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.

Superabsorbent bacterial cellulose film produced from industrial residue of cashew apple juice processing.

The industrial residue of cashew apple juice processing (MRC) was evaluated as an alternative medium for bacterial cellulose (BC) production by Komagataeibacter xylinus ATCC 53582 and Komagataeibacter xylinus ARS B42. The synthetic Hestrin-Schramm medium (MHS) was used as a control for growing and BC production. First, BC production was assessed after 4, 6, 8, 10, and 12 days under static culture. After 12 days of cultivation, K. xylinus ATCC 53582 produced the highest BC titer in MHS (3.1 g·L-1) and MRC (3 g·L-1), while significant productivity was attained at 6 days of fermentation. To understand the effect of culture medium and fermentation time on the properties of the obtained films, BC produced at 4, 6, or 8 days were submitted to infrared spectroscopy with Fourier transform, thermogravimetry, mechanical tests, water absorption capacity, scanning electron microscopy, degree of polymerization and X-ray diffraction. The properties of BC synthesized in MRC were identical to those of BC from MHS, according to structural, physical, and thermal studies. MRC, on the other hand, allows the production of BC with a high water absorption capacity when compared to MHS. Despite the lower titer (0.88 g·L-1) achieved in MRC, the BC from K. xylinus ARS B42 presented a high thermal resistance and a remarkable absorption capacity (14664 %), suggesting that it might be used as a superabsorbent biomaterial.

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.

ß-Galactosidase from Kluyveromyces lactis: Characterization, production, immobilization and applications - A review.

A review on the enzyme ß-galactosidase from Kluyveromyces lactis is presented, from the perspective of its structure and mechanisms of action, the main catalyzed reactions, the key factors influencing its activity, and selectivity, as well as the main techniques used for improving the biocatalyst functionality. Particular attention was given to the discussion of hydrolysis, transglycosylation, and galactosylation reactions, which are commonly mediated by this enzyme. In addition, the products generated from these processes were highlighted. Finally, biocatalyst improvement techniques are also discussed, such as enzyme immobilization and protein engineering. On these topics, the most recent immobilization strategies are presented, emphasizing processes that not only allow the recovery of the biocatalyst but also deliver enzymes that show better resistance to high temperatures, chemicals, and inhibitors. In addition, genetic engineering techniques to improve the catalytic properties of the ß-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the ß-galactosidase enzyme from K. lactis, aiming to improve existing bioprocesses or develop new ones.

The ß-galactosidase immobilization protocol determines its performance as catalysts in the kinetically controlled synthesis of lactulose.

In this paper, 3 different biocatalysts of ß-galactosidase from Kluyveromyces lactis have been prepared by immobilization in chitosan activated with glutaraldehyde (Chi_Glu_Gal), glyoxyl agarose (Aga_Gly_Gal) and agarose coated with polyethylenimine (Aga_PEI_Gal). These biocatalysts have been used to catalyze the synthesis of lactulose from lactose and fructose. Aga-PEI-Gal only produces lactulose at 50 °C, and not at 25 or 37 °C, Aga_Gly_Gal was unable to produce lactulose at any of the assayed temperatures while Chi_Glu_Gal produced lactulose at all assayed temperatures, although a lower yield was obtained at 25 or 37 °C. The pre-incubation of this biocatalyst at 50 °C permitted to obtain similar yields at 25 or 37 °C than at 50 °C. The use of milk whey instead of pure lactose and fructose produced an improvement in the yields using Aga_PEI_Gal and a decrease using Chi_Glu_Gal. The operational stability also depends on the reaction medium and of biocatalyst. This study reveals how enzyme immobilization may greatly alter the performance of ß-galactosidase in a kinetically controlled manner, and how medium composition influences this performance due to the kinetic properties of ß-galactosidase.

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.

Simultaneous hydrolysis of cheese whey and lactulose production catalyzed by ß-galactosidase from Kluyveromyces lactis NRRL Y1564.

ß-Galactosidase was produced by the yeast Kluyveromyces lactis NRRL Y1564 in cheese whey supplemented with yeast extract under the optimal temperature of 30 °C, delivering an enzymatic activity of 4418.37 U/gcell after 12 h of process. In order to develop more stable biocatalysts, the enzyme produced by fermentation was immobilized on 2.0% w/v chitosan activated with glutaraldehyde, epichlorohydrin or glycidol, producing a highly active and stable biocatalyst capable of hydrolyzing lactose and producing lactulose simultaneously. The biocatalyst obtained by immobilization in chitosan-glutaraldehyde showed high storage stabilities (100% of its activity when stored at 4 °C 105 days). Regarding the milk lactose hydrolysis by both the soluble and the immobilized enzyme, the conversions obtained were 38.0% and 42.8%, respectively. In this study, by using a biocatalyst deriving from enzyme immobilization to chitosan support, a lactulose production of 17.32 g/L was also possible.

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.

Recovery and purification of a Kluyvermyces lactis ß-galactosidase by Mixed Mode Chromatography.

Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis ß-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values ​​of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value.

Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and ß-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB).

Ultrasound-assisted production of biodiesel and ethanol from spent coffee grounds.

This study evaluates the production of biodiesel and ethanol from spent coffee grounds (SCG). The extraction of oil from SCG, biodiesel production and ethanol production processes were studied. The liquid-to-solid ratio and temperature were evaluated in the ultrasound-assisted extraction of the oil from SCG. The highest yield (12%) was obtained using 4 mL g(-1) liquid-to-solid ratio at 60°C for 45 min. The process to produce biodiesel showed a yield of 97% into fatty acid methyl esters (FAME). The highest glucose yield (192 mg g SCG(-1)) was obtained by hydrolysis with 0.4 mol L(-1) sulfuric acid at 121°C for 15 min. The hydrolysate was used as fermentation medium for ethanol production by Saccharomyces cerevisiae obtaining 19.0 g L(-1) at 10h of process of ethanol with a yield of ethanol and productivity of 0.50 g g(-1) and 1.90 g L(-1)h(-1), respectively. Spent coffee grounds were considered a potential feedstock for biodiesel and ethanol production.

Crude glycerol from biodiesel industry as substrate for biosurfactant production by Bacillus subtilis ATCC 6633

Glycerol, a co-product of the biodiesel industry, may be a suitable raw material for the production of high added-value compounds by the microorganisms. This study aimed to use the glycerol obtained from the biodiesel production process as the main carbon source for biosurfactant production by Bacillus subtilis ATCC 6633. Results indicated that the strain lowered the surface tension of the cell-free fermented broth to 31.5 ± 1.6 mN/m, indicating the production of biosurfactant. The critical micelle concentration (CMC = 33.6 mN/m) obtained was similar to the previously reported for biossurfactants isolated from other Bacillus. The produced biosurfactant was able to emulsify n-hexadecane and soybean oil.

Isolation, identification, and activity in vitro of killer yeasts against Colletotrichum gloeosporioides isolated from tropical fruits.

A total of 580 yeasts strains, isolated from Ceara State of Brasil, were evaluated for their ability to produce killer toxin. Of these strains, 29 tested positive for the killer phenotype and were further evaluated for their ability to control Colletotrichum gloeosporioides germination in vitro. All yeast strains that expressed the killer phenotype were characterized by sequencing the D1/D2 regions of the large subunit of the rRNA gene. Five yeast strains provided a significant reduction in mycelial growth and conidial germination of C. gloeosporioides in vitro, especially Meyerozyma guilliermondii, which was able to reduce the fungal mycelial growth on solid medium (potato dextrose agar (PDA)) by 60% and block 100% of conidia germination in liquid media (potato dextrose broth (PDB)). Filtering and autoclaving the liquid cultures had no effect on the growth of the pathogen. These results indicate the potential use of antagonist yeasts isolated from tropical fruits in the control of anthracnose caused by C. gloeosporioides in papaya. Further elucidation of main mechanisms involved on anthracnose control by these yeasts could be helpful for the development of biocontrol techniques related to the management of this disease in tropical fruits.

Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice.

In this work a low cost medium for the production of a biosurfactant by Bacillus subtilis LAMI005 and the kinetics of surfactin production considering the effect of initial substrate concentration were investigated. First, cashew apple juice supplementation for optimal production of biosurfactant by B. subtilis LAMI005 was studied. The medium formulated with clarified cashew apple juice and distilled water, supplemented with 1.0 g/L of (NH(4))(2)SO(4), proved to be the best among the nutrients evaluated. The crude biosurfactant had the ability to decrease the surface tension of water to 30 dyne/cm, with a critical micelle concentration (CMC) of 63.0 mg/L. Emulsification experiments indicated that this biosurfactant effectively emulsified kerosene (IE(24)=67%) and soybean oil (IE(24)=64%). Furthermore, the emulsion stability was always very high. It was shown by biochemical analysis, IR spectra, that there is no qualitative differences in the composition of the crude biosurfactant from a standard sample of surfactin from B. subtilis.

Ethanol production by fermentation using immobilized cells of Saccharomyces cerevisiae in cashew apple bagasse.

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82-37.83 g L(-1) in average value) and ethanol productivities (about 3.30-6.31 g L(-1) h(-1)) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L(-1)) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30-98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.

Clarified cashew apple juice as alternative raw material for biosurfactant production by Bacillus subtilis in a batch bioreactor.

Clarified cashew apple juice was evaluated as carbon source for surfactin production by Bacillus subtilis LAMI005 isolated from the tank of chlorination at the Wastewater Treatment Plant on Campus do Pici (WWTP-PICI) in the Federal University of Ceará, Brazil. The highest surfactin concentration using clarified cashew apple juice (CCAJ) supplemented with mineral medium (MM-CCAJ) was 123 mg/L, achieved after 48 h of fermentation. Almost 2-fold less than the amount produced using mineral medium supplemented with 10 g/L of glucose and 8.7 g/L of fructose (MM-GF). However, critical micelle concentration of the biosurfactants produced using MM-CCAJ was 2.5-fold lower than the one produced using MM-GF, which indicates it is a more efficient biosurfactant. Surface tension decreased from 38.50 +/- 0.0 to 29.00 +/- 0.0 dyne/cm when B. subtilis was grown on MM-CCAJ media (24.68% of reduction on surface tension) and remained constant up to 72 h. Emulsification index was 51.15 and 66.70% using soybean oil and kerosene, respectively. Surfactin produced in MM-CCAJ showed an emulsifying activity of, respectively, 1.75 and 2.3 U when n-hexadecane or soybean oil was tested. However, when mineral medium supplemented with 10 g/L of glucose (MM-G) was used an emulsifying activity of 2.0 and 1.75 U, with n-hexadecane and soybean oil, respectively, was obtained. These results indicate that it is feasible to produce surfactin from CCAJ, a renewable and low-cost carbon source.