Preparation of a heterogeneous biocatalyst through Thermomyces lanuginosus lipase immobilization on pore-expanded SBA-15.

Heterogeneous biocatalysts were prepared by adsorbing T. lanuginosus lipase (TLL) onto uncalcined (SBAUC-TLL) and calcined (SBAC-TLL) SBA-15, using ammonium fluoride as a pore expander to facilitate TLL immobilization. At an enzyme load of 1 mg/g, high immobilization yields (>90 %) and recovered activities (>80 % for SBAUC-TLL and 70 % for SBAC-TLL) were achieved. When increasing the enzyme load to 5 mg/g, the immobilization yield of SBAUC-TLL was 80 %, and the recovered activity was 50 %, while SBAC-TLL had a yield of 100 % and a recovered activity of 36 %. Crosslinking with glutaraldehyde (GA) was conducted to improve stability (SBAUC-TLL-GA and SBAC-TLL-GA). Although SBAC-TLL-GA lost 25 % of initial activity after GA modifications, it exhibited the highest thermal (t1/2 = 5.7 h at 65 °C), when compared to SBAC-TLL (t1/2 = 12 min) and the soluble enzyme (t1/2 = 36 min), and operational stability (retained 100 % activity after 5 cycles). Both biocatalysts presented high storage stability since they retained 100 % of initial activity for 30 days. These results highlight SBA-15's potential as an enzyme support and the protocol's efficacy in enhancing stability, with implications for industrial applications in the food, chemical, and pharmaceutical sectors.

Enzyme immobilization technology as a tool to innovate in the production of biofuels: A special review of the Cross-Linked Enzyme Aggregates (CLEAs) strategy.

This review emphasizes the crucial role of enzyme immobilization technology in advancing the production of two main biofuels, ethanol and biodiesel, with a specific focus on the Cross-linked Enzyme Aggregates (CLEAs) strategy. This method of immobilization has gained attention due to its simplicity and affordability, as it does not initially require a solid support. CLEAs synthesis protocol includes two steps: enzyme precipitation and cross-linking of aggregates using bifunctional agents. We conducted a thorough search for papers detailing the synthesis of CLEAs utilizing amylases, cellulases, and hemicellulases. These key enzymes are involved in breaking down starch or lignocellulosic materials to produce ethanol, both in first and second-generation processes. CLEAs of lipases were included as these enzymes play a crucial role in the enzymatic process of biodiesel production. However, when dealing with large or diverse substrates such as lignocellulosic materials for ethanol production and oils/fats for biodiesel production, the use of individual enzymes may not be the most efficient method. Instead, a system that utilizes a blend of enzymes may prove to be more effective. To innovate in the production of biofuels (ethanol and biodiesel), enzyme co-immobilization using different enzyme species to produce Combi-CLEAs is a promising trend.

Genipin and glutaraldehyde based laccase two-layers immobilization with improved properties: New biocatalysts with high potential for enzymatic removal of trace organic contaminants.

This research proposes the preparation of a two-layer laccase biocatalyst using genipin or/and glutaraldehyde as cross-linking agents. The multilayer biocatalysts were prepared using different combinations of genipin and glutaraldehyde in the individual preparation of the first and second laccase layers. First, chitosan was treated with genipin or glutaraldehyde, followed by the immobilization of the first laccase layer to form a single-layer biocatalyst. Then, the immobilized laccases were coated once again with genipin or glutaraldehyde, and a new laccase layer was immobilized onto the system, resulting in the final two-layer biocatalyst. Compared to the single-layer biocatalysts, catalytic activity increased 1.7- and 3.4-fold when glutaraldehyde coating was used to prepare the second laccase layer. However, adding a second layer did not always produce more active biocatalysts, since the two-layer biocatalysts prepared with genipin (GenLacGenLac and GluLacGenLac) presented a decrease in activity of 65% and 28%, respectively. However, these two-layer biocatalysts prepared with genipin maintained 100% of their initial activity after 5 cycles of ABTS oxidation. Nevertheless, the two-layer, genipin-coated biocatalyst resulted in a higher removal of trace organic contaminants, since it removed 100% of mefenamic acid and 66% of acetaminophen, compared with the glutaraldehyde-coated biocatalyst, which removed 20% of mefenamic acid, and 18% of acetaminophen.

Immobilization of Thermomyces lanuginosus lipase on a new hydrophobic support (Streamline phenyl™): Strategies to improve stability and reusability.

This paper establishes an efficient protocol for the immobilization of Thermomyces lanuginosus lipase (TLL) on a hydrophobic resin, Streamline phenyl. The biocatalyst produced by TLL immobilization on Streamline phenyl resin was named iTLL. In addition, strategies to improve stability and reusability of iTLL were performed using polyethylenimine (PEI) or/and glutaraldehyde (GA), producing iTLL-GA, iTLL-PEI, iTLL-PEI-GA biocatalysts. The immobilization yield was about 50%, using 1 mg/g of enzyme loading, and the immobilized enzyme activity was about 77 U/g, achieving about 100% of recovered activity. Desorption assays of the enzyme from the support using 0.6% cetyltrimethylammonium bromide (CTAB) and thermal and operational stability assays were performed. Although iTLL-PEI-GA lost about 50% of its initial activity after PEI and GA modifications, it was the most thermally and operationally stable (increases its stability about 66% if comparing with soluble enzyme at 65 ºC and maintenance 90% of its initial activity after 5 cycles of pNPB hydrolysis at 25 °C and pH 7.0). Furthermore, it showed almost no desorption of enzyme molecules with 24 h of CTAB incubation. Moreover, the streamline phenyl demonstrated a high TLL loading potential, with no diffusion limitations up to 14 mg/g. These characteristics allow future application of the iTLL-PEI-GA biocatalyst in fluidized bed reactors.

Synthesis of organic-inorganic hybrid nanoflowers of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL): Improvement of thermal stability and reusability.

Enzyme immobilization is used to improve the application of enzymes, allowing the reuse of biocatalysts and increasing their stability under reaction conditions. Immobilization of enzymes through structures, such as nanoflowers, is an innovative, simple, and low-cost method compared to other techniques. In this context, the main objective of this work is to synthesize hybrid biocatalytic nanostructures, similar to flowers, of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL). The production of nanoflowers occurred by precipitation of lipases with CuCl2 or CuSO4 salts for 72 h. However, challenges and obstacles were faced in obtaining effective and practical nanoflowers, such as nanoflowers' low thermal stability and reusability. To overcome these challenges, two conditions were tested: nanoflowers cross-linked with glutaraldehyde and nanoflowers and nanoparticles cross-linked with glutaraldehyde. This last biocatalyst prepared by CuSO4 precipitation showed better thermal stability (half-life about 230 and 233 min for CALB and TLL, respectively, under incubation at 60 °C and pH 7). The CALB biocatalyst retained 70 % of its initial activity (2.31 U) after 10 cycles of hydrolysis. Therefore, this work shows not only the problems and barriers of nanoflowers synthesis, but also the possibility of producing more stable and efficient biocatalysts using improved protocols.

Effect of enzymatic hydrolysis on digestibility and morpho-structural properties of hydrothermally pre-treated red rice starch.

The objective of this work was to evaluate the effects of enzymatic hydrolysis on digestibility and morphological and structural properties of hydrothermally pre-treated (HPT) red rice starch. The pre-treatments were performed in autoclave and cooking for the modification of rice grains and native starch. In vitro starch digestibility was performed consecutively and semi-simultaneously using α-amylase and amyloglucosidase. A first-order mathematical model was used to adjust the hydrolysis kinetic data, which made it possible to calculate the surface area, hydrolysis index, and glycemic index of the starch. Scanning electron microscopy images (SEM), Fourier transform infrared (FTIR) spectra and X-ray diffraction (XRD) were also performed to investigate the characteristics of the post-hydrolysis starch samples. The autoclaved starch HSS-A3, which was subjected to 121 °C/1.08 bar for 10 min, showed the highest in vitro digestibility values (80.08 %). Both starch samples showed increase of particle size and enzymatic digestibility after HPT. FTIR spectra of the starch samples showed that there was no appearance of new functional groups. However, XRD evidenced that HPT changed the intensity of the peaks and the type of crystallinity was changed for autoclaved starch (A3) from type A to Vh, with crystallinity ranging from 21.71 % to 26.42 %. The semi-simultaneous approach showed more advantages due to the highest in vitro digestibility as well as reducing the processing time and use of reagents.

Modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis.

The objective of this study was to evaluate the modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis. In vitro digestibility and the morphological, structural, functional, thermal, textural and rheological properties of red rice starch were evaluated. The starch submitted to autoclave (A3) obtained the highest hydrolysis yield (37.66 %) after 300 min. The morphological analysis showed that for the native starch, the granules presented a polyhedral shape and increased in diameter (2.36-394.12 µm) due to hydrothermal pre-treatments. α-Amylase (9 U mg-1) from Aspergillus oryzae modified the structure of red rice starch, presenting technological properties different from native starch. X-ray diffraction (XDR) were altered after the starch granules were cooked, showing a rupture in the amylose and amylopectin molecules, which justifies the greater absorption capacity of oil and milk. Cohesiveness, adhesiveness and apparent viscosity decreased according to HPT temperature and pressure, as well as α-amylase action.

Immobilization of Amano lipase AK from Pseudomonas fluorescens on different types of chitosan-containing supports: use in the kinetic resolution of rac-indanol.

Amano lipase AK from P. fluorescens was immobilized on different types of chitosan-containing supports. Chitosan lower molecular weight (2.5%), chitosan lower molecular weight/sodium alginate (2.5%/2.5%) and chitosan lower molecular weight/carrageenan (2.5%/2.5%) allowed the highest values of immobilization yields (IY) of 81, 81 and 83%, respectively. Best activity results were achieved using chitosan average molecular weight (5%) and chitosan lower molecular weight/sodium alginate (2.5%/2.5%) as support, with values of 1.40 and 1.30 UpNPB/ggel and with recovery activities of 45.75 and 35.6%, respectively. These derivatives were evaluated in the kinetic resolution of rac-indanol to obtain a key intermediate in the synthesis of a drug used in the treatment of Parkinson's disease. The most efficient derivatives in the kinetic resolution were lipase immobilized on chitosan average molecular weight (5.0%) and chitosan low molecular weight/sodium alginate, the latter leading to obtaining both (S)-indanol and (R)-indanyl acetate with > 99% ee and 50% conversion.

Further stabilization of lipase from Pseudomonas fluorescens immobilized on octyl coated nanoparticles via chemical modification with bifunctional agents.

The lipase from Pseudomonas fluorescens (PFL) was adsorbed on superparamagnetic NiZnFe2O4 octyl-nanoparticles via interfacial activation, producing the biocatalyst OCTYL-NANO-PFL. In order to further improve the stability of the immobilized lipase, the immobilized enzyme biocatalyst was chemically modified with different concentrations of diverse bifunctional molecules (glutaraldehyde (GA), divinylsulfone (DVS) or p-benzoquinone (BQ)). The concentrations of bifunctional agents were varied (0.5, 1, 2.5 and 5% (v/v for GA and DVS and w/v for BQ)). The results showed a greatly improved stability after chemical modification with all bifunctional molecules, mainly with 5% (v/v) GA or 1% (v/v) DVS. The biocatalysts OCTYL-NANO-PFL-GA 5% and -DVS 1% were about 60 folds more stable at pH 7 than the unmodified preparation and, at pH 5, >200 folds for 5% GA modified enzyme. The most stable BQ treated biocatalysts, OCTYL-NANO-PFL-BQ 0.5%, was about 8.3 more stable than OCTYL-NANO-PFL at pH 7, while was 20 fold more stable at pH 9.

Comparison of the immobilization of lipase from Pseudomonas fluorescens on divinylsulfone or p-benzoquinone activated support.

NiZnFe2O4 superparamagnetic nanoparticles were coated with silica by impregnation with tetraethoxysilane (TEOS) and further activated with divinylsulfone (DVS) and p-benzoquinone (BQ) for covalent immobilization lipase from Pseudomonas fluorescens (PFL), producing the biocatalysts TEOS-NANO-DVS-PFL and TEOS-NANO-BQ-PFL. The optimal conditions for enzyme immobilization were found to be pH 7 and 0.1 M of both activating reagents. PFL was also immobilized on TEOS nanoparticles without any activation as a reference (TEOS-NANO-PFL). Results indicated that TEOS could be released from the nanoparticles at alkaline pH value. Optimal TEOS-NANO-PFL exhibited a recovered activity of 55% and a t1/2(60°C) of just over 150 min; while TEOS-NANO-DVS-PFL showed 82% of activity recovered and t1/2(60°C) of 225 min; being the TEOS-NANO-BQ-PFL the biocatalyst offering the best results (89% of recovered activity and a half-life over 1440 min), the maximum enzyme load was ≈300 U/g.

Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead-350.

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac-indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead-350 (IB-350) and on glyoxyl-agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac-indanyl acetate and rac-(chloromethyl)-2-(o-methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB-350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB-IB-350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14-fold factor at pH 5-70°C and by a 11-fold factor in dioxane 30%-65°C) and that of the glyoxyl-agarose-CALB (e.g., by a 12-fold factor at pH 10-50°C and by a 21-fold factor in dioxane 30%-65°C). The CALB-IB-350 preparation (with 98% immobilization yield and activity versus p-nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac-indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e.p ) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:878-889, 2018.