Lipase Production Research Articles

Endomelanconiopsis endophytica Lipase Immobilized in Calcium Alginate for Production of Biodiesel from Waste Cooking Oil

The increasing global demand for biodiesel is due to the urgent need to replace fossil diesel with a fuel based on renewable energy sources. Although chemical catalysis is widely used to produce biodiesel, it uses harsh operating conditions, has high energy consumption, and generates unwanted byproducts. In this scenario, biocatalysis stands out as an efficient and environmentally friendly alternative to chemical catalysis. In biocatalysis, the use of immobilized enzymes plays an important role in the reduction in costs. In this sense, we investigated the use of the lipase produced by an Amazonian endophytic fungus in an immobilized form in the transesterification of waste cooking oil for biodiesel production. The fungus Endomelanconiopsis endophytica QAT_7AC demonstrated a high production of lipase. The lipolytic extract was precipitated in ethanol, which increased the specific enzyme activity. The lipolytic extract and the precipitated lipolytic extract were immobilized in calcium alginate beads. Immobilization efficiency was over 89%. The immobilized biocatalysts showed thermal stability and were used in the production of biodiesel using waste cooking oil and ethanol. It was possible to reuse them for up to four reaction cycles, with yields greater than 70%. These results prove the efficiency of immobilized biocatalysts in the production of biodiesel from waste oils.

Isolation, Characterization and Molecular Identification of Bacteria Associated with Green and Brown Seaweeds

Aims: The co-existence of bacterial communities along with macroalgae in marine environment develops a mutualistic relationship resulting in functional advantages for both the groups. The present research studied the composition of epiphytic bacteria associated with green (Ulva spp.) and brown seaweeds (Sargassum spp. and Padina spp.) sampled off Maharashtra coast, India, and their physiological and bioactive properties. Study Design: Green and brown seaweeds were collected from three locations along Maharashtra coast and analyzed for the associated bacteria. Place and Duration of Study: Seaweed samples analyzed in this study were from Kelwa, Manori and Ratnagiri along the Maharashtra coast. The analysis was performed during June to September 2021. Methodology: Bacteria from freshly collected seaweed samples were homogenized in saline, 10-fold serially diluted and plated on Zobell marine agar. Distinct colonies were selected and identified by a series of biochemical tests, followed by partial 16SrRNA gene sequencing. The physiological characteristics of the isolated bacteria were studied by screening for temperature and salinity tolerance, production of protease, lipase, agarose, amylase and biosurfactants. Results: The total seaweed bacterial count ranged from 4.5 ×103 to 2.9 × 104 CFU/g. Seventy-seven bacteria were isolated, 44 (57.14%) and 33 (42.85%) isolates from green and brown seaweeds respectively. The 16SrRNA sequencing of 20 representative isolates revealed the dominance of Bacillus spp., followed by Vibrio spp. Growth at 0°C was exhibited by all bacteria except Bacillus tequilensis, Bacillus altitudinis, Oceanobacillus iheyensis and one isolate of Vibrio spp. A majority of the isolates grew at 45°C. Vibrio spp. exhibited protease, amylase, gelatinase, and agarase activities, whereas the biosurfactant activity was commonly associated with Bacillus spp. Conclusion: The results of this study illustrate the occurrence of seaweed-associated beneficial bacteria exhibiting bioactive properties with potential biomedical applications.

Enhanced lipase production and characterization from Aeromonas media VBC8: Applications in biodegradation of lubricating oil waste

This study aimed to explore the potential of a novel indigenous strain for the improved production of lipase from castor oil-contaminated soil. Among the various isolates, Aeromonas media VBC8 was found to be the most effective for lipase production. The effect of different inducer oils (olive, peanut, soybean, rice bran, sunflower, coconut, sesame, and fish liver oil) on the biomass of A. media VBC8 and its lipase activity was determined. Among the various oils assessed, fish liver oil exhibited highest lipase activity, with 89 U/mL with 9.1 g/L of biomass. Furthermore, Box-Behnken Design was used to optimize the cultural conditions resulting in an enhanced lipase activity of 1156 U/mL. The lipase was purified through ammonium salt (60 w/v %) precipitation, desalting and ion exchange column, achieving a yield of 16 % and specific activity of 98.4 U/mL. The purified lipase remained active over a wide range of pH 4.0–11.0 and temperature of 10–80 °C with maximum activity at pH 8.0 and 40 °C. SDS-PAGE analysis revealed the lipase's molecular weight to be 94 kDa. The study also evaluated the role of crude and purified lipase in the biodegradability of lubricating oil waste, achieving a maximum fatty acid conversion of 39 and 76 %, respectively, after 7 h incubation at room temperature.

Molecular approach to the characterization of lipase encoding genes from Moraxella sp. SBE01

Lipase from Moraxella sp. SBE01 is an expression of the gene encoding lipase. Detection and characterization of the Moraxella sp. SBE01 lipase coding gene is necessary for large-scale lipase production through genetic engineering. This study aimed to observe the molecular weight, amino acid sequence, length, and conserved amino acids in the DNA encoding the lipase gene, with the goal of identifying and characterizing the lipase-coding gene from Moraxella sp. SBE01. The primer design process was conducted to amplify the lipase gene from Moraxella sp. SBE01 using specialized software for sequence alignment and phylogenetic analysis. Amplification was carried out using PCR with the designed primer, forward primer (GTC ATG ATG TAC TTC CAY GGN GGN GG), reverse primer (GGT TGC CGC CGG CDS WRT CNC C). PCR was carried out under pre-denatured conditions at 95°C (3 minutes), followed by 30 cycles of denaturation at 95°C, annealing at 66°C (30 seconds), 70°C elongations (1 minute) and final elongation of 70°C (10 minutes). The PCR results were electrophoresed using 1% agarose gel with a 1 kb DNA marker. The PCR results were sequenced and analyzed for gene and amino acid sequences and the type of lipase expressed. Sequencing resulted in 387 bp of the nucleotide sequence. The gene and amino acid sequences from Moraxella sp. SBE01 had high homology with the gene and amino acid sequences from Moraxella sp. strain TA144. The lipase gene encodes a protein consisting of 129 amino acids and contains a conserved HGG (His-Gly-Gly) motif, which is characteristic of lipases in family IV, also known as the hormone-sensitive lipase (HSL) family. This conserved sequence suggests that the lipase shares structural and functional similarities with other enzymes in the HSL family, playing a key role in lipid metabolism.

Lecithin-based mixed polymeric micelles for activity improvement of curcumin against Staphylococcus aureus

Considering cellular uptake promotion of lecithin and high expression of phospholipase in S. aureus, we designed curcumin (Cur)-loaded soy lecithin-based mPEG-PVL copolymer micelles (MPPC). The effect of soy lecithin on the anti-S. aureus activity of the formulation was studied with cur-loaded mPEG-PVL micelles (MPC without soy lecithin) as control. It was found that MPPC enhanced the water-solubility of Cur, and showed slow and sustained release behavior of Cur. Although MPPC had the same anti-S. aureus activity as Cur, its activity was significantly higher than MPC due to the cellular uptake promotion of soybean lecithin. It was noted that MPPC had good inhibition or destruction effect on biofilm, significant cell membrane damage, strong inhibition effect on protease or lipase production, and obvious induction effect on ROS expression when compared with Cur and MPC. So, the introduction of soy lecithin could improve the antibacterial activity of Cur. The lecithin-based micelles would offer potential to deliver antibacterial drugs for improved therapeutic action.

Optimization, Purification and Characterization of Lipase from Streptomyces sp. A3301, with Application of Crude Lipase for Cooking Oily Wastewater Treatment

Streptomyces sp. A3301, which produces lipase isolated by Panyachanakul et al. [1]. This optimization was done for the subsequent purification and characterization of the biological lipase produced by the isolate. The results showed that the strain produced lipase with a maximum activity of 321 U/mL using the optimal medium and conditions (1.5 % (w/v) xylose and 2 % (w/v) yeast extract, pH 7.0 at 30 °C, 150 rpm for 3 days). The specific activity of the purified lipase was 27,000 U/mg, which was 544 times the pre-purification level, based on hydrophobic chromatography. After ion-exchange chromatography, the specific activity was 5,600 U/mg, which was 113 times the pre-purification level, with a single-peak purification profile. The purified lipase had a single band based on SDS-PAGE analysis and the molecular mass was 45 kDa. The optimum temperature and thermo-stability of A3301 lipase were 60 and 30 - 55 °C, respectively. The optimum pH of the purified enzyme was pH 9.0, and the enzyme was stable in the pH range of 8.0 - 9.0. The purified lipase was stable in acetone, chloroform and toluene, with a high relative activity of 63 - 76 %. The immobilized strain was then applied to oily-wastewater treatment. The strain can remove oil and grease in synthetic wastewater containing oil at 1 - 3 % (v/v), with removal rates of 100, 99.82 and 99.68 %, respectively, after incubation for 6 days. It was then applied to oily-wastewater treatment from a restaurant, achieving the highest degradation rates of 98.53 % after treatment for 6 days. In addition, it also affected the Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) values decreasing. HIGHLIGHTS Optimization of lipase production by Streptomyces A3301. Purification and characterization of the lipase produced by the Streptomyces A3301. Application of the immobilized Streptomyces A3301 strain in the treatment of oily cooking wastewater. The immobilized Streptomyces A3301 strain has the capability to remove oil and grease in both synthetic mediums and restaurant wastewater. GRAPHICAL ABSTRACT

Enhancing the production and immobilization of cell‐bound lipase from yeast‐like fungus Magnusiomyces capitatusA4C for sustainable biodiesel production in a packed bed reactor

AbstractMagnusiomyces capitatus A4C, a mycelium‐forming and lipase‐producing yeast‐like fungus, was employed in a five‐level factorial design to optimize the collective and interactive influences of carbon, nitrogen and emulsifying sources and their possible effects on cell‐bound lipase (CBL) and cell biomass production. The cell culture of M. capitatus A4C was incubated along with biomass support particles (BSPs) to immobilize the enzyme while anchoring CBL on their surfaces. Among the BSPs tested, CBL immobilized on loofah sponge under optimized conditions showed a substantial hydrolytic activity of 12.7 U mL−1 and a cell‐loading capacity of 0.61 g g−1 of BSPs. Immobilized CBL was applied for biodiesel production via transesterification and esterification. The conversion percentage of triacylglycerides was approximately 100% at 24 h with the addition of water at 1:1 (v/v). The conversion of oleic acid into biodiesel via esterification was 100% at 48 h in the presence of 15% (v/v) isooctane. Further, biodiesel production was scaled up using a packed bed reactor. The batch production of biodiesel in a packed bed reactor through transesterification was 96.2%, with a circulation flow rate of 5.5 mL min−1 for 18 h. On the other hand, oleic acid conversion into biodiesel via esterification was 99.5%, with a circulation flow rate of 5.5 mL min−1 for 24 h. Further investigation revealed that the immobilized biocatalyst exhibited higher stability with esterification (85.3% fatty acid methyl ester) after ten repeated cycles.

Interference Between S. Aureus and P. Aeruginosa Clinical Isolates

General Background: Understanding microbial interactions between Pseudomonas aeruginosa and Staphylococcus aureus is crucial for clinical infections, as they often coexist and influence each other's growth and virulence. Specific Background: Both organisms are known for their ability to form biofilms and exhibit multidrug resistance (MDR), complicating treatment strategies in hospitalized patients. Knowledge Gap: Previous studies have explored the virulence of P. aeruginosa and S. aureus, but there is limited understanding of their direct in vitro interactions, particularly in protease and lipase production. Aims: The study examined the interaction between P. aeruginosa and S. aureus isolates in hospitalized patients' sputum, urine, and blood samples, focusing on virulence factors, biofilm formation, and antibiotic resistance patterns. Results: In Diyala, Iraq, 50 clinical isolates showed P. aeruginosa as protease producers, lipase producers, and biofilm producers, with significant MDR phenotypes in both species. Novelty: This study highlights the ability of P. aeruginosa to produce staphylolysin, offering novel insights into the antagonistic mechanisms that may suppress S. aureus in co-infections. Implications: The study emphasizes the significance of understanding microbial interactions in clinical infections, particularly in biofilm-associated MDR infections, to improve treatment outcomes and guide more effective therapeutic approaches. Highlights: P. aeruginosa staphylolysin inhibits S. aureus growth in vitro. Both bacteria produce biofilms and exhibit multidrug resistance. Microbial interactions impact infection severity and treatment strategies. Keywords: Microbial interactions, Pseudomonas aeruginosa, Staphylococcus aureus, Biofilm, Multidrug resistance

Characterization of a Polyphenol Oxidase and Lipase Produced by Microbes Derived from Palm Oil Sludge

Introduction: The waste-to-wealth concept was applied in this study. Our main objective was to identify and evaluate several microorganisms responsible for the production of important industrial enzymes using sludge from palm oil processing. We were able to isolate several bacteria that are tyrosinase producers: Bacillus cereus, Acinobacter seifertii, Klebsiella variicola and Pseudomonas stutzeri. Laccase producers Trametes polyzona, Bacillus subtilis, Bacillus pumilus and Staphylococcus condimenti, as well as lipase producers. Methods: In addition, we focused specifically on B. cereus because we knew that it produces tyrosinase. Lipase is another target enzyme, and S. condimenti was found to be a hyperproducer. Production conditions included a 24-hour incubation period at 40°C and a pH of 6.0, while typical substrates such as starch and coconut oil were used. Results: Preliminary protein purification studies identified a 43 kDa lipase that is active at pH 7.0 and 40°C. Salts such as NaCl, various detergents such as Triton X-100 and Tween-80 as well as many metal ions enhanced the activity and made the enzyme unique in its biological function. Conclusion: Even with EDTA (2.5 mM), which does not completely block its function, only 40% inhibition was observed. Only a few organic solvents such as butanol, acetone and DMF are involved in inhibiting its activity.

Statistical Optimization of Lipase Production by Aspergillus oryzae Using Olive Mill Wastewater

Among different sources of lipases, fungal lipases are hydrolytic enzymes owing much importance in industrial applications. However, the high production cost limits the industrial application of lipases. To address this issue, we have attempted to optimize lipase production by Aspergillus oryzae on olive mill wastewater (OMW) based medium. Media optimization is considered one of the best ways to economically produce a large quantity of lipase. The medium components were optimized by a statistical approach for optimal lipase production. The Plackett–Burman design was applied to identify the significant factors and response surface methodology to optimize medium components. The enzyme activity was highest under the following conditions: 3.49 g.L-1 glucose, 5.58 g.L-1 yeast extract and 0.72 g.L-1 CaCl2. A feasibility analysis of lipase production using the optimized data was performed, and at optimized conditions, lipase activity increased from 110.21 U.mL-1 to 212.53 U.mL-1, or an increase of 1.93 times.

إنتاج الليبازالبارد – النشط بواسطة فيوزاريوم سولاني

The current study aimed to the production and partial purification of a cold-active lipase by some fungi isolated from the olive oil processing wastes in Al-Gabal Al-Gharby, Libya. 31 fungal species from 12 genera were isolated. F.solani was the most prevalent comprising 94% of total Fusarium and 28.7% of total fungi, 102 fungal isolates were tested for their lipolytic activity on lipase production agar medium at 10 and 20°C. The most active isolates were Alternaria (2 isolates), Fusarium, and Penicillium (1isolate for each one). Molecular identification of the most active four isolates was carried out by their sequencing (ITS). The four powerful fungal strains' production of cold-active lipase was maximized by optimizing some nutritional and environmental factors. F. solani AUMC 16063 was able to produce the maximum amount of lipase activity (46.66U/mL/min) with specific activity (202.8U/mg), utilizing ammonium sulphate as a nitrogen source after 8 days of incubation at pH 3.0 and 15°C. However, at same condition after 6 days when yeast extract was employed as a nitrogen source, the generated cold-active lipase displayed the highest specific activity of (1550U/mg) and lipase activity (36.74U/ml/min). This is the first study in which the production, partial purification, maximized and characterization of a cold-active lipase enzyme by Fusarium solani.

Evaluation of the potential probiotic Bacillus subtilis isolated from darkbarbel catfish (Pelteobagrus fulvidraco) on growth performance, serum immunity, and disease resistance of Aeromonas hydrophila.

This study aimed to identify potential probiotic strains of Bacillus subtilis from healthy fish gut microbiota for application in aquaculture. The effects of dietary B. subtilis administration on growth performance, serum enzyme activity, immune gene expression, and disease resistance in darkbarbel catfish (Pelteobagrus fulvidraco) were investigated. The isolate, identified through gene sequencing and biochemical tests, demonstrated resilience to pH 3.0% and 6.0% bile, and exhibited extracellular protease, cellulose, lipase, and amylase production. Darkbarbel catfish were fed diets with varying B. subtilis concentrations (0 CFU/kg [T0], 107 CFU/kg [T1], 108 CFU/kg [T2], and 109 CFU/kg [T3]). After 8 weeks, significant increases (p < 0.05) were observed in final body weight, weight gain rate, specific growth rate, serum lysozyme, serum superoxide dismutase, alkaline phosphatase, and total antioxidant capacity, whereas malondialdehyde levels significantly decreased. Feeding darkbarbel catfish with B. subtilis diets increased immunoglobulin M (IgM) and C3 gene expression (p < 0.05), indicating a positive impact on the fish's immune system. The strain upregulated interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) expression and downregulated TNF-α and IL-1β, suggesting potential anti-inflammatory effects. Following a 7-day challenge with Aeromonas hydrophila, fish fed with B. subtilis exhibited lower mortality, with higher survival rates in the T2 and T3 groups. In conclusion, supplementing darkbarbel catfish diets with B. subtilis effectively enhances growth performance, immune response, and disease resistance.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the β-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

Specific gut microbiome's role in skin pigmentation: insights from SCARB1 mutants in Oujiang colour common carp.

Beyond the pivotal roles of the gut microbiome in initiating physiological processes and modulating genetic factors, a query persists: Can a single gene mutation alter the abundance of the gut microbiome community? Not only this, but the intricate impact of gut microbiome composition on skin pigmentation has been largely unexplored. Based on these premises, our study examines the abundance of lipase-producing gut microbes about differential gene expression associated with bile acid synthesis and lipid metabolism-related blood metabolites in red (whole wild) and white (whole white wild and SCARB1-/- mutant) Oujiang colour common carp. Following the disruption of the SCARB1 gene in the resulting mutant fish with white body colour (SCARB1-/-), there is a notable decrease in the abundance of gut microbiomes (Bacillus, Staphylococcus, Pseudomonas, and Serratia) associated with lipase production. This reduction parallels the downregulation seen in wild-type white body colour fish (WW), as contrasting to the wild-type red body colour fish (WR). Meanwhile, in SCARB1-/- fish, there was a downregulation noted not only at the genetic and metabolic levels but also a decrease in lipase-producing bacteria. This consistency with WW contrasts significantly with WR. Similarly, genes involved in the bile acid synthesis pathway, along with blood metabolites related to lipid metabolism, exhibited downregulation in SCARB1-/- fish. The SCARB1 knockout gene blockage led to significant alterations in the gut microbiome, potentially influencing the observed reduction in carotenoid-associated skin pigmentation. Our study emphasizes that skin pigmentation is not only impacted by genetic factors but also by the gut microbiome. Meanwhile, the gut microbiome's adaptability can be rapidly shaped and may be driven by specific single-gene variations.

Agricultural Biotechnological Potential of Bacillus velezensis C3-3 and Cytobacillus sp. T106 from Resource Islands of a Semi-arid Zone of La Guajira-Colombia

Resource islands are vegetative formations in arid and semi-arid ecosystems that harbor microorganisms facing extreme conditions. However, there is a limitation in the knowledge of the agricultural biotechnological potential of microorganisms present in these islands. This study aimed to determine the capacity of Bacillus velezensis C3-3 and Cytobacillus sp. T106 isolates from resource islands to promote plant growth and control the phytopathogen Rhizoctonia solani. The bacteria were sequenced, and both grew at 50 °C, resisted 5% NaCl, withstood UV exposure, and grew in extreme pH conditions. Sixty-six genes in C3-3 and 71 in T106 were identified associated with plant growth promotion, and C3-3 was shown to promote leaf growth in lettuce plants. This promotional effect was associated with the production of indole-3-acetic acid (IAA), phosphorus solubilization, and the presence of genes related to the assimilation of rhizosphere exudates. Both strains inhibited R. solani through the production of volatile compounds and antagonism. Forty-five and 40 of these genes in C3-3 and T106, respectively, were associated with the production of proteases, lipases, siderophores, antimicrobial compounds, degradation enzymes, and secretion systems. Notably, Cytobacillus sp. has not been previously reported as a biocontrol agent. This work contributes to the evidence of the biotechnological potential of semi-arid region bacteria, offering prospects for improving agricultural production in areas with limiting conditions.

Bioprospecting a mountain-derived phosphorus-solubilizing bacterium: Bacillus thuringiensis B3 as a plant-growth promoter in lettuce and tomato horticultural crops

The biogeographic and climatic conditions of islands like Cuba generate distinctive ecosystems as reservoirs of microbial biodiversity. This study explores the potential of the B3 strain, isolated from the Guamuhaya Mountain in Cuba, in Cuban urban horticulture, where the soil phosphorus availability poses a significant challenge to crop productivity. The B3 strain demonstrates remarkable phosphorus solubilization capabilities, along with nitrogen fixation and nutrient mobilization, production of growth regulators, siderophores, lipases, and both volatile and diffusible antimicrobial compounds. The strain's multifaceted functionalities indicate its potential to limit phytopathogen proliferation, contributing to plant health. The strain showed Bacilli morphological characteristics, confirmed through MALDI-ToF MS and MLSA, classify it as Bacillus thuringiensis. The strain's safety profile, demonstrated by negative results in hemolysis and DNA degradation tests, underscores its suitability for agricultural applications. The Bacillus thuringiensis B3 significantly enhanced germination of lettuce and tomato seeds, root structure and early plant growth, confirming its value in horticultural production. The integration of this strain into Cuban urban horticulture represents a step forward in sustainable agriculture with low environmental impact.

Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis

Lipases comprise the third most commercialized group of enzymes worldwide and those of microbial origin are sought for their multiple advantages. Agro-industrial waste can be an alternative culture medium for producing lipases, reducing production costs and the improper disposal of waste frying oil (WFO). This study aimed to produce yeast lipases through submerged fermentation (SF) using domestic edible oil waste as inducer and alternative culture medium. The optimal culture conditions, most effective inducer, and purification method for a new lipase from Moesziomyces aphidis BRT57 were identified. Yeast was cultured in medium containing green coconut pulp and WFO waste for 72 h. The maximum production of lipases in SF occurred in a culture medium containing WFO and yeast extract at 48 and 72 h of incubation, with enzyme activities of 8.88 and 11.39 U mL−1, respectively. The lipase was isolated through ultrafiltration followed by size exclusion chromatography, achieving a 50.46 % recovery rate. To the best of our knowledge, this is the first study to report the production and purification of lipases from M. aphidis, demonstrating the value of frying oil as inducer and alternative medium for SF, contributing to the production of fatty acids for biodiesel from food waste.

Enhancement on the lipase activity of M. circinelloides by ARTP mutagenesis for biodiesel production from woody plant oil

The objective is to enhance lipase activity of Mucor circinelloides via atmospheric and room temperature plasma (ARTP). ARTP was employed to treat spores of M. circinelloides for the selection of high lipase producers. With an irradiation time of 100 s at a lethal rate of 95 %, 18 mutants with higher lipase activity were initially selected. Measurements of the lipase activity revealed that a mutant strain LA-26 was characterized by a steady hereditary stability with lipase activity increase to 376.7 U/g. Moreover, the mutant LA-26 was immobilized as whole-cell biocatalyst used in solvent-free system to produce biodiesel. Response surface methodology (RSM) was used to optimize the reaction parameters. With the addition of 8 % biocatalyst, 10 % water content (w/w) and the stepwise addition of methanol (methanol to oil ratio of 4:1), the highest biodiesel (FAME) yield up to 98 % was achieved within 48 h. Immobilized LA-26 can be used for repeated batches, maintaining 70 % FAME yield after 6 cycles. The identified mutant strain LA-26 exhibits great potential for biodiesel production.

In Vitro Characterization and Identification of Potential Probiotic Yeasts Isolated from Zaopocu, a Traditional Fermented Dregs Vinegar from Hainan Island.

Recently, there has been an increasing interest in researching fermented food-derived yeasts as probiotics because they offer a natural and diverse source of potential strains with unique functional properties and health benefits. In this study, 13 yeast strains isolated from Zaopocu (ZPC), a traditional fermented dregs vinegar on Hainan Island, China, were evaluated for their probiotic characteristics in vitro. Yeast identification was conducted through 5.8S-ITS region sequencing, revealing Kodamaea ohmeri as the predominantly isolated species (ZPC_Y3, Y5, Y6, Y11), followed by Pichia kudriavzevii (ZPC_Y2, Y13, Y14), Rhodotorula mucilaginosa (ZPC_Y9, Y10), Pichia fermentans (ZPC_Y8, Y12), Pichia kluyveri (ZPC_Y4), and Pichia occidentalis (ZPC_Y1). Except for ZPC_Y4, ZPC_Y8, and ZPC_Y12, all isolated yeasts exhibited stable growth at 37°C. The survival rates of all test strains exceeded 60% under challenging conditions at pH = 2 and 0.3% bile salt, along with strong antioxidant activity (> 5 6%), notable autoaggregation (> 70%), and varying levels of cell hydrophobicity with xylene (ranging from 35.32 ± 8.57% to 89.73 ± 4.84%). In addition, all isolates showed resistance to multiple antibiotics, along with antagonistic activity, and were deemed safe as none exhibited hemolytic, gelatinase, or DNase activities. Significantly, two P. kudriavzevii strains (ZPC_Y2, Y14) exhibited the production of catalase, lipase, and β-galactosidase, along with the capacity to synthesize gamma-aminobutyric acid (GABA). In summary, this preliminary study represents the first attempt to identify and characterize potential probiotic yeast strains isolated from Zaopocu, providing a theoretical basis for exploring their application in developing novel therapeutic probiotics.

Harnessing a thermo-active and alkaline lipase from novel strain of Brevibacillus borstelensis gp-1, for application in biodiesel synthesis

A novel thermo-active and alkaliphilic strain of Gp-1, isolated from hot water springs of Ganeshpuri, Maharashtra, India, identified as Brevibacillus borstelensis based on 16SrDNA characterization, with accession no. MT292327, at NCBI database. Statistical optimization of lipase production was done using Plackett-Burman algorithm and response surface methodology. Fermentative production of Gp-1 lipase was followed by partial purification, with 24.10 fold purification. Gp-1 lipase has molecular mass of 31 kDa, as determined by SDS-PAGE. Gp-1 lipase exhibited maximal specific activity at pH 10 and 55 °C, with stability up to 70 °C. Kinetic parameters analysis revealed Gp-1 lipase has Km and Vmax values of 1.544 mM and 31.25 μM/mg. min, and specificity constant of 281.13 sec-1, with pNP caprylate as the base suited substrate. Gp-1 lipase was found to be retain more than 90% activity in presence of various organic solvents, and various commercial detergent formulations. The Gp-1 lipase was also tested for transesterification reactions, which revealed successful transesterification of coconut oil, olive oil and waste cooking oil, and conversion in esters. Hence, Gp-1 lipase stands out as a prospective candidate in biodiesel synthesis, a green energy alternative.