Insights on the role of blocking agent on the properties of the lipase from Thermomyces lanuginosus immobilized on heterofunctional support for hydroesterification reactions.

Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features.

An innovative multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells.

The use of multi-enzymatic systems for the industrial production of chemical compounds is currently considered an important green tool in synthetic organic chemistry. Gluconic acid is a multi-functional organic acid widely used in the chemical, pharmaceutical, food, textile, and construction industries. Its industrial production from glucose by fermentation using Aspergillus niger has drawbacks including high costs related to cell growth and maintenance of cell viability. This study presents an innovative one-step multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells in association with amylolytic enzymes. Using soluble starch as substrate, the following results were achieved for 96 h of reaction: 134.5 ± 4.3 g/L gluconic acid concentration, 98.2 ± 1.3 % gluconic acid yield, and 44.8 ± 1.4 gGA/gwhole-cells biocatalyst yield. Although the process has been developed using starch as raw material, the approach is feasible for any substrate or residue that can be hydrolyzed to glucose.

Enzymatic Synthesis of Fatty Acid Isoamyl Monoesters from Soybean Oil Deodorizer Distillate: A Renewable and Ecofriendly Base Stock for Lubricant Industries.

In this study, soybean oil deodorizer distillate (SODD), a mixture of free fatty acids and acylglycerides, and isoamyl alcohol were evaluated as substrates in the synthesis of fatty acid isoamyl monoesters catalyzed by Eversa (a liquid formulation of Thermomyces lanuginosus lipase). SODD and the products were characterized by the chemical and physical properties of lubricant base stocks. The optimal conditions to produce isoamyl fatty acid esters were determined by response surface methodology (RSM) using rotational central composite design (RCCD, 23 factorial + 6 axial points + 5 replications at the central point); they were 1 mol of fatty acids (based on the SODD saponifiable index) to 2.5 mol isoamyl alcohol, 45 °C, and 6 wt.% enzymes (enzyme mass/SODD mass). The effect of the water content of the reactional medium was also studied, with two conditions of molecular sieve ratio (molecular sieve mass/SODD mass) selected as 39 wt.% (almost anhydrous reaction medium) and 9 wt.%. Ester yields of around 50 wt.% and 70 wt.% were reached after 50 h reaction, respectively. The reaction products containing 43.7 wt.% and 55.2 wt.% FAIE exhibited viscosity indices of 175 and 163.8, pour points of -6 °C and -9 °C, flash points of 178 and 104 °C, and low oxidative stability, respectively. Their properties (mainly very high viscosity indices) make them suitable to be used as base stocks in lubricant formulation industries.

Stabilization and operational selectivity alteration of Lipozyme 435 by its coating with polyethyleneimine: Comparison of the biocatalyst performance in the synthesis of xylose fatty esters.

Differently modified Lipozyme 435 (L435) (immobilized lipase B from Candida antarctica) preparations were used as biocatalysts in the esterification reaction to synthesize sugar fatty acid esters (SFAEs) from xylose (acyl acceptor) and lauric/palmitic acids (acyl donors) in methyl ethyl ketone (MEK) solvent. The L435 treatment with polyethyleneimine (PEI) (2; 25; and 750 KDa) prevented the enzyme leakage in the crude sugar ester reaction product. The 2 KDa PEI coating of this enzyme preparation produced the highest enzyme stability in MEK, buffer solutions (pHs 5 and 7), and methanol aqueous phosphate buffer at pH 7. Using an excess of the acyl donor (1:5 xylose: fatty acid molar ratio), high xylose conversions (70-84%) were obtained after 24 h-reaction using both, non-modified and PEI (2 KDa) coated L435, but the PEI treated biocatalyst afforded a higher xylose modification degree. After 5 reuse cycles with the L435 coated with PEI 2 KDa, the xylose conversions only decreased 10%, while with the non-treated biocatalyst they decreased by 37%. The formation of SFAEs was confirmed by mass spectrometry, which showed the presence of xylose mono-, di-, and triesters. They exhibited emulsion capacities close to that of a commercial sucrose monolaurate.

Lipozyme 435-Mediated Synthesis of Xylose Oleate in Methyl Ethyl Ketone.

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates' molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.

Reviewing research on the synthesis of CALB-catalyzed sugar esters incorporating systematic mapping principles.

Rigorous evidence reviews must follow specific guidelines designed to improve transparency, reproducibility, and to minimize biases to which traditional reviews are susceptible. While evidence synthesis methods, such as systematic reviews and maps, have been used in several research fields, the majority of reviews published in the realm of chemical engineering are nonsystematic. In this study, we incorporated principles of systematic mapping to conduct a literature review covering research on the synthesis of sugar fatty acid esters (SFAE) with Candida antarctica lipase B (CALB). Our results showed that the simple monosaccharides were the most cited sugars among studies we conducted. The direct use of renewable raw materials and frequently available resources to produce alternative sugar esters (SE) was scarcely reported in our data set. We found that free fatty acids (FFA) were the most commonly cited acyl donors amongst all publications, with lauric, oleic, and palmitic acids accounting for ∼43% of the occurrences. Tertiary alcohols (ter-butyl alcohol (T-but) and 2-methyl-2-butanol (2M2B)) and ionic liquids were the most used solvents to synthesize SE. The co-occurence analysis of keywords involving solvent terms showed that most of the papers evaluated different solvents as reaction media (mostly in the form of a bisolvent system), also investigating the impact of their choice on sugar ester productivities. Given the potential of reviews informing us of research decisions, this article reveals trends and spaces across CALB-catalyzed SE synthesis research, in addition to introducing a new methodological perspective for developing reviews in the field of chemical engineering.

Immobilization of Eversa® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil.

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.

Stabilization of Glycosylated ß-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

Improving the Yields and Reaction Rate in the Ethanolysis of Soybean Oil by Using Mixtures of Lipase CLEAs.

Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from different sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch.

Evaluation of Strategies to Produce Highly Porous Cross-Linked Aggregates of Porcine Pancreas Lipase with Magnetic Properties.

The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation with protein co-feeders (bovine serum albumin and/or soy protein), use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor (approximately 65% of the equivalent free enzyme activity) and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches.

Performance of Different Immobilized Lipases in the Syntheses of Short- and Long-Chain Carboxylic Acid Esters by Esterification Reactions in Organic Media.

Short-chain alkyl esters and sugar esters are widely used in the food, pharmaceutical and cosmetic industries due to their flavor and emulsifying characteristics, respectively. Both compounds can be synthesized via biocatalysis using lipases. This work aims to compare the performance of commercial lipases covalently attached to dry acrylic beads functionalized with oxirane groups (lipases from Candida antarctica type B-IMMCALB-T2-350, Pseudomonas fluorescens-IMMAPF-T2-150, and Thermomyces lanuginosus-IMMTLL-T2-150) and a home-made biocatalyst (lipase from Pseudomonas fluorescens adsorbed onto silica coated with octyl groups, named PFL-octyl-silica) in the syntheses of short- and long-chain carboxylic acid esters. Esters with flavor properties were synthetized by esterification of acetic and butyl acids with several alcohols (e.g., ethanol, 1-butanol, 1-hexanol, and isoamyl alcohol), and sugar esters were synthetized by esterification of oleic and lauric acids with fructose and lactose. All biocatalysts showed similar performance in the syntheses of short-chain alkyl esters, with conversions ranging from 88.9 to 98.4%. However, in the syntheses of sugar esters the performance of PFL-octyl-silica was almost always lower than the commercial IMMCALB-T2-350, whose conversion was up to 96% in the synthesis of fructose oleate. Both biocatalysts showed high operational stability in organic media, thus having great potential for biotransformations.

Production of Whole-Cell Lipase from Streptomyces clavuligerus in a Bench-Scale Bioreactor and Its First Evaluation as Biocatalyst for Synthesis in Organic Medium.

This work evaluated a wild-type Streptomyces clavuligerus strain as a whole-cell lipase (Sc-WCL) producer by submerged fermentation. In an orbital shaker, lipase hydrolytic activity of 3000 U L-1, measured at pH 9.0 and 37 °C by using p-nitrophenyl palmitate as substrate, was achieved after 36 h fermentation using glycerol-free production medium in a baffled Erlenmeyer flask at 28 °C and pH 6.8. Maximum productivity of 52.5 U L-1 h-1 was achieved after 24 h in bioreactor using glycerol-free production medium at pH 6.8 and 28 °C, with agitation at 400 rpm and aeration at 1 vvm. Sc-WCL was shown to be more active at 60 °C and pH 10.7, while higher activity retention was observed at 30-40 °C after 1 h incubation at pH 10. Sc-WCL showed to have potential to be used as biocatalyst in hydrolysis and esterification reactions. In the hydrolysis of p-nitrophenyl palmitate, lyophilized Sc-WCL expressed a hydrolytic activity (330 units g-1 solid, measured at 37 °C and pH 9.0) around 100-fold higher than the ones declared by a supplier of lyophilized powders of mixtures of intracellular lipases from Thermus thermophiles and Thermus flavus (≥3.0 units g-1 solid, measured at 65 °C and pH 8.0). In the synthesis of butyl butyrate in anhydrous medium, 85% ester conversion was achieved at 37 °C after 8 h reaction. Thus, Sc-WCL showed to be a promising biocatalyst because it is cheaper than the isolated and purified lipases.

Gluconic acid production from sucrose in an airlift reactor using a multi-enzyme system.

Sucrose from sugarcane is produced in abundance in Brazil, which provides an opportunity to manufacture other high-value products. Gluconic acid (GA) can be produced by multi-enzyme conversion of sucrose using the enzymes invertase, glucose oxidase, and catalase. In this process, one of the byproducts is fructose, which has many commercial applications. This work concerns the batch mode production of GA in an airlift reactor fed with sucrose as substrate. Evaluation was made of the influence of temperature and pH, as well as the thermal stability of the enzymes. Operational conditions of 40 °C and pH 6.0 were selected, based on the enzymatic activity profiles and the thermal stabilities. Under these conditions, the experimental data could be accurately described by kinetic models. The maximum yield of GA was achieved within 3.8 h, with total conversion of sucrose and glucose and a volumetric productivity of around 7.0 g L(-1) h(-1).

Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized ß-glucosidase.

The ß-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles.

Immobilization and stabilization of xylanase by multipoint covalent attachment on agarose and on chitosan supports.

Xylanases have important applications in industry. Immobilization and stabilization of enzymes may allow their reuse in many cycles of the reaction, decreasing the process costs. This work proposes the use of a rational approach to obtain immobilized commercial xylanase biocatalysts with optimized features. Xylanase NS50014 from Novozymes was characterized and immobilized on glyoxyl-agarose, agarose-glutaraldehyde, and agarose-amino-epoxy support and on differently activated chitosan supports: glutaraldehyde-chitosan, glyoxyl-chitosan, and epoxy-chitosan. Two different chitosan matrices were tested. The best chitosan derivative was epoxy-chitosan-xylanase, which presented 100% of immobilization yield and 64% of recovered activity. No significant increase on the thermal stability was observed for all the chitosan-enzyme derivatives. Immobilization on glyoxyl-agarose showed low yield immobilization and stabilization degrees of the obtained derivative. The low concentration of lysine groups in the enzyme molecule could explain these poor results. The protein was then chemically modified with ethylenediamine and immobilized on glyoxyl-agarose. The new enzyme derivatives were 40-fold more stable than the soluble, aminated, and dialyzed enzyme (70 degrees C, pH 7), with 100% of immobilization yield. Therefore, the increase of the number of amine groups in the enzyme surface was confirmed to be a good strategy to improve the properties of immobilized xylanase.

Methods and supports for immobilization and stabilization of cyclomaltodextrin glucanotransferase from Thermoanaerobacter.

Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) was immobilized using different supports and immobilization methods to study the effect on activity recovery. The enzyme covalently attached into glyoxyl-silica showed low activity recovery of 1.5%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads yielded also low activity recovery, 3.83%, and the enzyme could easily leak from the support at low ionic strength, although the immobilization yield was satisfactory, approximately 76%. The CGTase encapsulated in a sol-gel matrix gave an activity recovery of 6.94% and maximum cyclization activity at 60 degrees C, at pH 6.0. The half-time life at 60 degrees C, pH 6.0, in the presence of substrate was 100 min, which was lower than that of the free enzyme. The best activity recovery in this work (6.94%) is approximately five times smaller than that obtained previously using glyoxyl-agarose as support and covalent immobilization. Thus, the best support and method we tested so far for immobilization of CGTase is covalent attachment on glyoxyl-agarose.