Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract

Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.     

Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Production,characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0–11.0) and moderate salinity (0–5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128?U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl₂?·?2H₂O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42?hr of incubation at 37°C. It was active over broad range of pH (7.0–12.0), temperature (15–100°C), and salinity (0–9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0–12.0 and salinities of 0–5% NaCl for 48?hr. Cu²⁺, Mn²⁺, Co²⁺, and Ca²⁺ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant–detergent-stable partially purified extracellular protease from A. caviae.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.     

Production,purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21?U/mL, which was ～2.3-fold higher than that of the wild strain (2.04?U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32?°C, pH 7.0, 150?rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25?mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30?kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ～21-fold, and the mean overall yield was ～6%. The purified endoglucanase was active up to 80?°C and showed a half-life of 214?min at 60?°C in the absence of substrate at pH 8.0. The apparent K_m value for the purified endoglucanase was 0.70?mg/mL, while the V_max value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30?U of proteinase K/mg. The addition of Mg⁺² and Ca⁺² (5?mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5?mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4?°C and 75 days at ?20?°C signify the suitability of this enzyme for industrial applications as detergent additive.     

Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p-dioxane, or acetonitrile/water at pH ? pK_a. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π-π* and n-π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one-term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x-ray diffraction studies.     

Effects of pH and phosphate on the production of struvite by Myxococcus xanthus

We studied the influence of pH and the phosphate content of the culture medium on the precipitation of struvite by Myxococcus xanthus, a bacterium that undergoes autolysis at the end of its exponential growth phase in liquid cultures. The best results were obtained with pH values between 7.2 and 8.0 and with a phosphate concentration of 10 mM. Our studies reveal for the first time that the precipitation of struvite always begins at the onset of autolysis and that culture conditions favoring the early occurrence of autolysis also enhance struvite production.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

Autolysis of disintegrated cells of the yeast saccharomyces cerevisiae

Optimum conditions for autolysis of disintegrated cells of S. cerevisiae are at 50–53°C and pH 5.5; the process is terminated after 6 h. In the presence of sodium chloride (3–5%) the autolysis is complete after 5 h. The yield of autolysis of disintegrated yeast cells is about 70% of autolytic product per yeast dry weight. The product obtained after centrifugation, filtration and drying has very good sensoric and physical properties.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Partial purification and some properties of lipoxygenase from Saccharomyces cerevisiae

A partially purified lipoxygenase extract was obtained from the yeast Saccharomyces cerevisiae by precipitation with solid (NH₄)SO₄ at 20% to 80% saturation. The enzyme had two pH optima, at pH 8.0 and 10.0, with respective apparent K _m values of 13 and 9.5 m. At both pH optima, the lipoxygenase demonstrated highest substrate specificity towards linoleic acid, followed by linolenic acid; although the enzyme had less specificity towards mono-linolein than di-linolein at pH 8.0, the reverse was true at pH 10.0.     

Assessment of spectroscopic parameters of solvated Eu(dmh)3phen organometallic complex in various basic and acidic solvents

We report on the comprehension of novel europium activated hybrid organic Eu(dmh)₃phen (Eu: europium, dmh: 2,6‐dimethyl‐3,5‐heptanedione, phen: 1,10 phenanthroline) organo‐metallic complexes, synthesized at different pH values by the solution technique. Photo physical properties of these complexes in various basic and acidic solvents were probed by UV–vis optical absorption and photoluminescence (PL) spectra. Minute differences in optical absorption peaks with variable optical densities were encountered with the variation in solvent from basic (chloroform, toluene, tetrahydrofuran) to acidic (acetic acid) media, revealing bathochromic shift in the absorption peaks. The PL spectra of the complex in various acidic and basic organic solvents revealed the position of the emission peak at 613 nm irrespective of the changes in solvents whereas the excitation spectrum almost matched with that of the UV–vis absorption data. The optical density was found to be maximum for the complex with pH 7.0 whereas it gradually decreased when pH was lowered to 6.0 or raised to 8.0 at an interval of 0.5, demonstrating its pH sensitive nature. Several spectroscopic parameters related to probability of transition such as absorbance A(λ), Napierian absorption coefficient α(λ), molecular absorption cross‐section σ(λ), radiative lifetime (τ₀) and oscillator strength (f) were calculated from UV–vis spectra. The relative intensity ratio (R‐ratio), calculated from the emission spectra was found to be almost the same in all the organic solvents. The optical energy gap, calculated for the designed complexes were found to be well in accordance with the ideal acceptance value of energy gap of the emissive materials used for fabrication of red organic light‐emitting diode (OLED). The relation between Stoke's shift and solvent polarity function was established by Lippert–Mataga plot. This remarkable independence of the electronic absorption spectra of Eu complexes on the nature of the solvent with unique emission wavelength furnishes its potential to serve as a red light emitter for solution processed OLEDs, display panels and solid‐state lighting.     

Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (K_m), and catalytic turnover rate (V_max/[E]_t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V_max/[E]_t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s⁻¹, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s⁻¹). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K_m. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.     

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon <Emphasis Type="Italic">Natrialba magadii</Emphasis>

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.     

Effect of salt on the stability of the pH 4.2 rA8 double helix in a series of organic/aqueous mixed solvents: A test of oligoelectrolyte theory

Temperature-dependent uv absorption spectroscopy has been used to investigate the salt dependence of the order–disorder transition for the pH 4.2 rA₈ double helix in 100% aqueous buffer and in a series of organic/aqueous mixed solvents. Melting temperature, T_m, data were obtained for the transitions in the different solvents by analysis of the uv melting curves. For the pure aqueous buffer solvent, the melting temperature was found to exhibit a reduced salt dependence (?t_m/? log Na⁺) when compared to the corresponding polymer. This reduction is explained in terms of end effects and is shown to be consistent with the theoretical treatments of oligoelectrolyte transitions developed by Record and Lohman [Biopolymers, 17 , 159–166 (1978)]. In the mixed solvents, the salt dependence of the melting temperature (?t_m/? log Na⁺) is shown to exhibit a linear dependence on the bulk dielectric constant of the medium for all of the hydroxyl-containing solvents studied. Significantly, N,N-dimethylformamide demonstrated different behavior.     

Characteristics of an extracellular protease isolated from<Emphasis Type="Italic">Bacillus subtilis</Emphasis> AG-1 and its performance in relation to detergent components

An extracellular protease isolated fromBacillus subtilis AG-1 was investigated with respect to various detergents and formulation components. The enzyme had optimum at pH 8.0 and 60 °C temperature while zymographic study revealed two activity bands of 24.9 and 18 kDa. It showed high stability towards non-ionic (Tween 20, Tween 80, Triton X-100) and anionic surfactants sodium dodycyl sulfate (SDS), retaining 100 and 71% of its original activity. Another distinctive feature of the enzyme was its efficient stability towards hydrogen peroxide (H₂O₂) and sodium perborate and different commercial detergent brands. AG-1 protease was also examined for its activity/performance in combination with different stabilizers like glycerol, propylene glycol and polyethylene glycol (PEG). Enzyme showed a promising activity in the presence of this polyols especially PEG (8000). Whilst its compatibility with different commercially available powder and liquid detergents was also very interesting. These results suggest AG-1 protease as a good detergent compatible and can be utilized in the formulation of an environment friendly bio-detergent.     

Functional enrichment of mannanase-treated spent brewer yeast

In this study, the effect of Bacillus amyloliquefaciens-produced β-mannanase on the nutrient diffusion (release) and antioxidant activity of spent brewer yeast (SBY) was investigated. Three pretreatments were performed: (1) autolysis at 50°C for 24?h; (2) autolysis at 50°C for 24?h, with the addition of β-mannanase during the autolysis; (3) autolysis at 50°C for 24?h, and the β-mannanase was added for another 12?h treatment. The pretreatments with the addition of β-mannanase caused significant cell wall degradation, markedly increased the yield of SBY extracts. More importantly, this study found that polysaccharides were degraded to be oligosaccharides with a considerable reduction in molecular weights. Meanwhile, pretreatment with the enzyme also exhibited a higher antioxidant activity in SBY extract compared to autolysis itself. The current study indicated that pretreatment (3) had a better effect than pretreatment (2) in terms of improving in antioxidant activity in SBY extract. These improved characteristics of SBY extracts isolated through enzymatic treatment appear to show promising features for their prospective use as natural functional agents.