Production Of Free Fatty Acids Research Articles

Antioxidative Potential and Ameliorative Effects of Rice Bran Fermented with Lactobacillus against High-Fat Diet-Induced Oxidative Stress in Mice

Rice bran is an important byproduct of the rice polishing process, rich in nutrients, but it is underutilized and often used as feed or discarded, resulting in a huge amount of waste. In this study, rice bran was fermented by Lactobacillus fermentum MF423 to obtain a product with high antioxidant activity. First, a reliable and efficient method for assessing the antioxidant capacity of the fermentation products was established using high-performance liquid chromatography (HPLC), which ensured the consistency of the batch fermentation. The fermented rice bran product (FLRB) exhibited significant antioxidant activity in cells, C. elegans, and hyperlipidemic mice. Transcriptome analysis of mouse livers showed that the expression of plin5 was upregulated in diabetic mice administered FLRB, thereby preventing the excessive production of free fatty acids (FFAs) and the subsequent generation of large amounts of reactive oxygen species (ROS). These studies lay the foundation for the application of rice bran fermentation products.

Antibacterial activity and mechanism of cell-free supernatants of Lacticaseibacillus paracasei against Propionibacterium acnes

Propionibacterium acnes (P. acnes) is an anaerobic and gram-positive bacterium involved in the pathogenesis and inflammation of acne vulgaris. This study particularly focuses on the antimicrobial effect of Lacticaseibacillus paracasei LPH01 against P. acnes, a bacterium that causes acne vulgaris. Fifty-seven Lactobacillus strains were tested for their ability to inhibit P. acnes growth employing the Oxford Cup and double dilution methods. The cell-free supernatant (CFS) of L. paracasei LPH01 demonstrated a strong inhibitory effect, with an inhibition zone diameter of 24.65 ± 0.27 mm and a minimum inhibitory concentration of 12.5 mg/mL. Among the CFS, the fraction over 10 kDa (CFS-10) revealed the best antibacterial effect. Confocal laser scanning microscopes and flow cytometry showed that CFS-10 could reduce cell metabolic activity and cell viability and destroy the integrity and permeability of the cell membrane. A scanning electron microscope revealed that bacterial cells exhibited obvious morphological and ultrastructural changes, which further confirmed the damage of CFS-10 to the cell membrane and cell wall. Findings demonstrated that CFS-10 inhibited the conversion of triglycerides, decreased the production of free fatty acids, and down-regulated the extracellular expression of the lipase gene. This study provides a theoretical basis for the metabolite of L. paracasei LPH01 as a potential antibiotic alternative in cosmeceutical skincare products.

Enzymatic Pretreatment of Slaughterhouse Wastewater: Application of Whole Lipolytic Cells of Rhizopus oryzae Produced from Residual Vegetable Oil

This study assessed the application of whole lipolytic cells in the pretreatment of slaughterhouse wastewater to reduce its lipid content. The fungal biomass of Rhizopus oryzae was evaluated in the hydrolysis of slaughterhouse wastewater containing high lipid concentrations, focusing on the biomass’s concentration and the effect of using an emulsifier and surfactant. The use of the whole-cells lipase of Rhizopus oryzae grown in a residual vegetable oil medium proved effective in the hydrolysis of slaughterhouse wastewater, generating concentrations of free fatty acids (FFA) ranging from 40.36 to 90.14 mM. The action of lipase in the hydrolysis of slaughterhouse residues indicated its effectiveness in pretreating lipid-rich liquid residues, potentially boosting the microbiota of this anaerobic treatment. The results showed that lipase activity without surfactant exhibited a similar performance to that of Triton X-100 in the hydrolysis of liquid residues. However, the combination of lipase and surfactant could represent a promising strategy to optimize free fatty acid production from slaughterhouse residues, strengthening anaerobic treatment processes and potentially enhancing the overall efficiency of waste management systems.

Identification of crucial roles of transcription factor IhfA on high production of free fatty acids in Escherichia coli

Transcription factor engineering has unique advantages in improving the performance of microbial cell factories due to the global regulation of gene transcription. Omics analyses and reverse engineering enable learning and subsequent incorporation of novel design strategies for further engineering. Here, we identify the role of the global regulator IhfA for overproduction of free fatty acids (FFAs) using CRISPRi-facilitated reverse engineering and cellular physiological characterization. From the differentially expressed genes in the ihfAL− strain, a total of 14 beneficial targets that enhance FFAs production by above 20 % are identified, which involve membrane function, oxidative stress, and others. For membrane-related genes, the engineered strains obtain lower cell surface hydrophobicity and increased average length of membrane lipid tails. For oxidative stress-related genes, the engineered strains present decreased reactive oxygen species (ROS) levels. These gene modulations enhance cellular robustness and save cellular resources, contributing to FFAs production. This study provides novel targets and strategies for engineering microbial cell factories with improved FFAs bioproduction.

Effects of water-soluble secondary antioxidants on the retention of carotene and tocols during hydrolysis of crude palm oil catalysed by Eversa® Transform 2.0 for alcohol-free production of palm phytonutrients concentrate

The liquid lipase Eversa® Transform 2.0 (ET 2.0) was used to catalyse the hydrolysis of crude palm oil (CPO) to concentrate and extract carotene and tocols, which is then followed by separation. Nonetheless, the production of free fatty acids (FFA) promotes auto-oxidation, resulting in significant loss of the palm carotenes and tocols during hydrolysis. Therefore, this study explores the effects of incorporating several secondary acid antioxidants namely ascorbic acid, citric acid and ethylenediaminetetraacetic acid (EDTA), on the production of FFA and retention of palm phytonutrients during the enzymatic hydrolysis of CPO. Generally, the results revealed that the addition of these secondary antioxidants notably enhanced the retention of the palm phytonutrients. However, citric acid at high concentrations were found to impose a negative effect on the production of FFA, primarily due to the acid denaturation of the lipase after at low pH conditions. It can be concluded that EDTA is the best performing antioxidant as it efficiently retained 98% of the palm carotene and 97% of the tocols after 24 h of reaction without disrupting the FFA production, remarkably at much lower concentrations (0.01 wt%) in comparison to the other antioxidants. It was deduced that the reason for this is mainly its low solubility in both oil and water phases of the reaction. Finally, separation of the FFA from the oil using vacuum distillation at 200 ℃ and 100 Pa for 1 h successfully enriched the palm carotene and tocols by 2.7-fold and 8.8-fold, respectively.

Conversion of Retinyl Palmitate to Retinol by Wheat Bran Endogenous Lipase Reduces Vitamin A Stability.

Wheat bran can be used as a cost-effective food ingredient to stabilise vitamin A. However, wheat bran endogenous enzymes have been shown to reduce vitamin A stability. In this study, we elucidated the mechanism for this negative effect in an accelerated storage experiment with model systems consisting of native or toasted wheat bran, soy oil and retinyl palmitate (RP). Both native and toasted wheat bran substantially stabilised RP. While RP was entirely degraded after ten days of storage in the absence of wheat bran, the RP retention after ten days was 22 ± 2% and 75 ± 5% in the presence of native and toasted bran, respectively. The significantly stronger stabilising effect of toasted bran was attributed to the absence of bran endogenous enzymes. In contrast to toasted bran systems, noticeable free fatty acid production was observed for native bran systems. However, this did not result in a pronounced lipid oxidation. Next to lipid hydrolysis, wheat bran lipase was shown to hydrolyse retinyl esters to the less stable retinol and fatty acids. This reaction could explain the major part, about 66 ± 5%, of the difference in RP stabilisation between native and toasted wheat bran.

Link between the Reduction of Ochratoxin A and Free Fatty Acids Production in Cocoa Beans Using Bacillus sp. and the Sensory Perception of Chocolate Produced There of

Link between the Reduction of Ochratoxin A and Free Fatty Acids Production in Cocoa Beans Using Bacillus sp. and the Sensory Perception of Chocolate Produced There of

Fabrication of lipase immobilized-carboxylated cellulose nanocrystals as biocatalyst for enzymatic hydrolysis of palm oil and its kinetical study

This research focuses on the production of free fatty acids (FFA), one of the essential components in the oleochemical industry, through the enzymatic hydrolysis process of palm oil at low temperature and pressure using cellulose nanocrystals (CNCs) as lipase immobilization support. Furthermore, the kinetic behavior of this hydrolysis reaction with the presence of lipase immobilized-CNCs (lipase@CNCs) was studied to find the optimum condition for designing the reactor. According to FTIR and TEM analyses, CNCs were successful in immobilizing lipases, as ascertained by the appearance of amide peaks from the formation of bonds between CNCs and lipases and the thickening of the surface of the CNC morphology after immobilization. As a result, the optimum immobilization conditions were obtained at a lipase concentration of 1.5 mg/mL, an immobilization time of 2 h, and the CNCs/EDC ratio of 1:3. Moreover, the Michaelis-Menten model with the inhibition product fitted with the experiment and lipase@CNCs has the highest Vmax value at a temperature of 40 °C of 15.53 mM/h with a KM of 99.80 mM and an Ea of 41.07 kJ/mol. The simulation results show that the semi-batch reactor can achieve a higher conversion at the beginning of the reaction with a conversion of 28%. Therefore, using lipase@CNCs in hydrolysis processes is expected to be one of the strategies that can overcome the limitations associated with existing methods in terms of high energy consumption.

Efficient Hydrolysis of Cottonseed Oil with Amano Lipase Entrapped on Hydrolysed Polyvinyl Alcohol Gel Pellets Optimized via Factorial Design

Abstract: The aim of this study was to obtain the optimum hydrolysis of cottonseed oil via immobilized lipases (Lipases Rhizopus oryzae, SL from Pseudomonas (Burkholderia) cepacia, Amano lipase from Mucor javanica, ANL from Aspergillus niger lipase, TL from Pseudomonas stutzeri, QLM from Alcaligenes sp., Triacylglycerol lipase (EC 3.1.1.3), PL from Alcaligenes sp., AL from lysosomal acid lipase, Candida antarctica (CA), and Candida cylindrea (CC)) on hydrolysed polyvinyl alcohol (PVA) gel pellets. : The production of free fatty acids by the hydrolysis of triglycerides from several sources is an important component in the economic exploitation of naturally produced renewable raw materials. : Herein, various lipases, including Rhizopus oryzea, SL, Amano lipase from Mucor javanica, ANL, TL, QLM, EC, PL, AL, CA, and CC were screened for optimum hydrolysis of cottonseed oil. Following the selection of lipase for hydrolysis of cottonseed oil, transparent PVA gel was synthesized by physical gelation method employing Dimethyl sulfoxide and a water system. In the last step, immobilization of selected lipase i.e. Amano lipase for cotton seed oil was carried out at 4oC for one hour. After successful synthesis and immobilization, the PVA gel was characterized by FTIR and SEM analysis. Finally, factorial design was employed for optimization of experimental variables such as pH, time and amount of cotton seed oil hydrolysis by lipase immobilized PVA. : The obtained results indicated that lipase Amano was found better with higher free fatty acids (FFA) yields (with 98.38%) in 5 hours using 3 g of cottonseed oil, suggesting that PVA immobilized Amano lipase is an efficient catalyst to hydrolyze the cottonseed oil. : The overall study indicated that hydrolysed PVA gel is an effective biocompatible solid support for immobilization of lipases to obtain higher FFA from cottonseed oil following hydrolysis reaction.

Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury.

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity, damaging the neurons. However, how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear. Herein, we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury. We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice. Lipid droplet accumulation could be induced by myelin debris in HT22 cells. Myelin debris degradation by phospholipase led to massive free fatty acid production, which increased lipid droplet synthesis, β-oxidation, and oxidative phosphorylation. Excessive oxidative phosphorylation increased reactive oxygen species generation, which led to increased lipid peroxidation and HT22 cell apoptosis. Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway, thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells. Motor function, lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury. The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Safety evaluation of the food enzyme triacylglycerol lipase from non-genetically modified Limtongozyma cylindracea strain MS-5-OF.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non-genetically modified yeast Limtongozyma cylindracea strain MS-5-OF by Meito Sangyo Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in five food manufacturing processes: brewing processes, baking processes, milk processing for cheese production, production of free fatty acids by hydrolysis and production of flavouring preparations from dairy products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 1.033 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2,084 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,017. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose.

Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l-1 of FFAs from glucose, with productivity of up to 0.27 g l-1 h-1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l-1 and 7.0 g l-1 FFAs from the glucose-xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l-1 in fed-batch fermentation in mixed glucose and xylose.

Production of Liquid Biofuel Precursors: Optimization and Regulation of Lipase Fermentation and Its Application in Plant Oil Hydrolysis Process

In the liquid biofuel production process, free fatty acids are important precursors for biodiesel and bio-aviation fuel. Enzymatic hydrolysis to produce free fatty acids has attracted more and more attention. However, enzymatic hydrolysis requires ensuring efficient expression of lipase and high product yield. In the shaking flask, the optimal addition amount of citric acid was 3 g/L, and the composition of composite amino acids was: histidine 0.1 mol/L, aspartic acid 0.03 mol/L and lysine 0.03 mol/L. On the basis of the shaking flask optimization, a 5 L fermentation tank was scaled up to obtain 25,000 U/mL of lipase after multiple batches of stable fermentation. This was the first time to cultivate Y. lipolytica using composite amino acid medium to produce high enzyme-activity lipase, demonstrating the industrial value of this lipase fermentation process. Finally, soybean oil was hydrolyzed to produce free fatty acids on a self-made static reactor. The optimized reaction conditions were: material flow rate 2813 mL/min, reaction temperature 45 °C, water content 25 wt% and lipase consumption 3 wt%. The yield of free fatty acids was 80.63% after 12 h under optimal reaction conditions in the self-made static reactor, 11.95% higher than that in the stirred reactor, which showed its industrial potential in the production of free fatty acids.

Effects of Individual Circulating FFAs on Plasma and Hepatic FFA Epoxides, Diols, and Epoxide-Diol Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Oxylipins, oxidation products of unsaturated free fatty acids (FFAs), are involved in various cellular signaling systems. Among these oxylipins, FFA epoxides are associated with beneficial effects in metabolic and cardiovascular health. FFA epoxides are metabolized to diols, which are usually biologically less active, by soluble epoxide hydrolase (sEH). Plasma epoxide-diol ratios have been used as indirect measures of sEH activity. This study was designed to examine the effects of acute elevation of individual plasma FFAs on a variety of oxylipins, particularly epoxides, diols, and their ratios. We tested if FFA epoxide-diol ratios are altered by circulating FFA levels (i.e., substrate availability) independent of sEH activity. Wistar rats received a constant intravenous infusion of olive (70% oleic acid (OA)), safflower seed (72% linoleic acid (LA)), and fish oils (rich in ω-3 FFAs) as emulsions to selectively raise OA, LA, and ω-3 FFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), respectively. As expected, olive, safflower seed, and fish oil infusions selectively raised plasma OA (57%), LA (87%), EPA (70%), and DHA (54%), respectively (p < 0.05 for all). Raising plasma FFAs exerted substrate effects to increase hepatic and plasma epoxide and diol levels. These increases in epoxides and diols occurred to similar extents, resulting in no significant changes in epoxide-diol ratios. These data suggest that epoxide-diol ratios, often used as indices of sEH activity, are not affected by substrate availability or altered plasma FFA levels and that epoxide-diol ratios may be used to compare sEH activity between conditions of different circulating FFA levels.

Green process to produce phytonutrients from crude palm oil: Effects of phenolic antioxidants on tocols and carotene retention during enzymatic hydrolysis

The enzymatic hydrolysis of crude palm oil (CPO), followed by separation, has been studied as an alcohol-free alternative to concentrate and extract palm carotene and tocols. However, a considerable amount of palm phytonutrients may be lost during hydrolysis due to the production of free fatty acids (FFAs), which promote auto-oxidation. In this research, the effects of incorporating phenolic antioxidants, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butyl hydroquinone (TBHQ), on the enzymatic hydrolysis of CPO and the retention of phytonutrients were evaluated. The addition of phenolic antioxidants did not affect the production of FFAs. Meanwhile, its addition positively impacted the phytonutrients preservation as the retentions of carotene and tocols were increased up to over 90 %; a great improvement compared to the 58 % carotene and 23 % tocols retentions without phenolic antioxidants. The results indicated that TBHQ is the best-performing phenolic antioxidant, owing to its low steric hindrance and higher solubility in the water phase. The inclusion of 0.08 wt% TBHQ in the reaction improved the carotenes and tocols retentions to 88 % and 98 %, respectively, after 24 h of hydrolysis. In conclusion, adding phenolic antioxidants considerably enhances the preservation of palm carotene and tocols during enzymatic hydrolysis. The results of this study revealed that the phenolic antioxidants performed in the following order: TBHQ > BHT > BHA.

Homologous Overexpression of Acyl-CoA Thioesterase 8 Enhanced Free Fatty Acid Accumulation in Oleaginous Fungus Mucor circinelloides WJ11

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.

Evaluation of Pentraxin 3 and Serum Amyloid A in the Gingival Crevicular Fluid of Patients with Periodontal Disease and Obesity.

Pentraxin 3 (PTX3) is associated with periodontal tissue inflammation, a condition that precedes alveolar bone resorption. It is also elevated in obese tissues and is a useful biomarker of proinflammatory status. Serum amyloid A (SAA) is a proinflammatory and lipolytic adipokine. Adipocytes strongly express SAA, which suggests that it may have a significant role in the production of free fatty acids and local and systemic inflammation. We statistically analyzed the gingival crevicular fluid (GCF) values of PTX3 and SAA in patients with periodontal disease, who were diagnosed with obesity, and compared them with the values of inflammatory markers from patients diagnosed with one of the diseases and with healthy patients. The patients with obesity and periodontitis had significantly higher levels of PTX3 and SAA than the patients diagnosed with either obesity or periodontitis. These two markers are involved in the association between the two pathologies, as evidenced by the correlations between these levels and some clinical parameters.

An allosteric modulator of the adenosine A1 receptor potentiates the antilipolytic effect in rat adipose tissue

The adenosine A1 receptor plays important roles in tuning free fatty acid (FFA) levels and represents an attractive target for metabolic disorders. Though remarkable progress has been achieved in the exploitation of effective (orthosteric) A1 receptor agonists in modulating aberrant FFA levels, the effect of A1 receptor allosteric modulation on lipid homeostasis is less investigated. Herein we sought to explore the effect of an allosteric modulator on the action of an A1 receptor orthosteric agonist in regulating the lipolytic process in vitro and in vivo. We examined the binding kinetics of a selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) in the absence or presence of an allosteric modulator (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)-phenyl]methanone (PD81,723) on rat adipocyte membranes. We also examined the allosteric effects of PD81,723 on mediating the CCPA-induced inhibition of cAMP accumulation, HSL (hormone-sensitive lipase) phosphorylation and FFA production in in vitro and in vivo models. Our results demonstrated that PD81,723 slowed down the dissociation of CCPA from the A1 receptor, which, consequently, potentiated the antilipolytic action of CCPA through downregulating the cAMP/HSL pathway. Our study exemplified the application of A1 receptor allosteric modulators as an alternative for metabolic disease treatments.

Enzymatic hydrolysis as a green alternative for glyceride removal from rice bran acid oil before γ-oryzanol recovery: Statistical process optimization

Rice bran acid oil (RBAO) is a byproduct of rice bran oil refinery, comprising of free fatty acids (FFAs), glycerides, and considerable amount of the natural antioxidant, γ-oryzanol. As a primary step to recover γ-oryzanol from RBAO, glycerides must be removed by conversion into more easily separable components such as FFAs. In this study, enzymatic hydrolysis was proposed as a green alternative to conventional base-catalyzed hydrolysis for glyceride removal from RBAO. The face-centered central composite rotatable design was used to investigate the effects of three independent variables: time, temperature, and water:RBAO ratio, and their interactions on the responses: glyceride removal, γ-oryzanol loss, and FFA production, and to determine the statistical models describing their relationships. In addition, by applying the desirability function approach, the optimal location was identified, corresponding to maximal glyceride removal with an imposed upper limit of 35% γ-oryzanol loss. At an established optimal condition of 22 h, 48.5 °C and 1:1 water:RBAO ratio, the experiments in which the enzymatic hydrolysis was carried out at 1x and 10x reaction scales confirmed the validity of the models: glyceride removal approached completion (99%), γ-oryzanol loss was as low as 32%, and the FFA production was 73–75%, falling within the 95% prediction interval. The results of this study demonstrated that the enzymatic hydrolysis is a promising method for glyceride removal prior to recovery of γ-oryzanol from RBAO, and that the statistical models gave accurate predictions of responses and would be useful further industrial design of the process.

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.