Arthrographis curvata and Rhodosporidium babjevae as New Potential Fungal Lipase Producers for Biotechnological Applications

Abstract Fungi have always attracted a lot of attention as they are able to produce a vast repertoire of enzymes that find a broad spectrum of uses in biotechnological and industrial fields. Undoubtedly, one of the most promising biocatalysts is the lipase, which has been widely used for the biotransformation of a number of commercial products due to its high stability, high catalytic efficiency, versatility and selectivity, making it one of the most attractive and best-studied enzymes. In this study we report the isolation and molecular identification of new lipase-producing fungi from different environmental samples from Morocco. The production and activity of extracellular lipases, at different parameters, was evaluated using the Rhodamine B agar, submerged fermentation and biochemical methods. Two fungal strains Arthrographis curvata and Rhodosporidium babjevae, were isolated and found to produce large amounts of lipases. The optimal activity of the extracellular lipase was detected at 40°C and pH 9.0 for A. curvata and at 40 °C and pH 8.0 for R. babjevae. This study add new information at the growing list of fungal species producing lipases with improved physicochemical proprieties which could constitute a new line of research for further studies and to be exploited for industrial or bioremediation purposes.

Evaluation of alkali and thermotolerant lipase from an indigenous isolated Bacillus strain for detergent formulation

Background: Lipases are used in detergent industries to minimise the use of phosphate-based chemicals in detergent formulations. The use of lipase in household laundry reduces environmental pollution and enhances the ability of detergent to remove tough oil or grease stains. Results: A lipase-producing indigenous Bacillus subtilis strain [accession no. KT985358] was isolated from the foothills of Trikuta mountain in Jammu and Kashmir, India. The lipase (BSK-L) produced by this strain expressed alkali and thermotolerance. Lipase has an optimal activity at pH 8.0 and temperature 37°C, whereas it is stable at pH 6.0­9.0 and showed active lipolytic activity at temperatures 30 to 60°C. Furthermore, lipase activity was found to be stimulated in the presence of the metal ions Mn2+, K+, Zn2+, Fe2+ and Ca2+. This lipase was resistant to surfactants, oxidising agents and commercial detergents, suggesting it as a potential candidate for detergent formulation. BSK-L displayed noticeable capability to remove oil stains when used in different washing solutions containing buffer, lipase and commercial detergent. The maximum olive oil removal percentage obtained was 68% when the optimum detergent concentration (Fena) was 0.3%. The oil removal percentage from olive oil-soiled cotton fabric increased with 40 U/mL of lipase. Conclusions: This BSK-L enzyme has the potential for removing oil stains by developing a pre-soaked solution for detergent formulation and was compatible with surfactants, oxidising agents and commercial detergents.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Enhancing production of lipase MAS1 from marine Streptomyces sp. strain in Pichia pastoris by chaperones co-expression

Background: A thermostable lipase MAS1 from marine Streptomyces sp. strain was considered as a potential biocatalyst for industrial application, but its production level was relatively low. Here, the effect of chaperones co-expression on the secretory expression of lipase MAS1 in Pichia pastoris was investigated. Result: Co-expression of protein disulfide isomerase (PDI), HAC1 and immunoglobulin binding protein could increase the expression level of lipase MAS1, whereas co-expression of Vitreoscilla hemoglobin showed a negative effect to the lipase MAS1 production. Among them, PDI co-expression increased lipase MAS1 expression level by 1.7-fold compared to the control strain harboring only the MAS1 gene. Furthermore, optimizing production of lipase MAS1 with Pichia pastoris strain X-33/MAS1-PDI in a 30-L bioreactor were conducted. Lower induction temperature was found to have a benefit effect for lipase MAS1 production. Lipase activity at 24 and 22°C showed 1.7 and 2.1-fold to that at 30°C, respectively. Among the induction pH tested, the highest lipase activity was obtained at pH 6.0 with activity of 440 U/mL after 144 h fermentation. Conclusion: Our work showed a good example for improving the production of recombinant enzymes in Pichia pastoris via chaperon co-expression and fermentation condition optimization.

Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi

Abstract A Plackett–Burman Factorial Design of 16 experiments was conducted to assess the influence of nine factors on the production of lipases by filamentous fungi. The factors investigated were bran type (used as the main carbon source), nitrogen source, nitrogen source concentration, inducer, inducer concentration, fungal strain (Aspergillus niger or Aspergillus flavus were selected as good lipase producers via submerged fermentation), pH and agitation. The concentration of the yeast extract and soybean oil and the pH had a significant effect (p < 0.05) on lipase production and were consecutively studied through a Full Factorial Design 23, with the concentration of yeast extract and pH being significant (p < 0.05). These variables were optimized using a central composite design, obtaining maximum lipolytic activities with the use of 45 g/L of yeast extract and pH 7.15. The statistical model showed a 94.12% correlation with the experimental data.

Utilization of coconut oil mill waste as a substrate for optimized lipase production, oil biodegradation and enzyme purification studies in Staphylococcus pasteuri

Background Oil and grease laden wastewaters pose hindrance to the treatment units and further threaten the receiving water bodies. Lipase-producing microbial strains are increasingly being exploited for the remediation of such effluents. Results When bacterial strains isolated from oil mill effluent were screened for their lipolytic activity, two isolates, COM-4A and COM-6B showed significant extracellular lipase activity. They were identified to be Staphylococcus pasteuri and Bacillus subtilis, respectively. S. pasteuri COM-4A was cultivated in nutrient media based on coconut oil mill waste (CMW), in which it showed good growth at concentrations up to 20 g/L. While growing in such media, it was capable of producing lipase and other important extracellular hydrolytic enzymes. Furthermore, the isolate was able to effectively biodegrade the CMW supplemented in the medium. Applying the Box Behnken Design of Response Surface Methodology, lead to a 1.4-fold increase in both lipase production and oil removal by the isolate. The lipase was purified 9.02-fold and the molecular weight of the monomeric enzyme was deduced to be around 56 kDa. Characterization of the enzyme revealed it to be alkaliphilic and moderately thermophilic in nature, with pH and temperature optima of 9.0 and 50°C, respectively. The enzyme was also quite stable in the presence of water-miscible organic solvents. Conclusion Hence, the COM-4A lipase could be considered to be suitable for a variety of industrial applications such as in detergent formulations and in biodiesel production as well, apart from the possibility of applying it for bioremediation of fat and oil contaminants.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

Lipase production and growth modeling of a novel thermophilic bacterium: aneurinibacillus thermoaerophilus strain AFNA

Aneurinibacillus thermoaerophilus strain AFNA as a novel isolated extracellular thermostable organic solvent tolerant lipase producing bacterium was employed in the present study. The lipase production of strain AFNA and its correlation with bacterial growth was studied via a modeling assessment by response surface methodology (RSM) and artificial neural network (ANN) techniques. The best achieved models were multilayer full feed forward incremental back propagation network and modified cubic response surface model (mRSM) using backward elimination. The highest lipase specific activity (13.1 Umg-1) and bacterial growth (OD600 = 3.0) were obtained at technically similar: growth temperature (53 and 53ºC), inoculum size (2.6 and 3.0 percent), agitation rate (118 and 115 rpm) and initial pH (7.0 and 7.2) but different medium volume (139 and 87 ml) and incubation period (48 and 38 hrs), respectively. In addition, the importance of effective parameters on the bacterial growth and lipase production was studied where pH and inoculum size were the most and the least effective factors, respectively. Significant correlation between lipase production and bacterial growth was observed when Bivariate correlation was employed to analyse the data. As a conclusion, lipase production was the result of a synergistic combination of effective parameters interactions and these parameters were in equilibrium.

Isolation and identification of a novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110

Lipases are particularly important due to the fact that they specifically hydrolyze acyl glycerol, oils and greases, which is of great interest for different industrial applications. In this study, several lipase-producing bacteria were isolated from wastewater of an oil processing plant. The strain possessing the highest lipase activity was identified both biochemically and sequencing of 16S rRNA gene. Then we increase lipase activity by improving conditions of production medium. Also, lipase from this strain was preliminarily characterized for use in industrial application. The 16S rRNA sequensing revealed it as a new strain of Pseudomonas aeruginosa and the type strain was KM110. An overall 3-fold enhanced lipase production [0.76 U mL[-1]] was achieved after improving conditions of production medium. The olive oil and peptone was found to be the most suitable substrate for maximum enzyme production. Also the enzyme exhibited maximum lipolytic activity at 45°C where it was also stably maintained. At pH 8.0, the lipase had the highest stability but no activity. It was active over a pH range of 7.0-10.0. The lipase activity was inhibited by Zn[2+] and Cu[2+] [32 and 27%, respectively] at ImM. The enzyme lost 29% of its initial activity in 1.0% SDS concentration, whereas, Triton X-100, Tween-80 and DMSO did not significantly inhibit lipase activity. Based on the findings of present study, lipase of P. aeruginosa KM110 is a potential alkaline lipase and a candidate for industrial applications such as detergent, leather and fine chemical industries

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Alkaline lipase production by Citrobacter freundii IIT-BT L139.

Around 150 lipase producing bacterial isolates were screened from the local soils enriched with oil. Citrobacrer freundii IIT-BT L139, an isolated microbial strain, produced lipase that had high activity (8.8 U/ml) at pH 9.0 and 40 degrees C. The 16S rDNA phylogenetic studies showed that Citrobacter freundii belongs to the family Enterobacteriaceae and later confirmed by the microbial identification. Suitable C and N sources for lipase production were deduced to be starch and peptone-urea, respectively. In a controlled fermenter (1 L), the lipase activity was found to increase by 36% (12 Uml(-1)). The variation of lipase activity, pH and dissolved oxygen (DO) during growth of the organism in the controlled batch fermenter were monitored. The rheological characteristics of the fermentation broth indicated that it behaved like a Newtonian fluid throughout the fermentation. The fermentation time was comparatively short (60 h). The lipase was also found to be substantially resistant to common detergents. This lipase was, thus, characterized as alkaline, thermostable and solvent stable, which was essentially desirable in pharmaceutical, detergent and other industrial applications or production.

Preliminary studies on production of extracellular lipase by an isolate from soil : Aspergillus flavus oryzae.

In our routine screening programme, using agar diffusion assay method, lipolytic activity was detected around a colony of a fungus. The fungus was isolated from a soil sample which was brought from a location near oil-mill. This lipolytic fungus was then identified to belong to Aspergillus flavus oryzae. A medium was then formulated and optimized which would not only support good growth but also would yield good extracellular lipolytic activity. It was observed that a conventional carbohydrate-protein containing medium supported good growth of the fungus but moderate lipase activity, whereas, a hydrocarbon containing medium, although supported relatively less growth, yielded much more lipase activity.

Isolation and identification of alkaline thermostable lipase producing microorganism and some properties of crude enzyme.

A bacterial strain isolated from soil and identified as Enterococcus faecalis was found capable of producing alkaline thermostable lipase. Optimum pH, temperature and time for enzyme substrate reaction were found to be 8.0, 60 degrees C and 10 min respectively. Phosphates and common surfactants have no or very little inhibitory effects on the activity of the enzyme, whereas bile salts are inhibitory to the enzyme activity. Maximum activity of the enzyme obtained so far is 54.6 IU/ml.

Constitutive lipase [s] from Thermophilic fungi isolated from mud and water samples collected from Aswan High Dam Lake[AHDl]

Twelve strains of thermophilic fungi isolated from mud and water samples of Aswan High Dam Lake [AHDL] were screened for their lipase[s] production. Four strains exhibited the highest lipolytic productivities, thus chosen for studying some factors affecting lipase[s] productivities. Maximum lipase[s] productivities simultaneously with mycelial dry weights were recorded for the four strains on 6th day of incubation at 45 degrees. Lack of lipid material in the production medium did not inhibit lipase[s] synthesis which refers to their constitutive nature. The present fungal lipases were able to hydrolyze both natural and synthetic glycerides. They were particularly active within a temperature range of 45-50 degrees and a pH range of 5.5-6.5. The involvement of the present fungi in the formation of milk products, butter flavors in food products and even in spoilage of groundnuts during storage is suggested

Disponibilidad digestiva in vitro de lipasa y tripsina en suplementos pancreáticos bajo diferentes grados de acidez / In vitro digestive disposability of lipasa in oral prancreatic supplements: a study under differente acidity parameters

Evaluamos el tiempo de desintegración y la actividad digestiva in vitro de ocho suplementos comerciales pancreáticos bajo condiciones de acidez similares a las gastroduodenales. Las muestras se sometieron a un proceso activo de desintegración durante 45 min a pH de 1, 3 o 6, continuándose el proceso a pH en 6, durante 135 min., la actividad de lipasa y tripsina se determinó cada 15 min por titulometría. A pH constante de 6, los productos sin capa entérica y el Creón tuvieron los tiempos de desintegración más cortos; a pH más ácidos, estos tiempos se alargaron, siendo mayores a 90 min en los productos con capa entérica. La actividad de lipasa fue mayor a pH constante de 6 en Creón, Pankreón y Cotazym-C, Onotón y Cotazym-B. Posterior a la exposición a Ph ácido, la biodisponibilidad enzimática disminuyó en todos los productos. El tiempo de desintegración y la activación de las enzimas por ácido deben tomarse en cuenta al prescribir suplementos pancreáticos.

Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions.

Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts. Addition of 2.0% ground nut oil yielded the highest productivity of enzyme. Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively. Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold. The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml). Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively. The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate.

Lipases of fungi isolated from Wadi Qena, eastern desert of Egypt

A screening program concerning lipolytic fungi of Wadi Qena of the Eastern Desert of Egypt was undertakenOf 105 fungal isolates, 42.8% were lipolytic of which 13.3%, 11.4%, 2.9% and 15.2% belonging to Aspergillus, Penicillium, Alternaria and other genera, respectivelyThe most potent lipolytic fungal species were Penicillium aurantiogriseum Dierck and Alternaria alternata [Fries] KeisslerOptimal conditions for maximum lipase[s] production were recorded as follows: Incubation period, 5 days, incubation temperature 30C +/- 2, the best lipid is coconut oil at the level of 0.2% [v/v], the best nitrogen source is peptone at the concentration of 0.3% [w/v]The best buffer is either phosphate or phosphate citrate [0.2 M and pH 5.8]The best divalent cation is Ca2+ at the concentration level of 50 ppm and shaking at 120 rpmUnder these conditions lipases yields increased up to 550 ug/ml in case of Paurantiogriseum and 1100 ug/ml in case of Alternaria alternataA suggestion was given to increase this yield by applying the production under large scale because the present lipases attack both simple and complex fats which gives some hope to apply them in the field of food technology or even as an adjuvant in industrial detergents

Metabolismo de quilomicros / Chylomicron metabolism

O fígado exerce um importante papel no metabolismo dos quilomicros. É responsável pela síntese de apolipoproteínas (ex., apo AI, apo CII, apo E), lecitina-colesterol-acil-tranferase e lipase de remover os remanescentes de quilomicros da circulaçäo sangüínea. Näo é surpresa portanto que o metabolismo de quilomicros esteja comprometido nas doenças hepáticas tais como hepatite aguda, cirrose e colestase. Assim, sendo, os autores revisaram os aspectos normais do metabolismo dos quilomicros os quais säo essencias para uma melhor compreensäo das alteraçöes lipoprotéicas observadas nas doenças hepáticas