant was observed after incubating for 30 h in Terrifc Broth (TB) medium. The keratinase exhibited outstanding resistance to metal ions and was surfactant-stable. Additionally, the enzyme displayed broad substrate specificity especially toward insoluble substrate feather meal because of its disulfide bond-reducing activity. Furthermore, the reducing power of the recombinant keratinase was investigated. It showed that the protein exhibited a relatively high reducing power, which was subsequently used in the biosynthesis of silver nanoparticles (AgNPs). The biosynthesized AgNPs were characterized by ultraviolet–visible (UV–vis) spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), as well as Fourier transform infrared spectroscopy (FTIR) and displayed obvious antibacterial activities toward Escherichia coli.

Konjac glucomannan (KGM) is a water-soluble polysaccharide obtained from the roots and tubers of konjac plants. Recently, a degraded product of KGM, depolymerized KGM (DKGM), has attracted attention because of its low viscosity, improved hydrophily, and favorable physiological functions. In this review, we describe the preparation of DKGM and its prebiotic effects. Other health benefits of DKGM, covering antioxidant and immune activity, are also discussed, as well as its safety. DKGM could be a candidate for use as a tool for the treatment of various diseases, including intestinal flora imbalance, and oxidative- and immune-related disorders. 魔芋葡甘露聚糖是从魔芋块茎中提取的一种高分 子水溶性多糖。近些年研究表明,其解聚产物, 除了具有高溶解性和低粘度等良好的理化性质 外,还具有调节微生物菌群结构、抗氧化、免疫 调节等多种生理活性。本文重点综述了解聚型葡 甘露聚糖的制备方法以及菌群调节功能。除此之 外,对其抗氧化、免疫调节功能以及安全性评价 也进行了全面的总结,为解聚型葡甘露聚糖的研 究与应用提供一定的依据与思路。

Untargeted and targeted metabolomics based on LC-MS/MS was applied here to investigate the changes in metabolites following Lactiplantibacillus plantarum(L. plantarum) fermentation of goji juice. The changes in physicochemical characteristics, free amino acid and organic acid levels were quantified. Total phenols, total flavonoids, and antioxidant activity were increased after these L. plantarumfermentation. 31 primary metabolites were screened out by comparison between two groups using multivariate statistical analysis. L. plantarumconsumed reducing sugar and most varieties of amino acids. Gamma amino butyric acid (GABA) and phenylacetaldehyde were significantly accumulated via precursor of glutamate and phenylalanine, respectively. Indole-3-lactic acid (ILA) was the characteristic secondary metabolite after fermentation. There were 6.25 and 4.19 mg/L ILA accumulated after LP-D and LP-F fermentation, respectively, whereas it was not detected in goji juice. The accumulation of tyrosol was strain-dependent. Part of the bitter-taste amino acids, including l-methinoine, l-phenylalanine and tryptophan, were converted into their corresponding acetyl-derivatives, which represented umami or kokumi taste. Overall, fermentation by L. plantarumstrains altered the metabolite profile and increased the levels of several biologically active substances of goji juice. These results deepen the understanding of L. plantarummetabolism, and the bioconversion associated with flavor and bioactive compounds unraveled here can be used as references in other plant-based materials for LAB fermentation.

ABSTRACTPhosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Surfactant-stable keratinases with good properties are promising candidates for extensive applications in detergent industries. A novel fungal keratinase-producing strain, Gibberella intermediaCA3-1, is described in this study. The keratinase production medium was optimized and composed of 10 g L−1of wool powder, 5 g L−1of tryptone, 10 g L−1of maltodextrin and 0.5 g L−1of NaCl. Keratinase activity was increased up to 109 U mL−1from 15 U mL−1by culture optimization. The optimal reaction pH and temperature of the enzyme were 9.0 and 60°C, respectively. The keratinase activity could be improved by sodium dodecyl sulphate (SDS), and it remained stable in the presence of several surfactants and commercial detergents. G. intermediakeratinase was proved to completely remove blood stains from cotton cloth when combined with detergents. These findings indicate that this fungal keratinase is a promising catalyst for the application in detergent industry. To our knowledge, this is the first report on keratinase production by Gibberellagenus.

Surfactant-stable keratinases with good properties are promising candidates for extensive applications in detergent industries. A novel fungal keratinase-producing strain, Gibberella intermediaCA3-1, is described in this study. The keratinase production medium was optimized and composed of 10 g L−1of wool powder, 5 g L−1of tryptone, 10 g L−1of maltodextrin and 0.5 g L−1of NaCl. Keratinase activity was increased up to 109 U mL−1from 15 U mL−1by culture optimization. The optimal reaction pH and temperature of the enzyme were 9.0 and 60°C, respectively. The keratinase activity could be improved by sodium dodecyl sulphate (SDS), and it remained stable in the presence of several surfactants and commercial detergents. G. intermediakeratinase was proved to completely remove blood stains from cotton cloth when combined with detergents. These findings indicate that this fungal keratinase is a promising catalyst for the application in detergent industry. To our knowledge, this is the first report on keratinase production by Gibberellagenus.

Keratinases specifically degrade insoluble keratin waste, thus contributing to environmental protection and sustainable biomass development. However, their industrial application is hindered by inefficient enzyme production and poor biomass generation. In this study, the heterologous expression of keratinase was found to have cytotoxicity and might block host cell growth due to its proteolytic property. To address this problem, an autoregulatory expression system based on quorum sensing was developed to synergistically regulate cell growth and keratinase production in Bacillus subtilis. The growth-dependent promoter PaprEwas chosen and shown to be effective in delaying keratinase production while promoting host cell proliferation. Copy number screening and core region mutations further balanced the two states. Carbon supplement optimization indicated that addition of 2% glucose facilitated biomass accumulation during the early stage of fermentation. Cell density increased to 15.6 (OD600 nm) from 8 with keratinase activity raised to 4200 U·mL–1from 1162 U·mL–1. Keratinase was then utilized in the bioconversion of feather waste to prepare soluble keratins, polypeptides, and amino acids. This study provides a powerful system for efficient production of keratinase and paves the way for keratin waste recycling.

Most available methods for modifying the catalytic properties of enzymes are costly and time-consuming, as they rely on the information of enzyme crystal structure or require handling large amounts of mutants. This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermediawas performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.

Pseudomonas putidaCGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putidanitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1h−1. P. putidashows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putidaand it represents the highest isonicotinic acid production reported so far.

This paper analyzed the concept and content of the road domain ecosystem ecological integration and its relationship with ecosystem health. Based on PSR framework, the ecological integrated measurement model was built, which divided the ecological integration into three subsystems, pressure, state and response. By constructing various subsystems indicators, the road domain ecosystem ecological integration was quantified by using the comprehensive evaluation method. On this basis, the ecological integration index was calculated along the highway at different distances of Yichang to Badong highway. The result shows that the road domain ecosystem ecological integration is relative to the distance on both sides of the highway. In the range of 0-5m, the ecological integration index is only 0.0408, which is gradually rising with distance increasing. To the range of 100-200m, the ecological integration index reaches 0.9343, which is close to the normal ecosystems level. This result is mainly related to the road construction disturbance. When it is closer to the road, the interference intensity is greater and the impact on the ecosystem is more obvious, which leads to the worse ecological integration. Conversely, the ecological integration is better.

This paper analyzed the impact of the mountain highway on road domain ecosystems, and defined the concept and content of the road domain ecosystem ecological integration and its relationship with ecosystem health. Based on PSR framework, the ecological integrated measurement model was built. By constructing various subsystems indicators, the road domain ecosystem ecological integration was quantified by using the comprehensive evaluation method. On this basis, this paper divided the ecological integration into four grades: poor, moderate, good and excellent. It provided a theoretical basis for the ecological protection and restoration of the mountain highway construction.

In order to investigate the difference of current toughness index standards for fiber reinforced concrete, two main groups of specimens were made to take bending toughness test with the requirements of corresponded standards, loading methods and loading speeds, which are ASTM C1018 in America, ACI 544 and JSCE G552 in Japan. United with software Origin, the load-deflection curves gathered from bending test was calculated with relative standards. The results show that the calculated toughness index value with ASTM C1018-98 in America is more accurate with three grades but the requested deflection of testing is much longer than others while ACI 544 and JSCE G552 in Japan are quite the contrary.