Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut.

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm.

Optimization of lipase production by Penicillium roqueforti ATCC 10110 through solid-state fermentation using agro-industrial residue based on a univariate analysis.

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are a class of enzymes with high industrial importance. An option for the production of this enzyme is through fungal growth via solid-state fermentation (SSF). Thus, this research presents a study of lipase production by Penicillium roqueforti ATCC 10110 through SSF using cocoa bran residues (Theobroma cacao) as a substrate. To achieve maximum lipase production, fermentation time (0 to 120 h) and palm oil (PO) percentage (0 to 50%) were optimized through analysis of one factor at a time (OFAT), with lipase activity as the response. The amount of cocoa was fixed (5 g), the incubation temperature was maintained at 27 °C, and the moisture content was established at 70%. For a 72 h incubation, the highest enzyme activity achieved using SSF without adding PO was 14.67 ± 1.47 U g-1, whereas with PO (30%), it was 33.33 ± 3.33 U g-1, thus demonstrating a 44% increase in enzyme activity. Through the OFAT methodology, it was possible to confirm that supplementation with palm residue was efficient and maximized the lipase of P. roqueforti ATCC 10110.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Application and characterization of crude fungal lipases used to degrade fat and oil wastes.

Aspergillus niger MH078571.1 and A. niger MH079049.1 were identified previously as the two highest Aspergillus niger strains producing lipase. Biochemical characterizations of lipase activity and stability for these two strains were examined and revealed that the optimal temperature is 45 °C at pH 8for A. niger MH078571.1 and 55 °C for MH079049.1. The lipase production of both strains was studied on medium contains waste oil, as a cheap source to reduce the industrial cost, showed that the optimal incubation period for the enzyme production is 3 days. Moreover, an experiment on lipase activates in organic solvents demonstrated that 50% of acetone is the best solvent for the two strains. In the presence of surfactants, 0.1% of tween 80 surfactant showed the best lipase activities. Furthermore, Mg2+ and Zn2+ ions enhanced the lipase activity of A. niger MH078571.1, while Na2+ and Cu2+ enhanced the enzyme activity of A. niger MH079049.1. Lipase activity was also tested for industrial applications such as integrating it with different detergents. Maximum lipase activity was obtained with 1% of Omo as a powder detergent for both strains. In liquid detergent, 0.1% of Fairy showed maximum lipase activity in A. niger MH078571.1, while the lipase in A. niger MH079049.1 was more effective in 1% of Lux. Moreover, the degradation of natural animal fat with crude enzyme was tested using chicken and sheep fats. The results showed that more than 90% of fats degraded after 5 days of the incubation period.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

Treadmill Running Changes Endothelial Lipase Expression: Insights from Gene and Protein Analysis in Various Striated Muscle Tissues and Serum.

Endothelial lipase (EL) is an enzyme capable of HDL phospholipids hydrolysis. Its action leads to a reduction in the serum high-density lipoprotein concentration, and thus, it exerts a pro-atherogenic effect. This study examines the impact of a single bout exercise on the gene and protein expression of the EL in skeletal muscles composed of different fiber types (the soleus-mainly type I, the red gastrocnemius-mostly IIA, and the white gastrocnemius-predominantly IIX fibers), as well as the diaphragm, and the heart. Wistar rats were subjected to a treadmill run: (1) t = 30 [min], V = 18 [m/min]; (2) t = 30 [min], V = 28 [m/min]; (3) t = 120 [min], V = 18 [m/min] (designated: M30, F30, and M120, respectively). We established EL expression in the total muscle homogenates in sedentary animals. Resting values could be ordered with the decreasing EL protein expression as follows: endothelium of left ventricle > diaphragm > red gastrocnemius > right ventricle > soleus > white gastrocnemius. Furthermore, we observed that even a single bout of exercise was capable of inducing changes in the mRNA and protein level of EL, with a clearer pattern observed for the former. After 30 min of running at either exercise intensity, the expression of EL transcript in all the cardiovascular components of muscles tested, except the soleus, was reduced in comparison to the respective sedentary control. The protein content of EL varied with the intensity and/or duration of the run in the studied whole tissue homogenates. The observed differences between EL expression in vascular beds of muscles may indicate the muscle-specific role of the lipase.

Regulatory Action of Plasma from Patients with Obesity and Diabetes towards Muscle Cells Differentiation and Bioenergetics Revealed by the C2C12 Cell Model and MicroRNA Analysis.

Obesity and type 2 diabetes mellitus (T2DM) are often combined and pathologically affect many tissues due to changes in circulating bioactive molecules. In this work, we evaluated the effect of blood plasma from obese (OB) patients or from obese patients comorbid with diabetes (OBD) on skeletal muscle function and metabolic state. We employed the mouse myoblasts C2C12 differentiation model to test the regulatory effect of plasma exposure at several levels: (1) cell morphology; (2) functional activity of mitochondria; (3) expression levels of several mitochondria regulators, i.e., Atgl, Pgc1b, and miR-378a-3p. Existing databases were used to computationally predict and analyze mir-378a-3p potential targets. We show that short-term exposure to OB or OBD patients' plasma is sufficient to affect C2C12 properties. In fact, the expression of genes that regulate skeletal muscle differentiation and growth was downregulated in both OB- and OBD-treated cells, maximal mitochondrial respiration rate was downregulated in the OBD group, while in the OB group, a metabolic switch to glycolysis was detected. These alterations correlated with a decrease in ATGL and Pgc1b expression in the OB group and with an increase of miR-378a-3p levels in the OBD group.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope.

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263 T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

Production strategies and biotechnological relevance of microbial lipases: a review.

Lipases are enzymes that catalyze the breakdown of lipids into long-chain fatty acids and glycerol in oil-water interface. In addition, they catalyze broad spectrum of bioconversion reactions including esterification, inter-esterification, among others in non-aqueous and micro-aqueous milieu. Lipases are universally produced from plants, animals, and microorganisms. However, lipases from microbial origin are mostly preferred owing to their lower production costs, ease of genetic manipulation etc. The secretion of these biocatalysts by microorganisms is influenced by nutritional and physicochemical parameters. Optimization of the bioprocess parameters enhanced lipase production. In addition, microbial lipases have gained intensified attention for a wide range of applications in food, detergent, and cosmetics industries as well as in environmental bioremediation. This review provides insights into strategies for production of microbial lipases for potential biotechnological applications.

CIDEC silencing attenuates diabetic nephropathy via inhibiting apoptosis and promoting autophagy.

OBJECTIVE: The role of cell death-inducing DFF45-like effector C (CIDEC) in insulin resistance has been established, and it is considered to be an important trigger factor for the progression of diabetic nephropathy (DN). We intend to explore whether CIDEC plays an important role in the regulation of DN and its potential mechanism. METHODS: High-fat diet and low dose streptozotocin were used to establish type 2 diabetic rat model. We investigate the role of CIDEC in the pathogenesis and process of DN through histopathological analysis, western blot and gene silencing. Meanwhile, the effect of CIDEC on renal tubular epithelial cells stimulated by high glucose was also verified. RESULTS: DM group exhibited glucose and lipid metabolic disturbance, with hypertrophy of kidneys, damaged renal function, increased apoptosis, decreased autophagy, glomerulosclerosis and interstitial fibrosis. CIDEC gene silencing improved metabolic disorder and insulin resistance, alleviated renal hypertrophy and renal function damage, decreased glomerular and tubular apoptosis, increased autophagy and inhibited renal fibrosis. At the cellular level, high glucose stimulation increased CIDEC expression in renal tubular epithelial cells, accompanied by increased apoptosis and decreased autophagy. CIDEC gene silencing can improve autophagy and reduce apoptosis. At the molecular level, CIDEC gene silencing also decreased the expression of early growth response factor (EGR)1 and increased the expression of adipose triglyceride lipase (ATGL). CONCLUSION: CIDEC gene silencing may delay the progression of DN by restoring autophagy activity and inhibiting apoptosis with the participation of EGR1and ATGL.

Crude glycerol impurities improve Rhizomucor miehei lipase production by Pichia pastoris.

Crude glycerol, a by-product of biodiesel production, was employed as the carbon source to produce lipase using Pichia pastoris. Under identical fermentation conditions, cell growth and lipase activity were improved using crude glycerol instead of pure glycerol. The impacts of crude glycerol impurities (methyl ester, grease, glycerol, methanol, and metal ions Na+, Ca2+, and Fe3+) on lipase production were investigated. Impurities accelerated P. pastoris entering the stationary phase. Na+, Ca2+, and grease in waste crude glycerol were the main factors influencing higher lipase activity. Through response surface optimization of Ca2+, Na+, and grease concentrations, lipase activity reached 1437 U/mL (15,977 U/mg), which was 2.5 times that of the control. This study highlights the economical and highly efficient valorization of crude glycerol, demonstrating its possible utilization as a carbon source to produce lipase by P. pastoris without pretreatment.

Sustainable Lipase Production by Diutina rugosa NRRL Y-95 Through a Combined Use of Agro-Industrial Residues as Feedstock.

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 °C and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations.

The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system.

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

Microbial lipase production: A deep insight into the recent advances of lipase production and purification techniques.

Importance of enzymes is ever-rising particularly microbial lipases holding great industrial worth owing to their potential to catalyze a diverse array of chemical reactions in aqueous as well as nonaqueous settings. International lipase market is anticipated to cross USD 797.7 million till 2025, rising at a 6.2% compound annual growth rate from 2017 to 2025. The recent breakthrough in the field of lipase research is the generation of new and upgraded versions of lipases via molecular strategies. For example, integration of rational enzyme design and directed enzyme evolution to attain desired properties in lipases. Normally, purification of lipase with significant purity is achieved through a multistep procedure. Such multiple step approach of lipase purification entails both conventional and novel techniques. The present review attempts to provide an overview of different aspects of lipase production including fermentation techniques, factors affecting lipase production, and purification strategies, with the aim to assist researchers to pick a suitable technique for the production and purification of lipase.

Efficient improvement of surface displayed lipase from Rhizomucor miehei in PichiaPink™ protease-deficient system.

Lipase from Rhizomucor miehei (RML) is a promising biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink™ as host to display RML efficiently. RML gene, GCW21 gene and α-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducution expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 °C and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li+, Na+, K+, and Mg2+ of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink™ protease-deficient system, which may promote the further research and development for the industrial application of RML.

Production, purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6.

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20-50 °C) and pH values (6-11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na+, Ba2+, Ca2+, and Mn2+). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s-1 mM-1, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.

Co-Expression of a Thermally Stable and Methanol-Resistant Lipase and Its Chaperone from Burkholderia cepacia G63 in Escherichia coli.

Biodiesel biosynthesis with enzymatic transesterification is considered green, sustainable, and environmentally friendly method. Lipase from Burkholderia cepacia G63 has excellent catalytic properties in biodiesel production. Lipase chaperones promote secretion and folding of enzymes, thereby enhancing enzymatic activity. In the current study, heterologous co-expression of lipase (lipA) and chaperone (lipB) was achieved in Escherichia coli through codon optimization. The enzymatic activity of purified and renatured lipAB was 2080.23 ± 19.18 U/g at 50 °C and pH 8.0. Moreover, lipAB showed increased resistance to pH and temperature changes, and lipAB retained stable catalytic properties after treatment with metal ions, organic solvents, and surfactants, namely Mg2+, methanol, and Triton-100X. Besides, using recombinant lipase lipAB as catalysts, biodiesel was synthesized using rapeseed oil under 50 °C for 72 h with a yield of 90.23%. Thus, the current study confirmed that co-expression of lipase and its chaperone is an effective strategy to enhance enzyme activity and improve the biochemical profile, meanwhile, showing that lipAB is a promising biocatalyst for biodiesel production.

Microbial lipase: a new approach for a heterogeneous biocatalyst.

Lipases are enzymes employed in several industrial process and their applicability can be increased if these biocatalysts are in the immobilize form. The objective of this work was to study the immobilization of lipase produced by submerged cultivation of Aspergillus sp. by hydrophobic interaction, evaluating its stability and reuse capacity. The immobilization process on octyl-sepharose (C8) and octadecyl-sepabeads (C18) carriers was possible after the removal of oil excess presented in the fermented broth. The results showed that the enzyme was isolated and concentrated in octyl-sepharose with 22% of the initial activity. To increase the amount of enzyme adsorbed on the carrier, 4 immobilization cycles were performed in a row, on the same carrier, with a final immobilization yield of 151.32% and an increase in the specific activity of 136%. The activity test with immobilized lipase showed that the immobilized enzyme maintained 75% of the initial activity after 20 cycles.

Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38.

A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.

Co-expression of Pseudomonas alcaligenes lipase and its specific foldase in Pichia pastoris by a dual expression cassette strategy.

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.