Melissa L. E. Gutarra

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

Lipase production by solid-state fermentation: cultivation conditions and operation of tray and packed-bed bioreactors.

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

Lipase production by solid-state fermentation in fixed-bed bioreactors

In the present work, packed bed bioreactors were employed with the aim of increasing productivity and scaling up of lipase production using Penicillium simplicissimum in solid-state fermentation. The influence of temperature and air flow rate on enzyme production was evaluated employing statistical experimental design, and an empirical model was adjusted to the experimental data. It was shown that higher lipase activities could be achieved at lower temperatures and higher air flow rates. The maximum lipase activity (26.4 U/g) was obtained at the temperature of 27°C and air flow rate of 0.8 L/min.

Lipase Production by Solid-State Fermentation

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.

Enzyme Surface Glycosylation in the Solid Phase: Improved Activity and Selectivity of Candida Antarctica Lipase B

Tailor‐made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL‐B) already immobilized on octyl‐Sepharose by interfacial activation. The chemical modification was performed in the N‐terminal amino acid enzyme residue by using low oxidized aldehyde–dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL‐B. These immobilized conjugates were compared with a genetically glycosylated version of CAL‐B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran‐1500 and also by the genetic glycosylation, retaining 90–96 % activity after 24 h at 55 °C. The catalytic activity of CAL‐B was improved by the incorporation of dextran polymers (Mw=1500 or 6000) more than twofold in the hydrolysis of p‐nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH 7. However, the activity of the genetically glycosylated CAL‐B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL‐B increased for all formed bioconjugates, with the Dextran‐1500–CAL‐B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1 % ee at pH 5). This glycosylated CAL‐B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80 % yield of monoglyceryl ester at 100 % conversion compared to 57 % yield obtained with unmodified CAL‐B or other polymer–lipase conjugates.

Lipase production and Penicillium simplicissimum morphology in solid‐state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.

Immobilization and Characterization of a Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus on Supports with Different Degrees of Hydrophobicity

We studied the immobilization of a recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic archaeon Pyrococcus furiosus on supports with different degrees of hydrophobicity: butyl Sepabeads and octadecyl Sepabeads. The enzyme was strongly adsorbed in both supports. When it was adsorbed on these supports, the enzyme showed 140 and 237% hyperactivation, respectively. The assessment of storage stability showed that the octadecyl Sepabeads immobilized enzyme showed 100% of residual activity after 30 days of storage. However, the greatest stability at 70°C was obtained in butyl Sepabeads immobilized enzyme, which retained 77% activity after 1 hour incubation. The maximum activity of the immobilized preparations was obtained with the pH between 6 and 7, at 70°C. Thus, this study achieved a new extremophilic biocatalyst with greater stability, for use in several biotechnological processes.

Optimization of Magnetosome Production and Growth by the Magnetotactic Vibrio Magnetovibrio blakemorei Strain MV-1 through a Statistics-Based Experimental Design

ABSTRACT The growth and magnetosome production of the marine magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 were optimized through a statistics-based experimental factorial design. In the optimized growth medium, maximum magnetite yields of 64.3 mg/liter in batch cultures and 26 mg/liter in a bioreactor were obtained.

Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support

The immobilization of Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74, on a new heterofunctional support consisted of phenylboronic acid and aldehyde groups (Borald) has been performed. This method occurs via a two step mechanism: first orientation by organoborane interaction at neutral pH and a consecutive multi-point covalent attachment by aldehyde reaction at alkaline pH. The enzyme was specifically immobilized on this support in 70% yield at pH 7, oriented by the reaction of the hydroxyl groups on the sugar moiety with boronic acid on the support, whereas commercial CAL-B from Novozymes (non-glycosylated) was hardly immobilized at this pH ( 99% final immobilization yield. The Borald-CAL-B preparation was a very stable biocatalyst in the presence of high amount of solvent or high temperature (e.g. more than 10 fold in the presence of 60% (v/v) acetonitrile). An improvement of the specific activity up to 5 fold for example in the hydrolysis of methyl phenyl acetate was obtained compared with a one-point covalent preparation. An ee of 89% towards R isomer was achieved with this new immobilized biocatalyst in the enantioselective hydrolysis of methyl mandelate.