High Substrate Concentrations Research Articles

Studying the rate of polyphenol oxidase activity in apples using a colorimeter

Salt is one of the oldest methods used to preserve food and help it stay fresh by preventing rotting due to its antibacterial properties. This study was therefore conducted to understand how the increase in salt concentration affects the rate of change of polyphenol oxidase activity in apples using a colorimeter. A colorimeter is a device used to measure absorbance of particular wavelengths of light by specific solutions, in this case, Granny Smith Apple juice. The results correlated with the hypothesis which is predicted that as the salt concentration of the dilutions increase, there will be a decrease in the rate of change of absorbance because the chlorine ions from the salt will inhibit the polyphenol oxidase activity and prevent further oxidation of phenols to quinones, limiting the polymerization and formation of melanin. The results confirm that salt is a non-competitive inhibitor of polyphenol oxidase because it slows down the catabolic processes in apples- mainly the breakdown of glucose. However, salt is a non-competitive inhibitor of PPO but as for lower concentrations, such as 0.25M and 0.50M, the higher concentration of substrates still mitigates the effect of salt as an inhibitor to a significant extent.

Effects of Increasing Doses of Condensed Tannins Extract from Cistus ladanifer L. on In Vitro Ruminal Fermentation and Biohydrogenation.

Simple SummaryRuminant edible products have been associated with adverse health effects, due to their high saturated fatty acids and low polyunsaturated fatty acids content, resulting from the extensive biohydrogenation conducted by rumen microbiota. Cistus ladanifer condensed tannins were able to change the lamb ruminal biohydrogenation, increasing the beneficial fatty acids production. The aim of this study was to test the effect of increasing doses of C. ladanifer condensed tannins extract (0, 25, 50, 75 and 100 g/kg dry matter) on in vitro rumen fermentation and biohydrogenation. The increasing doses of condensed tannins led to a moderate decrease of volatile fatty acids production, a pronounced depression in microbial odd and branched fatty acids and of dimethyl acetals production, and a minor effect on the biohydrogenation, which indicates that microbial growth was more inhibited than fermentative and biohydrogenation activities. The ability of C. ladanifer condensed tannins extract to modulate the biohydrogenation (BH) was not observed in the present study. However, the results obtained suggest a possible adaptative response of the microbial population to stress stimuli of condensed tannins and lipid supplementation.Cistus ladanifer (rockrose) is a perennial shrub quite abundant in the Mediterranean region, and it is a rich source in secondary compounds such as condensed tannins (CTs). Condensed tannins from C. ladanifer were able to change the ruminal biohydrogenation (BH), increasing the t11–18:1 and c9,t11–18:2 production. However, the adequate conditions of the C. ladanifer CTs used to optimize the production of t11–18:1 and c9,t11–18:2 is not yet known. Thus, we tested the effect of increasing the doses of C. ladanifer CT extract (0, 25, 50, 75 and 100 g/kg dry matter (DM)) on in vitro rumen BH. Five in vitro batch incubations replicates were conducted using an oil supplemented high-concentrate substrate, incubated for 24 h with 6 mL of buffered ruminal fluid. Volatile fatty acids (VFAs) and long chain fatty acids (FA) were analyzed at 0 h and 24 h, and BH of c9–18:1, c9, c12–18:2 and c9, c12, c15–18:3, and BH products yield were computed. Increasing doses of C. ladanifer CTs led to a moderate linear decrease (p < 0.001) of the VFA production (a reduction of 27% with the highest dose compared to control). The disappearance of c9–18:1 and c9,c12–18:2 as well as the production of t11–18:1 and c9, t11:18:2 was not affected by increasing doses of C. ladanifer CTs, and only the disappearance of c9, c12, c15–18:3 suffered a mild linear decrease (a reduction of 24% with the highest dose compared to control). Nevertheless, increasing the C. ladanifer CT dose led to a strong depression of microbial odd and branched fatty acids and of dimethyl acetals production (less than 65% with the highest dose compared to control), which indicates that microbial growth was more inhibited than fermentative and biohydrogenation activities, in a possible adaptative response of microbial population to stress induced to CTs and polyunsaturated fatty acids. The ability of C. ladanifer to modulate the ruminal BH was not verified in the current in vitro experimental conditions, emphasizing the inconsistent BH response to CTs and highlighting the need to continue seeking the optimal conditions for using CTs to improve the fatty acid profile of ruminant fat.

Enantioselective resolution of (±)-1-phenylethyl acetate using the immobilized extracellular proteases from deep-sea Bacillus sp. DL-1

Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve the stability of the enzyme, the immobilized extracellular proteases were preparated by using 60 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus sp. DL-1, shaking at 25 °C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7 mg extracellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on the resolution were investigated by single factor experiments. Under the optimal reaction conditions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5 (Tris-HCl), 5% (v/v) methanol and 45 °C for 2 h), (R)-1-phenylethanol was generated with the e.e.p being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction conditions (10-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.0 (PB), 5% (v/v) DMSO and 35 °C for 1.5 h), (S)-1-phenylethyl acetate was generated with the e.e.s being over 99% and the yield being 79%, respectively. Compared with the extracellular proteases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1 exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably, the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.

Acetate as substrate for l-malic acid production with Aspergillus oryzae DSM 1863

BackgroundMicrobial malic acid production is currently not able to compete economically with well-established chemical processes using fossil resources. The utilization of inexpensive biomass-based substrates containing acetate could decrease production costs and promote the development of microbial processes. Acetate is a by-product in lignocellulosic hydrolysates and fast pyrolysis products or can be synthesized by acetogens during syngas fermentation. For the fermentation of these substrates, a robust microorganism with a high tolerance for biomass-derived inhibitors is required. Aspergillus oryzae is a suitable candidate due to its high tolerance and broad substrate spectrum. To pave the path towards microbial malic acid production, the potential of acetate as a carbon source for A. oryzae is evaluated in this study.ResultsA broad acetate concentration range was tested both for growth and malic acid production with A. oryzae. Dry biomass concentration was highest for acetic acid concentrations of 40–55 g/L reaching values of about 1.1 g/L within 48 h. Morphological changes were observed depending on the acetate concentration, yielding a pellet-like morphology with low and a filamentous structure with high substrate concentrations. For malic acid production, 45 g/L acetic acid was ideal, resulting in a product concentration of 8.44 ± 0.42 g/L after 192 h. The addition of 5–15 g/L glucose to acetate medium proved beneficial by lowering the time point of maximum productivity and increasing malic acid yield. The side product spectrum of cultures with acetate, glucose, and cultures containing both substrates was compared, showing differences especially in the amount of oxalic, succinic, and citric acid produced. Furthermore, the presence of CaCO3, a pH regulator used for malate production with glucose, was found to be crucial also for malic acid production with acetate.ConclusionsThis study evaluates relevant aspects of malic acid production with A. oryzae using acetate as carbon source and demonstrates that it is a suitable substrate for biomass formation and acid synthesis. The insights provided here will be useful to further microbial malic acid production using renewable substrates.

Lactate and acetate applied in dual‐chamber microbial fuel cells with domestic wastewater

For about 20 years, microbial fuel cells (MFCs) are an emerging technology that has gained attention for its new wastewater treatment and energy generation, especially its ability to convert chemical energy from a broad range of substances into electricity. However, MFC has not been widely commercialized due to low efficiency. Studies have shown that substrate loading is an important factor in scaling up. Therefore, this study investigates the effect of substrate type and concentration on honeycomb MFCs (HCMFCs). The effect of different concentrations ranging from 10 to 40 mM of lactate and acetate (1:1 ratio) substrates was investigated. Power efficiency was analyzed using polarization and power density curves. Results showed that the performance of MFCs and biofilm formation is affected by the substrates. Scanning electron microscopy showed some changes in biofilm formation. Mixing lactate and acetate at 30 mM gave the best performance with a power density of 956.75 mW m−2 and chemical oxygen demand removal of 87.8%. Furthermore, effective substrate degradation, having COD removal of 91.4%, was observed with acetate. Highlights Substrate type and concentration affect MFCs performance significantly Mixed substrate promotes robust mix bacterial culture Substrate concentration of 30 mM gave the best power generation Adverse effect of high substrate concentration on power generation was confirmed Effective substrate degradation with COD removal of 91.4% was observed with acetate

The occurrence and ecology of microbial chain elongation of carboxylates in soils

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.

Recent insights, applications and prospects of xylose reductase: a futuristic enzyme for xylitol production

Xylose reductase (XR) is an intermediate inducible enzyme of xylose metabolism responsible for the reduction of xylose into xylitol. It is an intracellular enzyme present in various facultative bacteria, yeasts, molds and algae in their cytoplasm. The active site of enzyme is polar in nature which is responsible for acid–base catalysis. The enzyme is folded into (β/α)8 barrel and its substrate specificity is through C-terminal region. XR is an emerging industrially important food enzyme due to its major application in production of xylitol. Xylitol has shown remarkable applications in other industrial sectors such as food, biofuels, pharmaceutical, medical, odontological, textiles and cosmetics. This enzyme has been purified and characterized to understand its physico-chemical properties and stability. Further, robust microbial strains with tolerance towards high substrate concentration and toxic compounds are required to be developed for successful exploitation of XR at industrial level using agrowaste materials.

Hydrolysis pattern analysis of xylem tissues of woody plants pretreated with hydrogen peroxide and acetic acid: rapid saccharification of softwood for economical bioconversion

BackgroundWoody plants with high glucose content are alternative bioresources for the production of biofuels and biochemicals. Various pretreatment methods may be used to reduce the effects of retardation factors such as lignin interference and cellulose structural recalcitrance on the degradation of the lignocellulose material of woody plants.ResultsA hydrogen peroxide-acetic acid (HPAC) pretreatment was used to reduce the lignin content of several types of woody plants, and the effect of the cellulose structural recalcitrance on the enzymatic hydrolysis was analyzed. The cellulose structural recalcitrance and the degradation patterns of the wood fibers in the xylem tissues of Quercus acutissima (hardwood) resulted in greater retardation in the enzymatic saccharification than those in the tracheids of Pinus densiflora (softwood). In addition to the HPAC pretreatment, the application of supplementary enzymes (7.5 FPU cellulase for 24 h) further increased the hydrolysis rate of P. densiflora from 61.42 to 91.94% whereas the same effect was not observed for Q. acutissima. It was also observed that endoxylanase synergism significantly affected the hydrolysis of P. densiflora. However, this synergistic effect was lower for other supplementary enzymes. The maximum concentration of the reducing sugars produced from 10% softwood was 89.17 g L−1 after 36 h of hydrolysis with 15 FPU cellulase and other supplementary enzymes. Approximately 80 mg mL−1 of reducing sugars was produced with the addition of 7.5 FPU cellulase and other supplementary enzymes after 36 h, achieving rapid saccharification.ConclusionHPAC pretreatment removed the interference of lignin, reduced structural recalcitrance of cellulose in the P. densiflora, and enabled rapid saccharification of the woody plants including a high concentration of insoluble substrates with only low amounts of cellulase. HPAC pretreatment may be a viable alternative for the cost-efficient production of biofuels or biochemicals from softwood plant tissues.

Immobilization of Thermostable β-Glucosidase and α-l-Rhamnosidase from Dictyoglomus thermophilum DSM3960 and Their Cooperated Biotransformation of Total Flavonoids Extract from Epimedium into Icaritin

The thermostable GH3 family β-glucosidase DthBgl3 and thermostable GH78 family α-l-rhamnosidase DthRha from Dictyoglomus thermophilum DSM3960 were successfully immobilized by industrial amino resin 1000NH. The optimal reaction temperature and pH of 1000NH-DthBgl3 was 85 °C and pH 5.0. Over 65% residual enzyme activity maintained for 1000NH-DthBgl3 after a 3-h incubation under 85 °C, while about 20% residual enzyme activity maintained for free DthBgl3. The optimal reaction temperature and pH of 1000NH-DthRha was 95 °C and pH 6.5. Over 90% residual enzyme activity maintained for 1000NH-DthRha after a 3-h incubation under 90 °C, while about 22% residual enzyme activity maintained for free DthRha. Meanwhile, immobilized 1000NH-DthBgl3 and 1000NH-DthRha were successfully and could completely transform all major ingredient of 10 g/L total flavonoids extract from Epimedium (TFEE) into icaritin. After 15 cycles (45 h) of repeated use at 85 °C, 3 U 1000NH-DthBgl3 showed a molar conversion rate of 73.12%, initial activity of 30.09% and productivity of 124 mg/L/h, while 15 U 1000NH-DthRha showed a molar conversion rate of 88.50%, initial activity of 85.31% and productivity of 75 mg/L/h after ten cycles (60 h) of repeated use at 85 °C. The cooperation of two immobilized enzymes showed a molar conversion rate of 87.21% and productivity of 141 mg/L/h after 15 cycles of repeated use at 85 °C. This is the first report on immobilization of two thermostable glycosidases from thermophilic bacteria and their cooperated biocatalysis of all major ingredients of TFEE into icaritin with high productivity under a high substrate concentration.

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H2/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H2/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.

Biocatalysis for Rare Ginsenoside Rh2 Production in High Level with Co-Immobilized UDP-Glycosyltransferase Bs-YjiC Mutant and Sucrose Synthase AtSuSy

Rare ginsenoside Rh2 exhibits diverse pharmacological effects. UDP-glycosyltransferase (UGT) catalyzed glycosylation of protopanaxadiol (PPD) has been of growing interest in recent years. UDP-glycosyltransferase Bs-YjiC coupling sucrose synthase in one-pot reaction was successfully applied to ginsenoside biosynthesis with UDP-glucose regeneration from sucrose and UDP, which formed a green and sustainable approach. In this study, the his-tagged UDP-glycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports. The affinity adsorption significantly improved the capacity of specific binding of the two recombinant enzymes, and the dual enzyme covalently cross-linked by the acetaldehyde groups significantly promoted the binding stability of the immobilized bienzyme, allowing higher substrate concentration by easing substrate inhibition for the coupled reaction. The dual enzyme amount used for ginsenoside Rh2 biosynthesis is Bs-YjiC-M315F: AtSuSy = 18 mU/mL: 25.2 mU/mL, a yield of 79.2% was achieved. The coimmobilized M315F/AtSuSy had good operational stability of repetitive usage for 10 cycles, and the yield of ginsenoside Rh2 was kept between 77.6% and 81.3%. The high titer of the ginsenoside Rh2 cumulatively reached up to 16.6 mM (10.3 g/L) using fed-batch technology, and the final yield was 83.2%. This study has established a green and sustainable approach for the production of ginsenoside Rh2 in a high level of titer, which provides promising candidates for natural drug research and development.

Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential.

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis–Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

Efficient Biosynthesis of Vanillin from Isoeugenol by Recombinant Isoeugenol Monooxygenase from Pseudomonas nitroreducens Jin1

Currently, the biotechnological preparation of fragrances using natural materials attracted growing attention. Enzymatic synthesis of vanillin from isoeugenol by recombinant isoeugenol monooxygenase from Pseudomonas nitroreducens Jin1 was systematically investigated herein. With series of work on the construction of recombinant E. coli over-expressing isoeugenol monooxygenase, optimization of the culture conditions for enzyme production and reaction process for converting isoeugenol into vanillin, an increase of 22-fold in the enzyme activity (2050 U/L) was obtained, and the conversion was significantly increased at high substrate concentration with the aid of magnetic chitosan membrane for product isolation in situ. Under optimal conditions, the product concentration and space-time yield reached 252 mM and 115 g/L/d, respectively, and vanillin was obtained in 82.3% yield and > 99% purity in the gram preparative scale. The developed bioprocess showed application potential for efficient preparation of vanillin from inexpensive natural resources.

Phase II stanozolol metabolism study using the zebrafish water tank (ZWT) model

Stanozolol (STAN) is an androgen anabolic steroid often misused in sports competitions and prohibited at all times by the World Anti-Doping Agency (WADA). It can be long term detected by the analysis of human urine for traces of intact glucuronide metabolites. The Zebrafish Water Tank (ZWT) experimental setup can produce phase I STAN metabolites. In the present study, we investigated the in vivo phase II metabolism of STAN through the ZWT model to determine whether the ZWT produces metabolites relevant for doping control. We added STAN to a 200 mL recipient containing eight fish at 32 ± 1 °C. We analyzed the noninvasive samples (recipient water) both with and without pretreatment using Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in positive ionization mode. Our data show that four hydroxylated-sulfate and four hydroxylated-glycoconjugate metabolites were formed, two of the last ones being 3’OH-STAN-Glucuronide and 16β-OH-STAN-Glucuronide. Additionally, two STAN-Glucuronide derivatives were produced: one was confirmed to be 17epi-STAN-N-Glucuronide, and the other was presumed to be STAN-O-Glucuronide. After eight hours of the experiment, STAN-O-Glucuronide was the most intense phase II metabolite produced. The accumulation curves suggest that high concentrations of fish and substrate in water are required to form phase II metabolites.

Endoscopic Carbon Dioxide Insufflation into the Pancreatic Duct: is it Possible to Manage the Acute Pancreatitis by Decreasing Local pH to Suppress Enzymatic Self-Digestion?...161

Background: the main clinical symptoms of the acute pancreatitis (AP) used to be explained by a local and a systemic action of activated pancreatic enzymes. Methods. To evaluate a conceptual possibility of the management of AP, the self-digestive enzyme suppression by the local рН lowering via the endoscopic carbon dioxide insufflation into the pancreatic duct has been simulated numerically. For the main patterns of a duct system structure, the СО2 diffusion, рН distributions, kinetic constants of trypsin and lipase have been calculated. Results. The СО2 diffusion zone volume-averaged рН has been revealed to vary within 7,16-7,44. For such a decrease, the lipase activity been assessed as nearly intact, but the trypsin proteolysis - as suppressed per 28-46%, and per 12-24% of its initial level for a low and a high substrate concentration, respectively. The diagnostic insufflation of 10 ml of CO2 within the time of 10 s been estimated to expand the near duct diffusion front for only 0,84-1,04 mm, which is not enough for embracing the entire wall of the main pancreatic duct to provide its anti-trypsin protection. To get it, the insufflation should be prolonged up to ~1 min, providing ~2,3 mm diffusion zone depth. Subsequent distribution of CO2 within the gland volume has been shown to furnish the volume-averaged рН value for ~ 7,69-7,95, and to inhibit the proteolysis per 4-15% for a low, and per 0-5% for a high substrate concentration. As predicted, the diffusion zone spread up to the gland walls would require the prolongation up to ~ 11-73 min depending on a specific surface area of the duct system. Conclusion. A possibility of the pancreatic enzyme suppression by endoscopic CO2 insufflation into the pancreatic duct for AP management has been numerically assessed and conceptually confirmed.

Enzymatic Synthesis of Glucose‐ and Xylose Laurate Esters Using Different Acyl Donors, Higher Substrate Concentrations, and Membrane Assisted Solvent Recovery

AbstractGlucose‐ and xylose laurate esters are enzymatically synthesized using equimolar substrate concentrations in 2‐methyl‐2‐butanol, comparing free lauric acid with methyl‐ and vinyl‐laurate as acyl donors. All reactions result in ≥70% acyl donor conversions after 72 h but the activated donors are also partially hydrolyzed to lauric acid, highlighting the difficulty in controlling water presence in this particular reaction system. The esterification of xylose generates a complex product profile, with several regioisomers of monoesters and diesters. The esterification of glucose is quite selective, forming mainly the 6‐O monoester (≥96%) with a small presence of two diester isomers (4%). Increasing substrate concentration up to 800 millimoles kg−1 results in lower conversion values (down to 58%) but shows that the reaction proceeds successfully even in the presence of high amounts of insoluble glucose. However, the reaction is less selective and the proportion of diester increases, becoming up to 46% (molar fraction) of the final product. Solvent recovery after esterification can be achieved by organic solvent nanofiltration through a polymeric membrane able to retain ≥80% of all reaction substrates and products.Practical Applications: The use of high substrate concentrations during the enzymatic synthesis of sugar ester biosurfactants leads to product titers that are more industrially appealing, without the need to find a solvent that can solubilize all initial substrate. The sustainability of the enzymatic conversion at mild temperatures can be enhanced by recycling of the reaction solvent through organic solvent nanofiltration, an energy efficient alternative to other traditional methods like distillation.

Increased substrate availability reveals the potential of scentless lisianthus flowers in producing fragrant benzenoid-phenylpropanoids.

Lisianthus (Eustoma grandiflorum), a leading plant in the cut flower industry, is scentless. Here we show that lisianthus flowers have potential to produce several fragrant benzenoid-phenylpropanoids when substrate availability is not limited. To enable hyperaccumulation of substrates for the production of volatile benzenoid-phenylpropanoids, lisianthus commercial hybrid "Excalibur Pink" was transformed via floral dipping with a feedback-insensitive Escherichia coli DAHP synthase (AroG*) and Clarkia breweri benzyl alcohol acetyltransferase (BEAT), under constitutive promoters. The T1 progeny of "Excalibur Pink" plants segregated into four visual phenotypes, with pink or white colored petals and multiple or single petal layers. Interestingly, transformation with AroG* and BEAT caused no significant effect in the pigment composition among phenotypes, but did increase the levels of down-stream fragrant volatile benzenoids. All the transgenic lines exclusively accumulated methyl benzoate, a fragrant benzenoid, either in their petals or leaves. Furthermore, feeding with benzyl alcohol resulted in the accumulation of two novel benzenoids, benzyl acetate (the product of BEAT) and benzoate, as well as a dramatic increase in the concentrations of additional benzenoid-phenylpropanoid volatiles. Presumably, the degree of benzaldehyde overproduction after benzyl alcohol feeding in both leaves and flowers revealed their reverse conversion in lisianthus plants. These findings demonstrate the concealed capability of lisianthus plants to produce a wide array of fragrant benzenoid-phenylpropanoids, given high substrate concentrations, which could in turn open opportunities for future scent engineering.

High-Yield Biosynthesis of Glucosylglycerol through Coupling Phosphorolysis and Transglycosylation Reactions.

Glucosylglycerol is a powerful osmolyte that has attracted attention as a useful moisturizing ingredient in the cosmetic industry. This study demonstrates two artificially designed synthetic routes for manufacturing glucosylglycerol by combining phosphorolysis and transglycosylation reactions. The overall Gibbs energy change of the synthetic routes was negative, indicating that they are thermodynamically favorable. In vitro biosystems were constructed through combining the phosphorolysis ability of sucrose/maltose phosphorylase and the transglycosylation capacity of glucosylglycerol phosphorylases from different organisms. A near-stoichiometric conversion of sucrose and glycerol with a high product yield of 98% was achieved under optimal reaction conditions. The large-scale glucosylglycerol production of this biosystem was investigated under a high concentration of substrates (2 mol/L sucrose and 2.4 mol/L glycerol), and the titer reached 1.78 mol/L (452 g/L) with a productivity of 24.3 g/L/h. To the best of our knowledge, this value presented the highest glucosylglycerol production level until now, which indicated a great industrial application potential for glucosylglycerol manufacturing.

Understanding the interactive influence of hydrolytic conditions on biocatalytic production of fructooligosaccharides from inulin

Statistical optimization of hydrolytic conditions for the production of fructooligosaccharides (FOSs) from pure inulin using Aspergillus tritici endoinulinase was carried out in a batch system. FOSs yield 99.19% was obtained under the optimized hydrolytic conditions i.e. inulin concentration (7.3%), enzyme load (65 IU), hydrolysis time (13 h) and agitation (100 rpm). The closeness of value of co-efficient of determination (R2) to 1, good agreement between model's predicted and experimental values, low percentage error (<5%), high adequate precision (>4%) and F value (11,634.32), and low Lack of fit (0.60) of the designed model authenticates its fitness. High substrate concentration, low enzyme load and short hydrolysis span justifies efficiency of developed process for the preparation of FOSs from inulin using fungal endoinulinase. TLC chromatographic and densitometry studies confirmed the synthesis of short-chain length FOSs. FOSs preparation contained 33.85% GF2 (ketose), 24.50% GF3 (nystose), 7.26% GF4 (fructofuranosylnystose) and 33.58% FOSs of DP5–9.

Caffeic acid modulates methane production and rumen fermentation in an opposite way with high-forage or high-concentrate substrate in vitro.

Plant secondary metabolites, including tannins, saponins and phenolic acids, possess potential methane (CH4 ) inhibition bioactivity. Caffeic acid (CA), as one of the typical phenolic acids, serves as a promising rumen CH4 inhibitor, but the underlying mechanisms and investigations with typical formulated rations are still not well documented. Therefore, a batch culture study was conducted to investigate the effects of CA on methanogenesis, rumen fermentation and growth of ruminal microorganisms when high-forage or high-concentrate substrates are fermented. After 48 h incubations, adding CA up to 40 g kg-1 dry matter linearly reduced (P < 0.05) the disappearance of dry matter, neutral detergent fiber (NDFD), total gas, methanogenesis, total volatile fatty acid and 16S rDNA copy numbers of Ruminococcus albus and Butyrivibrio fibrisolvens, and increased 16S rDNA copy numbers of methanogens for the high-forage treatment. For the high-concentrate treatment, CA exerted opposite effects (P < 0.05) on the above variables, except that CA did not affect (P > 0.05)16S rDNA copy numbers of methanogens or R. albus. Caffeic acid inhibited in vitro methanogenesis and rumen fermentation with high-forage substrate incubation. Contrarily, CA benefited in vitro fermentation and enhanced methanogenesis with high-concentrate substrate incubation. It suggests that CA modulates methanogenesis and rumen fermentation mainly by affecting the growth of cellulolytic bacteria in vitro. © 2020 Society of Chemical Industry.