High Esterase Research Articles

Cloning, Expression, Purification and Enzymatic Characterization of Low-temperature Cholesterol Esterase from Marine Panthenia Agglutinosa

BACKGROUND: The low-temperature cholesterol esterase is primarily used in industries such as papermaking and healthcare. OBJECTIVE: To discover a microorganism with high cholesterol esterase activity and tolerance to low temperatures, leading to the promotion of the sustainable utilization of marine cold-adapted microbial resources and fostering industrial development. MATERIALS AND METHODS: This study isolated a strain producing low-temperature cholesterol esterase from marine samples in the China Bohai Sea. The strain was identified through 16S rDNA sequencing and named Panthenia agglutinosa Y03. The cholesterol esterase gene (PaChe) from P. agglutinosa Y03 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme PaChe was purified and characterized. The structure of PaChe was predicted using AlphaFold2, and molecular docking was performed with cholesterol linoleate as the ligand. RESULTS: The enzyme protein has a molecular weight of 56.35 KDa, a theoretical pI of 7.24, lacks a signal peptide, and exhibits structural features of the α/β hydrolase superfamily protein. The concentration of the purified PaChe is 0.5 mg/mL, with a specific activity of 42.7 U/mg. The optimal working temperature is 30 °C, and the enzyme retains activity at 4 °C , demonstrating weaker thermal stability. The optimal pH is 7, and the enzyme maintains over 70% activity at pH 9. Na +, Ca 2+ and Mg 2+ are the primary activators, while Ba 2+, Fe 2+, Mn 2+, Cu 2+ and chemical agents such as SDS as inhibitors, with Cu2+ exhibiting particularly significant inhibitory effects. CONCLUSION: This study establishes the theoretical groundwork for the development and utilization of a novel lowtemperature cholesterol esterase.

Screening and evaluation of high stress tolerance, high esterase activity and safety of Oenococcus oeni strains adapt to challenging conditions in Northwest China wine

Screening and evaluation of high stress tolerance, high esterase activity and safety of Oenococcus oeni strains adapt to challenging conditions in Northwest China wine

Microplastics Biofragmentation and Degradation Kinetics in the Plastivore Insect Tenebrio molitor.

The insect Tenebrio molitor possesses an exceptional capacity for ultrafast plastic biodegradation within 1 day of gut retention, but the kinetics remains unknown. Herein, we investigated the biofragmentation and degradation kinetics of different microplastics (MPs), i.e., polyethylene (PE), poly(vinyl chloride) (PVC), and poly(lactic acid) (PLA), in T. molitor larvae. The intestinal reactions contributing to the in vivo MPs biodegradation were concurrently examined by utilizing aggregated-induced emission (AIE) probes. Our findings revealed that the intestinal biofragmentation rates essentially followed the order of PLA > PE > PVC. Notably, all MPs displayed retention effects in the intestine, with PVC requiring the longest duration for complete removal/digestion. The dynamic rate constant of degradable MPs (0.2108 h-1 for PLA) was significantly higher than that of persistent MPs (0.0675 and 0.0501 h-1 for PE and PVC, respectively) during the digestive gut retention. Surprisingly,T. molitor larvae instinctively modulated their internal digestive environment in response to in vivo biodegradation of various MP polymers. Esterase activity and intestinal acidification both significantly increased following MPs ingestion. The highest esterase and acidification levels were observed in the PLA-fed and PVC-fed larvae, respectively. High digestive esterase activity and relatively low acidification levels inT. molitor larvae may, to some extent, contribute to more efficient MPs removal within the plastic-degrading insect. This work provided important understanding of MPs biofragmentation and intestinal responses to in vivo MPs biodegradation in plastic-degrading insects.

Inoculation of Zaopei with biofortification: Inducing variation in community structure and improving flavor compounds in Nongxiangxing baijiu.

The deterioration of the quality of raw liquor caused by the low content of ethyl hexanoate in Nongxiangxing baijiu has become a pervasive problem in the baijiu industry. Therefore, this study attempted to increase the synthesis of ethyl hexanoate by microorganisms with high esterase activity to increase Zaopei fermentation. The results showed that biofortification was a feasible and important way to improve the quality of the raw liquor and increase the ethyl hexanoate content. Adding Bacillus subtilis, Staphylococcus epidermidis, and Millerozyma farinosa for biofortified fermentation disturbed the microbial community structure of Zaopei and increased the abundance of Wickerhamomyces, Saccharomyces, and Thermoascus. The contents of ethyl hexanoate, ethyl valerate, ethyl caprylate, and ethyl heptanoate also increased noticeably in baijiu. The results of E-nose and sensory analysis tested and verified that the baijiu in the fortified group had better flavor characteristics.

Flow cytometric assessments of metabolic activity in bacterial assemblages provide insight into ecosystem condition along the Buffalo National River, Arkansas

The Buffalo National River (BNR), on karst terrain in Arkansas, is considered an extraordinary water resource. Water collected in Spring 2017 along BNR was metagenomically analyzed using 16S rDNA, and for 17 months (5/2017–11/2018), bacterial responses were measured in relation to nutrients sampled along a stretch of BNR near a concentrated animal feed operation (CAFO) on Big Creek. Because cell count and esterase activity can increase proportionally with organic enrichment, they were hypothesized to be elevated near the CAFO. Counts (colony forming units; CFUs) were different among sites for 73 % of the months; Big Creek generated highest CFUs 27 % of the time, with the closest downstream site at 13.3 %. Esterase activity was different among sites 94 % of the time, with Big Creek exhibiting lowest activity 71 % of the time. Over the months, activity was similar across sites at ~70 % active, except at Big Creek (56 %). The α-diversity of BNR microbial consortia near a wastewater treatment plant (WWTP) and the CAFO was related to distance from the WWTP and CAFO. The inverse relationship between high CFUs and low esterase activity at Big Creek (r = −0.71) actuated in vitro exposures of bacteria to organic wastewater contaminants (OWC) previously identified in the watershed. Exponential-phase Escherichia coli (stock strain), Streptococcus suis (avirulent, from swine), and S. dysgalactiae (virulent, from silver carp, Hypophthalmichthys molitrix) were incubated with atrazine, pharmaceuticals (17 α-ethynylestradiol and trenbolone), and antimicrobials (tylosin and butylparaben). Bacteria were differentially responsive. Activity varied with exposure time and OWC type, but not concentration; atrazine decreased it most. Taken together - the metagenomic taxonomic similarities along BNR, slightly higher bacterial growth and lower bacterial esterase at the CAFO, and the lab exposures of bacterial strains showing that OWC altered metabolism - the results indicated that bioactive OWC entering the watershed can strongly influence microbial processes in the aquatic ecosystem.

White grape variety Maraština as a promising source of non-Saccharomyces yeasts intended as starter cultures

Non-Saccharomyces wine yeasts are recognized for their ability to enhance the aroma complexity of wines. New trends in winemaking have highlighted the concept of microbiological terroir thus encouraging the use of indigenous non-Saccharomyces starter cultures in mixed fermentations with Saccharomyces cerevisiae to overcome the lack of authenticity and provide the wines with unique regional character and distinct aromatic profiles. In this study, 257 indigenous non-Saccharomyces isolates were obtained for the first time from 11 Maraština vineyards located within the winegrowing region of Dalmatia (Croatia). The isolates were identified at the molecular level and characterized for desirable oenological traits such as growth at different temperatures, high ethanol and SO2 tolerance, and for negative features such as high hydrogen sulphide (H2S) and acetic acid production as well as hydroxycinnamic acid decarboxylase (HCDC) activity. Among 27 identified species, those from Aureobasidium genera prevailed, followed by Metschnikowia, Hanseniaspora and Lachancea. The representatives from different species showed high SO2 (up to 250 mg/L) and ethanol (up to 12%) tolerance, low acetic acid, and medium-low H2S production. The HCDC activity was detected exclusively among Metschnikowia, Aureobasidium and Kabatiella species. Some regionally distinct characteristics within the isolates from the same species were observed. The encouraging insights into potential new starter cultures emerged after the evaluation of enzymatic activities of selected isolates. Most of them showed high leucine and valine arylamidase activity, whereas Hanseniaspora and Metschnikowia isolates were additionally characterized by high β-glucosidase and esterase activity, respectively.

Probiotic Properties of Pediococcus pentosaceus JBCC 106 and Its Lactic Acid Fermentation on Broccoli Juice.

To understand the biological roles of Pediococcus pentosaceus strains as probiotics isolated from the traditional Korean fermented food, Jangajji, Pediococcus pentosaceus was selected based on its high cinnamoyl esterase (CE) and antioxidant activities. The acid and bile stability, intestinal adhesion, antagonistic activity against human pathogens, cholesterol-lowering effects, and immune system stimulation without inflammatory effects were evaluated. Nitric oxide (NO) levels were measured in co-culture with various bacterial stimulants. Fermentation ability was measured by using a broccoli matrix and the sulforaphane levels were measured. Resistance to acidic and bilious conditions and 8% adherence to Caco-2 cells were observed. Cholesterol levels were lowered by 51% by assimilation. Moreover, these strains exhibited immunomodulatory properties with induction of macrophage TNF-α and IL-6 and had microstatic effects on various pathogens. Co-culture with various bacterial stimulants resulted in increased NO production. Fermentation activity was increased with the strains, and higher sulforaphane levels were observed. Therefore, in the future, the applicability of the selected strain to broccoli matrix-based fermented functional foods should be confirmed.

Enzymatic extraction of ferulic acid from brewer’s spent grain: Effect of physical pretreatments and optimization using design of experiments

Brewers' grains (BSG), the main waste product of the beer industry, are widely available and rich in fiber and bioactive compounds. Ferulic acid (FA) is the main phenolic compound in BSG, it has antioxidant and antibacterial properties that make it a valuable product for cosmetic, food and pharmaceutical industries. Alkaline hydrolysis is the common technology for extracting FA from biomass, this technology needs to be substituted as it is non-selective and requires harsh conditions making purification more complicated and expensive. This research work presents enzymatic hydrolysis as a green alternative for FA extraction. Five commercial enzymes were screened and compared for their hydrolytic efficiency to release FA. Prior to the enzymatic hydrolysis, autoclave was selected as the most effective pretreatment. Depol 740L was selected as the desired enzyme based on its high feruloyl esterase activity (0.4 U/mL). The enzymatic extraction parameters (pH, temperature and enzyme dosage) were optimized using response surface methodology. The highest FA recovery, 1.06 ± 0.01 mg/g dry weight (43.13% in comparison with the alkaline hydrolysis), could be achieved at pH 5.27; temperature 60 °C and 1.72% enzyme. The optimal extraction conditions were found to be sufficient within 22 h, as determined by a kinetic study.

Pseudomonas forestsoilum sp. nov. and P. tohonis biocontrol bacterial wilt by quenching 3-hydroxypalmitic acid methyl ester.

Bacterial wilt caused by Ralstonia solanacearum ranks the second top important bacterial plant disease worldwide. It is also the most important bacterial disease threatening the healthy development of Casuarina equisetifolia protection forest. 3-hydroxypalmitic acid methyl ester (3-OH PAME) functions as an important quorum sensing (QS) signal regulating the expression of virulence genes in R. solanacearum, and has been regarded as an ideal target for disease prevention and control. To screen native microorganisms capable of degrading 3-OH PAME, samples of C. equisetifolia branches and forest soil were collected and cultured in the medium containing 3-OH PAME as the sole carbon source. Bacteria with over 85% degradation rates of 3-OH PAME after 7-day incubation were further separated and purified. As a result, strain Q1-7 isolated from forest soil and strain Q4-3 isolated from C. equisetifolia branches were obtained and identified as Pseudomonas novel species Pseudomonas forestsoilum sp. nov. and P. tohonis, respectively, according to whole genome sequencing results. The degradation efficiencies of 3-OH PAME of strains Q1-7 and Q4-3 were 95.80% and 100.00% at 48 h, respectively. Both strains showed high esterase activities and inhibited R. solanacearum exopolysaccharide (EPS) and cellulase production. Application of strains Q1-7 and Q4-3 effectively protects C. equisetifolia, peanut and tomato plants from infection by R. solanacearum. Findings in this study provide potential resources for the prevention and control of bacterial wilt caused by R. solanacearum, as well as valuable materials for the identification of downstream quenching genes and the research and development of quenching enzymes for disease control.

Radiation-Chemical Methods of Hydrogel Dressing Structure Modification

The objects of the study are polymeric composites synthesized by mixing chemically and structurally complementary natural (Agar-Agar [AA], Chitosan [CS]) and Synthetic (Poly-N-Vinylpyrrolidone [PVP]) macromolecules in the presence of low molecular weight substrates (Polyethylene Glycol [PEG-400], Glycerol [GL]) as plasticizer to regulate the surface properties and volume characteristics of hydro-gel materials with functionally specified operational parameters, such as high adhesive affinity to damaged tissues, biological compatibility with body and organ fluids, high esterase and antimicrobial activity

Plastisphere and microorganisms involved in polyurethane biodegradation

Rapid accumulation of end-of-life polyurethanes (PUR) in the environment is a global crisis. While biodegradation of PUR has been reported, the process is slow, and the microbiology involved in PUR biodegradation is poorly understood. This study reported the microbial community involved in PUR biodegradation (designed as PUR-plastisphere) in estuary sediments, and isolation and characterization of two PUR-utilizing isolates. PUR foams were pretreated with oxygen plasma (referred as p-PUR foams) to mimic weathered conditions before embedded in microcosms containing estuary sediments. After 6 months of incubation, a substantial loss of ester/urethane bonds on the embedded p-PUR foams was observed, according to Fourier transform infrared (FTIR) spectroscopy. Analysis of PUR-plastisphere showed two dominant genera, Pseudomonas (2.7 %) and Hyphomicrobium (3.0 %), along with many unknown genera in Sphingomonadaceae (9.2 %), and predicted hydrolytic enzymes such as esterases and proteases. Purpureocillium sp., and Pseudomonas strain PHC1 (designated as strain PHC1 hereafter), isolated from the PUR plastisphere, can grow on Impranil (a commercial water-borne PUR) as a sole nitrogen or carbon source. High esterase activities were detected in the spent Impranil-containing media, and a significant loss of ester bonds of the spent Impranil was also observed. After 42 days of incubation, the strain PHC1-inoculated p-PUR foam showed a noticeable development of biofilm as observed via scanning electron microscopy (SEM), and disappearance of ester and urethane bonds of the PUR as detected by FTIR, supporting the role of strain PHC1 in biodegradation of the p-PUR foam. Also, the FTIR spectra observed for the sediment-embedded p-PUR foams was similar to those for the strain PHC1-inoculated p-PUR foams, suggesting the potential role of the dominant species of Pseudomonas in PUR-plastisphere. The results of this study showed the promise of rapid biodegradation of PUR foam through inoculating with a PUR-utilizing isolate, Pseudomonas strain PHC1.

Resistance to Pyrethroids in the Malaria Vector Anopheles albimanus in Two Important Villages in the Soconusco Region of Chiapas, Mexico, 2022.

Chiapas State comprises the largest malaria foci from Mexico, and 57% of the autochthonous cases in 2021, all with Plasmodium vivax infections, were reported in this State. Southern Chiapas is at constant risk of cases imported due to migratory human flow. Since chemical control of vector mosquitoes is the main entomological action implemented for the prevention and control of vector-borne diseases, this work aimed to investigate the susceptibility of Anopheles albimanus to insecticides. To this end, mosquitoes were collected in cattle in two villages in southern Chiapas in July-August 2022. Two methods were used to evaluate the susceptibility: the WHO tube bioassay and the CDC bottle bioassay. For the latter, diagnostic concentrations were calculated. The enzymatic resistance mechanisms were also analyzed. CDC diagnostic concentrations were obtained; 0.7 μg/mL deltamethrin, 12 μg/mL permethrin, 14.4 μg/mL malathion, and 2 μg/mL chlorpyrifos. Mosquitoes from Cosalapa and La Victoria were susceptible to organophosphates and to bendiocarb, but resistant to pyrethroids, with mortalities between 89% and 70% (WHO), and 88% and 78% (CDC), for deltamethrin and permethrin, respectively. High esterase levels are suggested as the resistance mechanism involved in the metabolism of pyrethroids in mosquitoes from both villages. Mosquitoes from La Victoria might also involve cytochrome P450. Therefore, organophosphates and carbamates are suggested to currently control An. albimanus. Its use might reduce the frequency of resistance genes to pyrethroids and vector abundance and may impede the transmission of malaria parasites.

Function and safety evaluation of Staphylococcus epidermidis with high esterase activity isolated from strong flavor Daqu

This study was aimed at evaluating the safety of Staphylococcus epidermidis YTPW-4 isolated from strong flavor Daqu and its beneficial functional characteristics in Baijiu-making. The content of ethyl caproate catalyzed by crude enzyme solution of Staphylococcus epidermidis YTPW-140 was 583.09 mg/100 mL, and the activity of esterase was 2.13 U/mL. Vancomycin, erythromycin, and gentam were sensitive to the strain, which was negative for the causative gene, hemolytic activity, and biofilm formation capacity. In addition, the strain was selected for laboratory-scale fermentation using HS-SPME-GCMS and statistical analysis to confirm its role in the production of flavor compounds. Propyl-3-methylbutyrate, hexanoic acid, and ethyl acetate had higher contents after fermentation than other flavor compounds, at 530.40 μg/L, 471.07 μg/L, and 297.33 μg/L, respectively. There was also a small amount of ethyl hexanoate in these products. Esterase could increase the levels of short-chain free fatty acids, which were precursors of flavor compounds. Most importantly, the isolate maintained viability under simulated pit conditions, and was resistant to pH 3.5 and 8% ethanol. These results indicated that the strain had the potential to be used as a starter to improve the quality of Nongxiangxing baijiu.

Biochemical Evaluation of Cotton Genotypes using Soluble Protein, Esterase (EST), Peroxidase (POX) And Polyphenol Oxidase (PPO) and their Role in Plant Disease Resistance

Isozyme analysis is a powerful biochemical technique that has numerous applications in plant biology. It has long been used by geneticists to study the population genetics. The isozyme esterase, peroxidase and polyphenol oxidase were standardized for upland cotton (Gossypium hirsutum L.) germplasm lines collected from all over the country. The knowledge of nature and magnitude of genetic diversity present in the germplasm is most important pre-requisite for the success of any breeding program. The thirty-four cotton germplasm lines were screened for prime three isozymes based on quantification assay and qualitative PAGE profiling. Among the material, the genotype AKH – 24 (190.60 mg ml-1), AKH – 053 (189.42 mg ml-1) and VIKAS (184.53 mg ml-1) recorded high protein content, whereas the enzymatic activities of esterase, peroxidase and polyphenol oxidase exhibited remarkable differences along with the protein content. The genotype LRA–5166 exhibited high esterase (462.68 mM mg protein-1 min-1) and peroxidase activity (250.97mM mg protein-1 min-1), while AKH – 24 recorded the maximum polyphenol oxidase activity (131.45 mM mg protein-1min-1). The banding pattern of biochemical markers revealed that the maximum number of bands were recorded in esterase analysis (fifteen) followed by protein (twelve) whereas, only five bands each were detected in peroxidase and polyphenol oxidase analysis indicating limited polymorphism. The Relative Mobility (Rm) values were ranged from 0.083 to 0.883 (protein), 0.100 to 0.971 (esterase), 0.033 to 0.283 (peroxidase) and 0.048 to 0.206 (polyphenol oxidase). The present study demonstrated that cotton genotypes could be differentiated by their quantity and quality through electrophoretic banding profiles. These results could be of practical value for cultivar identification, purity testing along with associated prediction of pest and disease resistance. However, the major constraint is that these biochemical markers do not able to reproduce the similar kind of variation pattern, but can provide strong distinguishing polymorphism each time.

Screening of Lactiplantibacillus plantarum with High Stress Tolerance and High Esterase Activity and Their Effect on Promoting Protein Metabolism and Flavor Formation in Suanzhayu, a Chinese Fermented Fish.

In this study, three Lactiplantibacillus plantarum, namely 3-14-LJ, M22, and MB1, with high acetate esterase activity, acid, salt, and high-temperature tolerance were selected from 708 strains isolated from fermented food. Then, L. plantarum strains MB1, M22, and 3-14-LJ were inoculated at 107 CFU/mL in the model and 107 CFU/g in actual Suanzhayu systems, and the effects during fermentation on the physicochemical properties, amino acid, and volatile substance were investigated. The results showed that the inoculated group had a faster pH decrease, lower protein content, higher TCA-soluble peptides, and total amino acid contents than the control group in both systems (p < 0.05). Inoculation was also found to increase the production of volatile compounds, particularly esters, improve the sour taste, and decrease the bitterness of the product (p < 0.05). L. plantarum M22 was more effective than the other two strains in stimulating the production of isoamyl acetate, ethyl hexanoate, and ethyl octanoate. However, differences were discovered between the strains as well as between the model and the actual systems. Overall, the isolated strains, particularly L. plantarum M22, have good fermentation characteristics and have the potential to become excellent Suanzhayu fermenters in the future.

Construction of esterase-responsive hyperbranched polyprodrug micelles and their antitumor activity in vitro

Abstract This research constructs an esterase-responsive hyperbranched polyprodrug nano pharmaceutical and investigates their antitumor activity. Polyprodrug micelle was prepared by one-pot method based on glutathione (GSH), doxorubicin (DOX), and polyethylene glycol (PEG) under the catalyst of N,N-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and 1-hydroxybenzotriazole (HOBt). The polyprodrug was characterized by nuclear magnetic resonance (NMR), Fourier transform infrared spectrometer (FT-IR), ultraviolet-visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), and transmission electron microscope (TEM), respectively. The antitumor activity of polyprodrug micelle was evaluated by Hela cell and the distributions of micelles in cells were observed by fluorescent microscope. The NMR and FT-IR confirmed that the DOX-GSH-PEG polyprodrug was successfully synthesized. The drug loading rate is 10.21% and particle size is 106.4 ± 1 nm with a narrowed polydispersity (PDI = 0.145). The DLS showed that the micelles were stable during 7 days at 25°C. The drug release results showed that the micelles could be esterase-responsive disrupted, and the drug release rate could reach 43% during 72 h. Cell uptake and cell viability demonstrated that the micelles could distribute to cell nuclei during 8 h and induce cell apoptosis during 48 h. Overall, these hyperbranched polyprodrug micelles prepared by one-pot method could be esterase-responsive disrupted and release the antitumor drugs in a high esterase environment for cancer therapy in vitro. These results confirm that DOX-GSH-PEG is an effective nanomedicine in vitro and the endogenous-based strategy with one-pot synthesis to construct esterase-responsive polyprodrug would probably be a preferred choice in the future.

Responsive oligochitosan nano-vesicles with ursodeoxycholic acid and exenatide for NAFLD synergistic therapy via SIRT1

To explore effective therapeutic strategy on nonalcoholic fatty liver disease (NAFLD), the amphiphilic oligochitosan derivative containing ursodeoxycholic acid (UDCA) was synthesized and named as UBC, which could self-assemble and encapsulate exenatide (Exe) to obtain Exe-UBC nano-vesicle. Exe-UBC could be uptaken by fatty-acid cultured cells and release UDCA and Exe responsive to the high esterase concentration. In vitro experiments demonstrated that Exe-UBC activated the expression level of SIRT1 with inhibited expression of PGC-1β and PPAR-γ and consequently exerted synergistic bioaction immediately on reducing lipidosis. After a month of Exe-UBC treated through intravenous injection, the body weight of high-fat diet feeding C57BL/6 mice recovered to ordinary level, and their lipid contents in the liver declined significantly. The recovery in hepatic function indexes like TG, AST, and ALT further revealed the superiority of Exe-UBC vesicles. These results suggested that the co-delivery of UDCA and Exe via Exe-UBC could be a potent platform for NAFLD treatment.

Dual functional roles of a novel bifunctional β-lactamase/esterase from Lactococcus garvieae

A novel bifunctional β-lactamase/esterase (LgLacI), which is capable of hydrolyzing β-lactam-containing antibiotics including ampicillin, oxacillin, and cefotaxime as well as synthesizing biodiesels, was cloned from Lactococcus garvieae. Unlike most bacterial esterases/lipases that have G-x-S-x-G motif, LgLacI, which contains S-x-x-K catalytic motif, has sequence similarities to bacterial family VIII esterase as well as β-lactamases. The catalytic properties of LgLacI were explored using a wide range of biochemical methods including spectroscopy, assays, structural modeling, mutagenesis, and chromatography. We confirmed the bifunctional property of LgLacI hydrolyzing both esters and β-lactam antibiotics. This study provides novel perspectives into a bifunctional enzyme from L. garvieae, which can degrade β-lactam antibiotics with high esterase activity.

Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations

PurposePolysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions.MethodsTwo polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity.ResultsThe addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions.ConclusionsThese results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

Screening and identifying microorganisms with feruloyl esterase activity in Chinese sesame-flavour baijiu fermentation materials (Jiupei)

Baijiu is a national beverage in China. Some functional compounds have been identified in baijiu, one of which is ferulic acid. One route for in situ ferulic acid synthesis is catalysed by feruloyl esterase from microorganisms using baijiu raw materials as substrates. However, few studies have focused on microorganisms obtained from baijiu that have feruloyl esterase activity. In this work, microorganisms with feruloyl esterase activity were screened in the fermented raw materials of baijiu. Four different microorganisms with feruloyl esterase activity were identified, and one strain, designated J1, showed high feruloyl esterase activity. The strain J1 belonged to the genus Acetobacter and showed the closest relationship with Acetobacter fabarum and Acetobacter lovaniensis. The feruloyl esterase production capacity of J1 was confirmed, and the fermentation conditions were optimised. The optimal enzyme production conditions for the highest feruloyl esterase activity required an initial fermentation pH of 7.0 at 30 °C, an inoculation volume of 10 %, and culturing for 24 h.