Semi-rational design for enhancing thermostability of Culex pipiens acetylcholinesterase and sensitivity analysis of acephate.

Acetylcholinesterase (AChE) has emerged as a significant biological recognition element in the biosensor field, particularly for the detection of insecticides. Nevertheless, the weak thermostability of AChE restricts its utilization due to the complexities associated with production, storage, and application environments. By evaluating the binding affinity between representative AChE and insecticides, an AChE from Culex pipiens was screened out, which displayed a broad-spectrum and high sensitivity to insecticides. The C. pipiens AChE (CpA) was subsequently expressed in Escherichia coli (E. coli) as a soluble active protein. Furthermore, a three-point mutant, M4 (A340P/D390E/S581P), was obtained using a semi-rational design strategy that combined molecular dynamics (MD) simulation and computer-aided design, which exhibited a four-fold increase in half-life at 40 °C compared to the wild-type (WT) enzyme. The mutant M4 also demonstrated an optimal temperature of 50 °C and a melting temperature (Tm) of 51.2 °C. Additionally, the sensitivity of WT and M4 to acephate was examined, revealing a 50-fold decrease in the IC50 value of M4. The mechanism underlying the improvement in thermal performance was elucidated through secondary structure analysis and MD simulations, indicating an increase in the proportion of protein helices and local structural rigidity. MD analysis of the protein-ligand complexes suggested that the enhanced sensitivity of M4 could be attributed to frequent specific contacts between the organophosphorus (OP) group of acephate and the key active site residue Ser327. These findings have expanded the possibilities for the development of more reliable and effective industrial enzyme preparations and biosensors.

Isolation and Identification of Indole Alkaloids from Aspergillus amstelodami BSX001 and Optimization of Ultrasound-Assisted Extraction of Neoechinulin A.

This study aimed to investigate the alkaloid secondary metabolites of Aspergillus amstelodami BSX001, a fungus isolated from Anhua dark tea, and to improve the extraction yield of the active ingredients by optimizing the extraction process. The structural characterization of the compounds was investigated using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The antioxidant activity of echinulin-related alkaloids was evaluated by determining the total reducing power and DPPH radical scavenging capacity. The extraction process of the compound with optimum activity was optimized by a single-factor test and response surface methodology (RSM) combined with Box-Behnken design (BBD). The optimized result was validated. Finally, a new alkaloid 8-hydroxyechinulin (1), and four known alkaloids, variecolorin G (2), echinulin (3), neoechinulin A (4), and eurocristatine (5), were isolated. Echinulin-related compounds 1, 3, and 4 possessed certain antioxidant activities, with IC50 values of 0.587 mg/mL, 1.628 mg/mL, and 0.219 mg/mL, respectively, against DPPH radicals. Their total reducing power at a concentration of 0.5 mg/mL was 0.29 mmol/L, 0.17 mmol/L, and 4.25 mmol/L. The extraction process of neoechinulin A was optimized with the optimum extraction parameters of 72.76% methanol volume fraction, 25 mL/g solid-liquid ratio, and 50.8 °C soaking temperature. Under these conditions, the extraction yield of neoechinulin A was up to 1.500 mg/g.

Surface-displayed phenolic acid decarboxylase for increased vinylphenolic pyranoanthocyanins in blueberry wine.

During the fruit wine production, phenolic acid decarboxylase (PAD) converts free hydroxycinnamic acid into 4-vinyl derivatives that can then react spontaneously with anthocyanins, generating more stable pyranoanthocyanins that are responsible for the color stability of fruit wine. Nevertheless, the low PAD activity in yeast under the winemaking conditions has largely limited the generation of 4-vinyl derivatives. To bridge this gap, we expressed PAD from Bacillus amyloliquefaciens in Pichia pastoris and surface-displayed it on Saccharomyces cerevisiae. As a result, S. cerevisiae surface-displayed PAD (SDPAD) exhibited an enhanced thermal stability and tolerance to acidic conditions. Fermentation experiments showed that SDPAD can significantly increase the content of vinylphenolic pyranoanthocyanins and thus maintain the color stability of blueberry wine. Our study demonstrated the feasibility of surface display technology for color stability enhancement during the production of blueberry wine, providing a new and effective solution to increase the content of vinylphenolic pyranoanthocyanins in the fruit-based wines.

Effect of in vitro simulated digestion on the physicochemical properties and pancreatic lipase inhibitory activity of fucoidan.

BACKGROUND: Fucoidan has an anti-obesity effect. However, there are few studies on its mechanism. In this study, we investigated the in vitro and in silico inhibitory properties of fucoidan against pancreatic lipase for the first time. We examined the changes in composition, structure, and pancreatic lipase inhibition of fucoidan during in vitro digestion. RESULTS: Simulated saliva-gastrointestinal digestion resulted in a slight decrease in the molecular weight of fucoidan but no significant changes in the monosaccharide composition, sulfate content, and functional groups. Moreover, the digestion process significantly increased the inhibition of pancreatic lipase by fucoidan. The study on the type of inhibition showed that the inhibition of pancreatic lipase by fucoidan belonged to mixed inhibition with competitive inhibition. Molecular docking analysis showed that fucoidan could bind to the active site of pancreatic lipase. CONCLUSION: This study indicates that fucoidan can be a potential functional food for anti-obesity. © 2024 Society of Chemical Industry.

Detection of insecticides by Tetronarce californica acetylcholinesterase via expression and in silico analysis.

The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica (TcA) in Escherichia coli (E. coli) as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. KEY POINTS: • T. californica AChE was expressed solubly in prokaryotic system. • The biochemical properties of free/immobilized enzyme were characterized. • The sensitivity of enzyme to insecticides was evaluated in vitro and in silico.

[Protopanaxadiol-type ginsenoside hydrolases and their application in the preparation of ginsenoside Compound K: a review].

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

High-Value Bioconversion of Ginseng Extracts in Betaine-Based Deep Eutectic Solvents for the Preparation of Deglycosylated Ginsenosides.

Deep eutectic solvents (DES), as a green alternative to traditional organic solvents in biocatalysis, not only activate proteins but even increase the efficiency of enzymatic reactions. Here, DES were used in a combinatorial enzyme-catalyzed system containing ß-glucosidase BGLAt and ß-galactosidase BGALAo to produce deglycosylated ginsenosides (De-g) from ginseng extracts (GE). The results showed that DES prepared with betaine and ethylene glycol (molar ratio, 1:2) could significantly stimulate the activity of the combinatorial enzymes as well as improve the acid resistance and temperature stability. The DES-based combinatorial enzyme-catalyzed system could convert 5 g of GE into 1.24 g of De-g (F1, F2, 20 (S)-PPT, and CK) at 24 h, which was 1.1 times that of the buffer sample. As confirmed by the spectral data, the changes in the conformations of the combinatorial enzymes were more favorable for the binding reaction with the substrates. Moreover, the constructed DES-based aqueous two-phase system enabled the recovery of substantial amounts of DES and De-g from the top phase. These results demonstrated that DES shows great application as a reaction solvent for the scale-up production of De-g and provide insights for the green extraction of natural products.

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K.

Ginsenoside compound K (CK) is an emerging functional food or pharmaceutical product. To date, there are still challenges to exploring effective catalytic enzymes for enzyme-catalyzed manufacturing processes and establishing enzyme-catalyzed processes. Herein, we identified three ginsenoside hydrolases BG07 (glucoamylase), BG19 (ß-glucosidase), and BG23 (ß-glucosidase) from Aspergillus tubingensis JE0609 by transcriptome analysis and peptide mass fingerprinting. Among them, BG23 was expressed in Komagataella phaffii with a high volumetric activity of 235.73 U mL-1 (pNPG). Enzymatic property studies have shown that BG23 is an acidic (pH adaptation range of 4.5-7.0) and mesophilic (thermostable < 50 °C) enzyme. Moreover, a one-pot combinatorial enzyme-catalyzed strategy based on BG23 and BGA35 (ß-galactosidase from Aspergillus oryzae) was established, with a high CK yield of 396.7 mg L-1 h-1. This study explored the ginsenoside hydrolases derived from A. tubingensis at the molecular level and provided a reference for the efficient production of CK.

Heterologous expression of GH5 chitosanase in Pichia pastoris and antioxidant biological activity of its chitooligosacchride hydrolysate.

Chitosanase was widely used in the production of bioactive chitooligosacchride (CHOS) due to their safety, controllability, environmental protection, and biodegradability. Studies showed that the bioactivity of CHOS is closely related to its degree of polymerization. Therefore, the production of ideal polymerized CHOS becomes our primary goal. In this study, the glycosyl hydrolase (GH) family 5 chitosanase was successfully expressed heterologously in Pichia pastoris. After 96 h of high-density fermentation, the chitosanase activity reached 90.62 U·mL-1, the protein content reached 9.76 mg·mL-1. When 2% chitosan was hydrolyzed by crude enzyme (20 U/mL), the hydrolysis rate reached 91.2% after 8 h, producing a mixture of CHOS with 2-4 desirable degrees of polymerization (DP). Then, the antioxidant activity of CHOS mixture was investigated, and the results showed that the antioxidant effect was concentration-dependent and had great application potential in the field of nutrition.

Biochar-bacteria-plant partnerships: Eco-solutions for tackling heavy metal pollution.

Over the past 30 years, the ever-rising demands of the modern and growing population have led to the rapid development of agricultural and industrial sectors worldwide. However, this expansion has exposed the environment to various pollutants including heavy metal (HM)s. Almost all HMs are serious toxicants and can pose serious health risks to living organisms in addition to their bioaccumulative and non-biodegradable nature. Different techniques have been developed to restore the ecological functions of the HM-contaminated soil (HMCS). However, the major downfalls of the commonly used remediation technologies are the generation of secondary wastes, high operating costs, and high energy consumption. Phytoremediation is a prominent approach that is more innocuous than the existing remediation approaches. Some microbes-plant interactions enhance the bioremediation process, with heavy metal resistant-plant growth promoting bacteria (HMRPGPB) being widely used to assist phytoremediation of HMs. However, the most common of all major microbial assisted-phytoremediation disturbances is that the HM-contaminated soil is generally deficient in nutrients and cannot sustain the rapid growth of the applied HMRPGPB. In this case, biochar has recently been approved as a potential carrier of microbial agents. The biochar-HMRPGPB-plant association could provide a promising green approach to remediate HM-polluted sites. Therefore, this review addresses the mechanisms through which biochar and HMRPGPB can enhance phytoremediation. This knowledge of biochar-HMRPGPB-plant interactions is significant with respect to sustainable management of the HM-polluted environment in terms of both ecology and economy, and it offers the possibility of further development of new green technologies.

Bioconversion of Flavonoid Glycosides from Hippophae rhamnoides Leaves into Flavonoid Aglycones by Eurotium amstelodami.

The flowering process has been reported to play crucial roles in improving the flavor and efficacy of fermented tea. Hippophae rhamnoides leaves containing many beneficial ingredients are a suitable plant source for tea processing. In this study, we isolated a ß-glucosidase-producing fungus Eurotium amstelodami BSX001 from the fermented tea and used Hippophae rhamnoides leaves (HRL) as a substrate to explore the detailed process of bioconversion of some important functional factors. The results show that the contents of total phenolic compounds and flavonoids increased significantly after seven days, especially flavonoid aglycones (e.g., quercetin, kaempferol, and isorhamnetin). Such compounds greatly enhance the antioxidative activity of fermented products. Metabolic analysis of the standard compounds (rutin, quercetin-3-glucoside, kaempferol-3-glucoside, quercetin, isorhamnetin-3-glucoside, isorhamnetin, and kaempferol) further confirmed the effective biotransformation by E. amstelodami. Mechanisms of the bioconversion could be involved in deglycosylation, dihydroxylation, and O-methylation. Our findings expand the understanding of tea fermentation process and provide further guidance for the fermented tea industry.

Production, structural characterization and gel forming property of a new exopolysaccharide produced by Agrobacterium HX1126 using glycerol or d-mannitol as substrate.

A strain Agrobacterium HX1126 was isolated from soil sample near the canal in Wuxi. Glycerol was used as carbon source for the production of a new exopolysaccharide which was named PGHX. PGHX composed mainly of galactose, with lower amounts of arabinose and aminogalactose. It was found that this strain could use d-mannitol as carbon source to produce PGHX too. A method for the preparation of crude PGHX was proposed and the crude PGHX can be formed in a gel formation when 30 g/L was put into the boiling water for 10 min, with an achieved gel strength of 957 g/cm(2). The concentration of proteins in the crude product was considered to be an important parameter which directly influence the gel forming property. The highest production of PGHX (24.9 g/L) was obtained under the nitrogen depletion condition. The structure of the product was confirmed by NMR and FTIR.

Isolation and characterization of curdlan produced by Agrobacterium HX1126 using α-lactose as substrate.

A strain Agrobacterium HX1126 was isolated from soil sample near the canal in Wuxi. α-lactose was used as the sole carbon source for the production of an exopolysaccharide which was named PLHX. The highest production of PLHX (21.4g/L) was obtained under nitrogen depletion. PLHX composed mainly of glucose, with lower amounts of galactose and aminogalactose. The structure of the product was confirmed by NMR and FTIR and was identified as curdlan. This exopolysaccharide formed a gel when 30g/L was put in boiling water for 10min, with an achieved gel strength of 831g/cm(2). Moreover, a hypothesis for higher gel strength production is proposed. The gel forming property makes this exopolysaccaride a good potential application in the food, pharmaceutical and cosmetic industries.

Biodegradation of acetochlor by a newly isolated Pseudomonas strain.

A novel microbial strain JD115 capable of degrading acetochlor was isolated from the sludge of acetochlor manufacture and was identified as Pseudomonas aeruginosa species. This strain was able to grow on acetochlor as the sole source of both carbon and nitrogen. The biodegradation of acetochlor by strain JD115 could be described either by the pseudo-first-order or by the second-order kinetics models, while the latter gave a better performance. The strain optimally degraded acetochlor at a pH value of 7.0 and a temperature of 37 °C. Additional nutriments could greatly enhance the degradation rate of acetochlor up to 95.4% in the presence of 50 mg acetochlor l(-1). The metabolite analyses by GC-MS presumed that catechol was an intermediate product of acetochlor, which was finally degraded for 5 days of incubation. This study highlights the potential use of this strain for the bioremediation of an acetochlor-polluted environment.

Acetone, butanol, and ethanol production from gelatinized cassava flour by a new isolates with high butanol tolerance.

To obtain native strains resistant to butanol toxicity, a new isolating method and serial enrichment was used in this study. With this effort, mutant strain SE36 was obtained, which could withstand 35g/L (compared to 20g/L of the wild-type strain) butanol challenge. Based on 16s rDNA comparison, the mutant strain was identified as Clostridium acetobutylicum. Under the optimized condition, the phase shift was smoothly triggered and fermentation performances were consequently enhanced. The maximum total solvent and butanol concentration were 23.6% and 24.3%, respectively higher than that of the wild-type strain. Furthermore, the correlation between butanol produced and the butanol tolerance was investigated, suggesting that enhancing butanol tolerance could improve butanol production. These results indicate that the simple but effective isolation method and acclimatization process are a promising technique for isolation and improvement of butanol tolerance and production.

Elimination of the formation of biofilm in industrial pipes using enzyme cleaning technique.

Currently, there is a growing demand in how to eliminate the biofilm formed in industrial pipelines, especially in food, fermentation, and water treatment industry. However, the traditional techniques for CIP (cleaning in place) are usually ineffective, superficial, halfway, and do not clean or sterilize microbes located in the inner layers of the biofilm. A recent strategy for removing the biofilm in pipes is employing enzymes to clean it in the circulating water system under an optimal condition. However, how to operate and control the whole cleaning process is difficult. Here, we will introduce the strategy of enzyme cleaning to make it more appropriated and effective.•A modification of CIP method is proposed for higher efficiency by using N-acetylmuramide glycanohydrolase as catalysts whose optimal pH and temperature is 10 ± 1 and 45 ± 2 °C, respectively.•The initial efficiency of enzyme cleaning was evaluated by testing the content of ATP in water sample using Clean-Trace™ (3M Corporation).•Lastly, the terminal water was tested with SLYM-BART™ (HACH Corporation) to find out whether there were biofilm-forming bacteria, such as Pseudomonas aeruginosa (Lakretz et al. (2011) [1]), Pseudomonas fluorescens (O'Toole and Kolter (1998) [2]), iron bacterium, etc.

Enhanced lycopene content in Blakeslea trispora by effective mutation-screening method.

The zygomycete fungus Blakeslea trispora is usually used as a natural source of lycopene and ß-carotene. In this study, the B. trispora (-) strain, a major mating type for lycopene production, was treated with N(+) ion implantation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid (CTA). After several rounds of screening, four mutants with higher yield of lycopene and biomass were isolated. Among these mutants, I5 obtained with N(+) ion implantation showed a maximum lycopene yield (28.8 mg/g), which was 64 % higher than the parent strain (17.5 mg/g) in the production of lycopene. The results indicated that N(+) ion implantation is more suitable for B. trispora (-) than NTG treatment, and the addition of lovastatin promoted the generation of positive mutant and CTA amplified the color differences between colonies.

A facile and efficient pretreatment of corncob for bioproduction of butanol.

The present study developed a combined ball milling-aqueous swelling (CBMAS) pretreatment to accelerate the hydrolysis of corncob. The enzymatic hydrolysis of microcrystalline cellulose carried out in the plates and flasks indicated that the response of enzymatic hydrolysis to CBMAS was quite evident. The fermentable reducing sugars of hydrolysates from CBMAS-pretreated corncob was 59.8 g/L, which was 1.3 and 1.7 folds higher than those from diluted acid and alkaline pretreated corncob hydrolysates, respectively. Simultaneous CBMAS pretreatment and enzymatic hydrolysis was also conducted, reducing the processing time from 66 h to 28 h. The enzymatic hydrolysates from CBMAS-pretreated corncob could be directly utilized as the substrate for butanol fermentation without detoxication. Under the optimal conditions, fermentable sugars in the corncob hydrolysate were completely consumed to generate 9.52 g/L butanol.

Acetone, butanol, and ethanol production from cane molasses using Clostridium beijerinckii mutant obtained by combined low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction.

In order to obtain mutant strains showing higher solvent tolerance and butanol production than those of wild-type strains, the butanol-producing strain Clostridium beijerinckii L175 was subjected to mutagenesis using a combined method of low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction. With this effort, mutant strain MUT3 was isolated. When it was used for butanol fermentation in P2 medium, the production of butanol was 15.8±0.7 g/L 46% higher than the wild-type strain. Furthermore, after optimization of butanol production from cane molasses with MUT3, the maximum butanol production of 14.9±0.5 g/L were obtained in crew-capped bottles. When ABE production by MUT3 was carried out in a bioreactor, the production of butanol and total solvent were 15.1±0.8 g/L and 22.1±0.9 g/L, respectively. The remarkable butanol production and solvent tolerance of MUT3 make it promising for butanol production from cane molasses.

Repetitive domestication to enhance butanol tolerance and production in Clostridium acetobutylicum through artificial simulation of bio-evolution.

To improve butanol tolerance and production in Clostridium acetobutylicum, a novel approach was developed in this study, which was called artificial simulation of bio-evolution (ASBE) based on the evolutionary dynamics and natural selection. Through repetitive evolutionary domestications, a butanol-tolerant strain C. acetobutylicum T64 was obtained, which could withstand 4% (v/v) (compared to 2% of the wild-type) butanol and was accompanied by the increase of butanol production from 12.2g/L to 15.3g/L using corn meal as substrate. Fermentation was also carried out to investigate the relationship between butanol tolerance and ABE production, suggesting that enhancing butanol tolerance could increase butanol production but unlikely improve total ABE production. These results also indicated that the ASBE would be an available and feasible method used in biotechnology for enhancement of butanol tolerance and production.