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The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources and are easy to modify, due to the high variety of functional groups on their surfaces. However, although many lipases have been immobilized on biomaterials and have shown potential for application in biocatalysis, special features are required when the biocatalyst is used in non-conventional media, for example, in organic solvents, which are required for most reactions in organic synthesis. In this article, we discuss the use of biomaterials for lipase immobilization, highlighting recent developments in the synthesis and functionalization of biomaterials using different methods. Examples of effective strategies designed to result in improved activity and stability and drawbacks of the different immobilization protocols are discussed. Furthermore, the versatility of different biocatalysts for the production of compounds of interest in organic synthesis is also described.

A lipolytic fermented solid was produced by solid-state fermentation of Rhizopus microsporus CPQBA 312-07 DRM on a mixture of sugarcane bagasse and sunflower seed meal and used, in a packed-bed bioreactor, to pretreat a high-fat wastewater from a meat and sausage processing factory located in São José dos Pinhais, State of Paraná, Brazil. With a hydraulic residence time of 24 h, this pretreatment not only reduced the wastewater’s oil and grease content by up to 96 %, but also increased its 5-day biochemical oxygen demand to chemical oxygen demand (BOD5/COD) ratio. This ratio was only 0.19 in the raw wastewater, indicating poor biodegradability, but increased to 0.55 in the pretreated wastewater, indicating that it had a sufficiently high biodegradability to be sent to a traditional anaerobic digestion or activated sludge process. After 96 days of operation of the packed bed, a microbiological analysis showed that R. microsporus was still present and viable in the fermented solid. Our work shows that a continuous packed-bed bioreactor containing fermented solid produced by R. microsporus has good potential for the treatment of high-fat wastewater.

A set of experiments to investigate the enzymatic oxidation of 5-o-caffeoylquinic acid with polyphenol oxidases was carried out in the temperature range from 18 to 39 °C. The kinetic data were obtained in a batch isothermal reactor made of quartz and operated at an optimum pH (approx. 7.0) for enzyme activity, using citrate-phosphate buffer. The decay of 5-o-caffeoylquinic acid concentrations was experimentally monitored with a spectrophotometer at 323 nm. A coefficient of molar absorptivity equal to 1.96·103 mM–1m–1 was found for 5-o-caffeoylquinic acid, based on absorbance measurements with standard aqueous solutions. The kinetic experimental results were used to calculate the parameters Km and vmax of the Michaelis-Menten model, which were 0.24 mM and 2.77·10–4 mM/s at 25 °C, respectively. The effects of temperature on the maximum velocity of oxidation of 5-o-caffeoylquinic acid and on the rate of enzyme deactivation were well described using the Arrhenius equation.

The filamentous fungus Rhizopus microsporus CPQBA 312-07 DRM was grown in solid-state cultivation and the fermented solid produced was used to hydrolyze triacylglycerols in a high-fat dairy wastewater. For the solid-state cultivation, a mixture of sunflower seed meal and sugarcane bagasse (1:3 by mass on dry basis) was selected. After 18 h of culture, the fermented product had an activity, measured titrimetrically against triolein, of 26 U per gram of dry solids. This substrate mixture does not suffer from compaction and therefore can be used in large scale solid-state cultivation bioreactors. When used to pretreat a high-fat dairy wastewater, with an oil and grease level above 1300 mg/L, the fermented solid reduced the oil and grease level to below 300 mg/L after 72 h at 35 °C. Further work is required to improve the production of lipolytic activity in the solid-state cultivation step and to find the optimum pretreatment time in the wastewater pretreatment step.

We undertook a preliminary characterization of the tyrosinase produced by a strain of Lentinula boryana from Brazil, with a view to evaluate its potential for biotechnological applications. The enzyme was similar to other fungal tyrosinases in many respects. When the crude extract was characterized, the tyrosinase activity was optimal at pH=6 and was not particularly thermostable, with half-lives of about 10 min and 1 min at 50 and 60 °C, respectively. We purified the enzyme with ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE Sepharose column, obtaining a yield of 33 % and a 5.3-fold enrichment. The purified preparation gave three bands on SDS-PAGE, with molecular masses of 20, 27 and 47 kDa. This preparation showed substrate inhibition kinetics with L-DOPA (3,4-dihydroxy-L-phenylalanine), with a KM of 1.9 mM and a KI of 72 mM. Under the same reaction conditions, a commercial mushroom tyrosinase followed Michaelis-Menten kinetics, with a KM of 0.51 mM. Although the present study did not identify properties that would make the tyrosinase of L. boryana more suitable in biotechnological applications than tyrosinases from other mushrooms, it has made a contribution by showing that the enzyme suffers substrate inhibition by L-DOPA, something that has not previously been reported for mushroom tyrosinases.

Over the last decade there has been a significant interest in developing biotechnological applications of tyrosinases. These applications include the production of L-DOPA (3,4-dihydroxyphenyl-L-alanine) from L-tyrosine, the production of cross-linked protein networks for use as novel food additives and the detection of phenolic compounds in wastewater or their removal from it. Much of the research into these applications has involved mushroom tyrosinases. We review the potential biotechnological applications of mushroom tyrosinases and evaluate the state of knowledge about their production, recovery and immobilization. We conclude that much more research is necessary in these areas if mushroom tyrosinases are to fulfill their biotechnological potential.

In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50 °C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30 °C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

We compare isotherms for soybeans and soybeans fermented with Rhizopus oryzae, showing that in solid-state fermentation the biomass affects the isotherm of the fermenting solids. Equations are developed to calculate, for a given overall water content of the fermenting solids, the water contents of the biomass and residual substrate, as well as the water activity. A case study, undertaken using a mathematical model of a well-mixed bioreactor, shows that if water additions are made on the basis of the assumption that fermenting solids have the same isotherm as the substrate itself, poor growth can result since the added water does not maintain the water activity at levels favorable for growth. We conclude that the effect of the microbial biomass on the isotherm of the fermenting solids must be taken into account in mathematical models of solid-state fermentation bioreactors.

Biocatalysis has become a useful alternative to chemical transformations for the production of a range of compounds with applications in the food, feed, chemical and pharmaceutical industries. However, it is not necessarily an easy task to obtain the desired levels of performance in terms of rate, yield and selectivity of the reaction. One strategy for optimizing biocatalyst performance is to use non-conventional media, such as non-aqueous heterogeneous systems. In this article, we highlight some of the current trends in biocatalysis in such systems, focusing on reverse micelles, supercritical fluids and ionic liquids.

A wild fungal strain isolated from soybean oil and identified as Penicillium aurantiogriseum initially presented a volumetric lipase activity of 0.4 U/mL in submerged culture in a medium containing 0.5 % yeast extract and 1 % olive oil. Studies were undertaken to improve lipase production. The effect of nitrogen source was studied by adding casein peptone, meat peptone, yeast extract or ammonium sulfate to a medium containing potassium nitrate and other mineral salts. The best yield, of 13 U/mL after 72 h, was obtained with the medium supplemented with ammonium sulfate. With the ammonium sulfate concentration increased to double the C/N ratio from 2.5 to 5, a lipolytic activity of 18 U/mL was obtained. Olive, corn, soy and sunflower oils were tested as carbon sources in this medium, with olive oil at 1 % giving a lipolytic activity of 25 U/mL after 48 h, the highest yield obtained in this study. Enzyme production was best at 29 °C, within a range tested from 26 to 32 °C. These results are promising because this strain produces lipase in an inexpensive inorganic medium and we succeeded in increasing the lipolytic activity 62-fold over the initial values obtained with the non-optimized medium.

A mathematical model is developed for converting between the two measurement bases commonly used in the construction of growth profiles in solid-state fermentation, namely absolute mass ratio m(dry biomass)/m(initial dry matter) and relative mass ratio m(dry biomass)/m(dry matter). These are not equivalent, due to the loss of dry matter as CO2 during the fermentation. The model is equally applicable to any biomass component used in indirect measurements of growth, such as protein. Use of the model to convert absolute mass ratio of the biomass profiles for the growth of Rhizopus oligosporus to a relative basis gave profiles that agreed well with the experimentally determined relative biomass profiles. This agreement was obtained for three different fermentations using the same set of parameter values in the model, namely a yield coefficient of m(protein)/m(dry substrate) = 0.2 g/g and a maintenance coefficient of zero, giving confidence in the reliability of the model. The model was then used to show that the measurement basis used can affect the form of the curve and therefore can also affect the conclusion drawn about the type of kinetics shown by the organism, with the extent of this effect depending on the length of time that growth occurs and the values of the yield and maintenance coefficients. This work shows that great care must be taken in drawing conclusions about growth kinetics in solid-state fermentation.

A mathematical model is developed for converting between the two measurement bases commonly used in the construction of growth profiles in solid-state fermentation, namely absolute mass ratio m(dry biomass)/m(initial dry matter) and relative mass ratio m(dry biomass)/m(dry matter). These are not equivalent, due to the loss of dry matter as CO2 during the fermentation. The model is equally applicable to any biomass component used in indirect measurements of growth, such as protein. Use of the model to convert absolute mass ratio of the biomass profiles for the growth of Rhizopus oligosporus to a relative basis gave profiles that agreed well with the experimentally determined relative biomass profiles. This agreement was obtained for three different fermentations using the same set of parameter values in the model, namely a yield coefficient of m(protein)/m(dry substrate) = 0.2 g/g and a maintenance coefficient of zero, giving confidence in the reliability of the model. The model was then used to show that the measurement basis used can affect the form of the curve and therefore can also affect the conclusion drawn about the type of kinetics shown by the organism, with the extent of this effect depending on the length of time that growth occurs and the values of the yield and maintenance coefficients. This work shows that great care must be taken in drawing conclusions about growth kinetics in solid-state fermentation.

In this work a method of analyzing the performance of solid-state fermentation bioreactors is described. The method is used to investigate the optimal value for the spacing between the cooling plates of the Zymotis bioreactor, using simulated fermentation data supplied by a mathematical model. The Zymotis bioreactor has good potential for those solid-state fermentation processes in which the substrate bed must remain static. The current work addresses two design parameters introduced by the presence of the internal heat transfer plates: the width of the heat transfer plate, which is governed by the amount of heat to be removed and the pressure drop of the cooling water, and the spacing between these heat transfer plates. In order to analyze the performance of the bioreactor a productivity term is introduced that takes into account the volume occupied within the bioreactor by the heat transfer plates. As part of this analysis, it is shown that, for logistic growth kinetics, the time at which the biomass reaches 90 % of its maximum possible value is a good estimate of the optimum harvesting time for maximizing productivity. Application of the productivity analysis to the simulated fermentation results suggests that, with typical fast growing fungi ( = 0.324 h–1), the optimal spacing between heat transfer plates is of the order of 6 cm. The general applicability of this approach to evaluate the productivity of solid-state bioreactors is demonstrated.

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

Immobilized and stabilized lipases are important biocatalytic tools. In this paper, different tailor-made bifunctional supports were prepared for the immobilization of a new metagenomic lipase (LipC12). The new supports contained hydrophobic groups (different alkyl groups) to promote interfacial adsorption of the lipase and aldehyde groups to react covalently with the amino groups of side chains of the adsorbed lipase. The best catalyst was 3.5-fold more active and 5000-fold more stable than the soluble enzyme. It was successfully used in the regioselective deacetylation of peracetylated d-glucal. The PEGylated immobilized lipase showed high regioselectivity, producing high yields of the C-3 monodeacetylated product at pH 5.0 and 4 °C.

Crystals of Mg/Al layered double hydroxide were synthesized by alkaline precipitation and treated in an aqueous solution of glutamic acid. The glutamate ions were not intercalated into the interlayer space, but were detected in the material by Fourier transform infrared spectroscopy, suggesting that only the external surfaces of crystals were modified with glutamate ions. The resulting hybrid material was tested as a support for immobilization of the enzyme laccase (Myceliophthora thermophila). The immobilized enzyme preparation was characterized by electronic paramagnetic resonance spectroscopy and by assays of catalytic activity. The activity of the immobilized laccase was 97% of the activity in the free enzyme. Layered double hydroxide is a suitable support for use in remediation of soil studies.