Preparative Biochemistry & Biotechnology最新文献

This work investigates the effects of two elicitors, yeast extract (YE) and methyl jasmonate (MeJA), on the growth, oleanolic acid (OA) biosynthesis, and biological activities (antioxidant and antibacterial) of Polyscias fruticosa suspension cells. The results showed that while both elicitors inhibited cell growth, with YE having a stronger inhibitory effect, they significantly increased both OA content and the overall biological activity of the cell extracts. The degree of growth inhibition was directly proportional to the elicitor concentration and inversely related to the time of addition. YE proved more effective than MeJA, leading to a higher OA accumulation (78.55 mg/flask) and superior antioxidant activity, as evidenced by a lower IC₅₀ (0.55 mg/mL) and a higher FRAP value (0.46 µmol Fe²⁺/g). Additionally, extracts from YE-treated cells showed stronger antibacterial activity against both Escherichia coli and Staphylococcus aureus compared to the MeJA-treated extracts. The study concludes that there is a tradeoff between cell growth and the biosynthesis of bioactive compounds. Optimizing both the type of elicitor and its application timing is crucial to balance biomass yield with the production of key compounds. YE proved to be a superior elicitor to MeJA in enhancing both OA content and the overall biological activity of the extracts, providing a scientific basis for the future production of highly potent bioactive compounds from P. fruticosa cell cultures.

Clitoria ternatea has a long history of traditional use in treating cardiovascular, neurological, and cancer-related ailments. To support its historical claims, this study aimed to compare the two varieties of C. ternatea whole flower, i.e., blue and white, to assess their chemical constituents for potential impact on medicinal properties. The major compounds detected in blue and white C. ternatea whole flower were trilinolein and ternatin A1, whereas in white flower: carda-4,20(22)-dienolide and trans-traumatic acid were detected by GC-MS. In blue variety, the calcium level was found to be 5.326 mg/Kg in ICP-OES. The levels of sodium and potassium were 4.78 and 12.62 weight % in EDAX, 9.57 and 16.05 mg/Kg in flame photometry of blue flower, respectively. FTIR spectra confirmed the presence of functional groups such as primary amines, carboxylic acid and alkanes in the blue color variety, whereas the white color variety contains only the alkane group. The XRD showed the presence of a brominated compound which could be responsible for forming the most stable complex with another organic compound. At 50 µg/mL, the methanol extract of the blue color variety exhibited higher antioxidant potential in all of the tests as compared to the white variety.

The Malaysian fish sausage (Keropok Lekor, KL) is a fish snack made mainly from fish, sago flour, and seasonings. In KL production, the fish bones, innards, skins, and heads are usually removed, and only the flesh is used. The by-products are normally discarded, causing disposal problems and environmental pollution. The by-products can however be converted into high-value antioxidative peptides (APs) by microbial or enzymatic processing. After the bioconversion, the complicated, lengthy, and expensive bioseparation including filtration, precipitation, dialysis, and chromatography are commonly employed in purifying the APs. Thus, researchers are interested in developing alternative bioseparation technology. In this study, the feasibility of using precipitation techniques and aqueous two-phase system (ATPS) to isolate the APs from the protein hydrolysate produced from KL by-products by Lactobacillus casei fermentation was evaluated. Four types of precipitation were evaluated: (1) salt precipitation using ammonium sulfate ((NH₄)₂SO₄) and sodium chloride (NaCl), (2) solvent precipitation using ethanol and acetone, and (3) combination of salts and solvent precipitation, and (4) pH-shift precipitation. Amongst the precipitation techniques investigated, the pH-shift precipitation (pH 7.5) gave the highest purification fold (P_F,181.1). Both solvent and salt-solvent precipitations gave the lowest P_F (1.5 and 1.6, respectively). The salt precipitation (60%, w/v (NH₄)₂SO₄) achieved 2.0 for P_F. Besides, exceptionally high P_F (3,140.4) was obtained using ATPS (bottom phase of 15% (w/w) PEG2000 with 10% (w/w) phosphate system). Overall, the ATPS showed superior performance in enriching the AP compared to the precipitation techniques. The effectiveness of the AP purification may be increased by combining the ATPS with precipitation techniques. The study laid the groundwork for developing an effective bioprocessing, which is important to valorize the KL by-products to the high-value APs.

Isothermal nucleic-acid testing is ideal for on-site diagnostics, but large-scale lyophilization of reagents is hindered by three bottlenecks: costly in-situ capping of vials or PCR tubes, activity loss during non-in-situ transfer/sealing caused by ambient humidity and exposure time, and wild-type T7 RNA polymerase inactivation due to lyophilization-induced structural changes. We overcame these obstacles by optimizing the freeze-drying protocol, screening for robust enzyme variants, and refining the protectant recipe. Sealing at 15% RH prior to stoppering proved optimal; a lyophilization-tolerant T7 RNA polymerase mutant termed V5 was isolated; and a blend of 3%(w/v) PEG8000 plus 0.5%(w/v) mannitol markedly enhanced stability. The resulting cake contains 3%(w/w) residual moisture, reconstitutes in <1 s, and retains full activity after 13 months at 4 °C and for approximately 2 months at 25 °C. These specifications enable short-term, cold-chain-independent field deployment of isothermal nucleic-acid detection reagents.

Environmental concerns over synthetic plastics highlight the need for sustainable alternatives, among which bacterial polyhydroxyalkanoates (PHAs) have emerged as promising. However, their commercialization is limited, owing to their higher production cost and lower applicability. Hence, this study employs ANN-GA-based optimization of a novel in-situ fed-batch process using a self-isolated Bacillus sp. PhNs9 utilizing untreated sugarcane molasses as a cost-effective substrate. The bioreactor scale production at optimized conditions resulted in a PHA production of 3.47 ± 0.11 g/L, whose GC-MS and DSC analysis revealed it to be Polyhydroxybutyrate-co-valerate (PHBV). This PHBV was successfully blended with in-situ extracted neem oil and threadlets to form a biocomposite, which was found to be antibacterial, non-cytotoxic, and biodegradable with significantly higher tensile strength. The HR-MS analysis of the extracted neem oil revealed the presence of bioactive compounds like nimbin, azadirachtin, and Salannin, providing medicinal properties. The techno-economic analysis of the whole process resulted in the cost of PHBV production and extraction as ₹1.03/g, extraction of neem oil as ₹5.75/mL, which further resulted in the biocomposite preparation cost to be ₹0.30 cm^-2. This accentuates the commercial feasibility of the developed biocomposite as an eco-friendly alternative to synthetic plastics.

The current study focuses on enhancing microbial dextran production using a renewable and cost-effective Saccharum officinarum juice medium (SOJM) feedstock and compares it with a chemically defined medium (CDM). The optimized medium variables using SOJM (sucrose: 200 g/L; yeast extract: 10 g/L; KH₂PO₄: 50 g/L; time: 2 d) and CDM (sucrose: 200 g/L; yeast extract: 40.5 g/L; KH₂PO₄: 50 g/L; time: 1 d) for predicting the dextran production were found to be 80 and 96 g/L, respectively. The economic productivity of produced dextran from SOJM and CDM was 0.06 and 0.18 $ dextran per $ nutrient per h, respectively. The functional, structural characteristics, and molecular mass distribution of the produced dextran using SOJM and CDM were analyzed using various characterization techniques. The average molecular mass of dextran using SOJM (before optimization: 2519.7 kDa; after optimization: 3474.3 kDa) is greater than that of CDM (before optimization: 2119.7 kDa; after optimization: 2884.2 kDa). These findings demonstrated that the optimization approach significantly enhanced both yield and average molecular mass using CDM and SOJM. Owing to its antioxidant activity, the produced dextran could be extensively employed in various healthcare applications. Furthermore, the produced dextran is involved in the preparation of an immobilization matrix with calcium alginate for the entrapment of cellulase. The prepared matrix (dex-cel-alg) exhibited greater immobilization efficiency (86.9%) than the alginate matrix (35.5%), illustrating its suitability for the hydrolysis of cellulose to fermentable sugars for biofuel applications.

Melanogenesis is tightly regulated by tyrosinase, and its inhibition represents a key strategy in developing skin-whitening agents. In this study, p-hydroxyphenethyl anisate (HP) was identified as a potent melanogenesis inhibitor through a zebrafish-based screening without affecting embryo viability at concentrations up to 10 μM. The biological activity of HP was further validated in isobutyl-1-methylxanthine (IBMX)-stimulated B16-F1 murine melanoma cells. HP treatment significantly suppressed IBMX-induced melanin synthesis without reducing cell viability. Mechanistically, HP markedly inhibited tyrosinase activity and reduced tyrosinase protein levels. Furthermore, HP suppressed tyrosinase mRNA expression, as well as the expression of Mitf, a key transcriptional regulator of tyrosinase. These findings indicate that HP inhibits melanogenesis through transcriptional downregulation of Mitf, hence its downstream tyrosinase, leading to reduced protein expression and enzymatic activity. Seeking the source of HP, we applied the purification process guided by HPLC peak-tracing and successfully isolated HP from a concentrated Chinese medicine Qiang Huo. Starting from herbal powder, a methanol extract was fractionated by LH-20 column chromatography, and the target compound was purified using preparative C18 HPLC. Collectively, our study identifies HP as a promising natural compound with potent and nontoxic anti-melanogenic activity, supporting its potential application in cosmetic or therapeutic skin depigmentation.

A statistical method was utilized in this study to enhance Lentinus edodes (shiitake) secondary metabolites, thereby improving its anticancer properties. The importance of employing the statistical method in this study stemmed from the initial statistical optimization of the substrate in submerged conditions, and the subsequent transfer of that optimal setting to the solid-state culture medium. The Plackett-Burman (PB) screening indicated that wheat starch, meat peptone, and vitamin B₆ had a more pronounced impacted on polysaccharide production. The optimal substrates for polysaccharide production, identified using response surface methodology (RSM) after screening, was revealed to contain 48.56 g/L wheat starch, 15 g/L meat peptone, and 300 µg B₆ vitamins. Shiitake polysaccharide solutions exhibited shear-thinning behavior, enhanced viscosity, and gel-like properties at higher concentrations. Shiitake polysaccharide also demonstrated superior emulsifying capacity for oil-in-water emulsions, suggesting its potential as an effective stabilizer in food formulations. The fruit body of shiitake had the highest diversity in flavonoids and phenolic acids and anticancer activity against MDA-MB-231 breast cancer cell. The IC50 levels of extracted polysaccharide, fruiting body, fruit stipe, fruit pileus, mycelium from the control sample, and mycelium cultivated in MgO nanoparticles-enriched culture medium were 225, 182, 194, 244, 202, and 138 μg/mL, respectively. The statistical methods used in this study have successfully optimized the bioactive compound content in both the biomass and fruiting body of shiitake, yielding a promising natural source of polysaccharides and anticancer agents.

This study investigates the immobilization of Rhizomucor miehei lipase in organogel systems formulated with gelatin, chitosan, or alginate, incorporating different surfactants and organic solvents, both with and without glutaraldehyde activation. Among the systems tested, gelatin/SDS combinations demonstrated superior thermal stability, whereas chitosan-based matrices exhibited the highest catalytic performance, achieving up to 80% conversion in the esterification of oleic acid at a 1:1 molar ratio. When applied to the esterification of pequi and buriti oils, the biocatalyst performance was influenced by the oils' viscosity and lipid composition. The presence of sago starch in the reaction medium enhanced conversion rates, likely due to its role in modulating the interfacial environment and reducing water activity. This effect was less evident with pequi oil, possibly owing to its sensitivity to ethanol. Catalytic assays confirmed high initial activity, whereas reusability and operational stability tests revealed significant mass loss and limited activity retention during long-term operation, mainly due to physical disintegration of the chitosan-SDS matrix. Despite this limitation, the study provides mechanistic insight into the instability of ionically assembled organogels under real oil conditions. Overall, the findings highlight the complex interplay between support, solvent, substrate, and reaction conditions.

The study uses solid-state fermentation to explore the co-production of industrial enzymes (amylases, cellulases, xylanases, and phytases). The experiments examine the key cultivation parameters, such as fermentation time (2 to 7 days), incubation temperature (25 to 35 °C), medium components, and the effects of inducers. Maximum enzyme activities were observed at 30 °C after 6 days of incubation, using paddy straw as a primary substrate. Supplementing paddy straw with rice bran significantly enhanced the activities of amylase, CMCase, FPase, and xylanase by 1.94x, 1.36x, 1.45x, and 2.14x, respectively using fungal strain Lasiodiplodia theobromae strain RBEE-GCS103. Further, enzyme production in enamel-coated metallic trays increased enzyme activities by 20-35% compared to production in 250 ml flasks. The present study highlights L. theobromae strain RBEE-GCS103 as a novel and potential co-producer for the industrially important enzyme utilizing underutilized agro-waste.