ANCUT1, a novel thermoalkaline cutinase from Aspergillus nidulans and its application on hydroxycinnamic acids lipophilization.

One of the four cutinases encoded in the Aspergillus nidulans genome, ANCUT1, is described here. Culture conditions were evaluated, and it was found that this enzyme is produced only when cutin is present in the culture medium, unlike the previously described ANCUT2, with which it shares 62% amino acid identity. The differences between them include the fact that ANCUT1 is a smaller enzyme, with experimental molecular weight and pI values of 22 kDa and 6, respectively. It shows maximum activity at pH 9 and 60 °C under assayed conditions and retains more than 60% of activity after incubation for 1 h at 60 °C in a wide range of pH values (6-10) after incubations of 1 or 3 h. It has a higher activity towards medium-chain esters and can modify long-chain length hydroxylated fatty acids constituting cutin. Its substrate specificity properties allow the lipophilization of alkyl coumarates, valuable antioxidants and its thermoalkaline behavior, which competes favorably with other fungal cutinases, suggests it may be useful in many more applications.

Challenges in the production and use of probiotics as therapeuticals in cancer treatment or prevention.

Probiotics were defined as microbial strains that confer health benefits to their consumers. The concept has evolved during the last 20 years, and today metabolites produced by the strains, known as postbiotics, and even dead cells, known as paraprobiotics, are closely associated to them. The isolation of commensal strains from human microbiome has led to the development of next generation probiotics. This review aims to present an overview of the developments in the area of cancer prevention and treatment, intimately related to advances in the knowledge of the microbiome role in its genesis and therapy. Strain identification and characterization, production processes, delivery strategies, and clinical evaluation are crucial to translate results into the market with solid scientific support. Examples of recent tools in isolation, strain typification, quality control, and development of new probiotic strains are described. Probiotics market and regulation were originally developed in the food sector, but these new strategies will impact the pharmaceutical and health sectors, requiring new considerations in regulatory frameworks.

Improved antimicrobial spectrum of the N-acetylmuramoyl-L-alanine amidase from Latilactobacillus sakei upon LysM domain deletion.

The gene encoding N-acetylmuramoyl-L-alanine amidase in Latilactobacillus sakei isolated from a fermented meat product was cloned in two forms: its complete sequence (AmiC) and a truncated sequence without one of its anchoring LysM domains (AmiLysM4). The objective of this work was to evaluate the effect of LysM domain deletion on antibacterial activity as well the biochemical characterization of each recombinant protein. AmiC and AmiLysM4 were expressed in Escherichia coli BL21. Using a zymography method, two bands with lytic activity were observed, which were confirmed by LC-MS/MS analysis, with molecular masses of 71 kDa (AmiC) and 66 kDa (AmiLysM4). The recombinant proteins were active against Listeria innocua and Staphylococcus aureus strains. The inhibitory spectrum of AmiLysM4 was broader than AmiC as it showed inhibition of Leuconostoc mesenteroides and Weissella viridescens, both microorganisms associated with food decomposition. Optimal temperature and pH values were determined for both proteins using L-alanine-p-nitroanilide hydrochloride as a substrate for N-acetylmuramoyl-L-alanine amidase activity. Both proteins showed similar maximum activity values for pH (8) and temperature (50 °C). Furthermore, structural predictions did not show differences for the catalytic region, but differences were found for the region called 2dom-AmiLysM4, which includes 4 of the 5 LysM domains. Therefore, modification of the LysM domain offers new tools for the development of novel food biopreservatives.

Omics in traditional vegetable fermented foods and beverages.

For a long time, food microbiota has been studied using traditional microbiological techniques. With the arrival of molecular or culture-independent techniques, a strong understanding of microbiota dynamics has been achieved. However, analyzing the functional role of microbial communities is not an easy task. The application of omics sciences to the study of fermented foods would provide the metabolic and functional understanding of the microbial communities and their impact on the fermented product, including the molecules that define its aroma and flavor, as well as its nutritional properties. Until now, most omics studies have focused on commercial fermented products, such as cheese, wine, bread and beer, but traditional fermented foods have been neglected. Therefore, the information that allows to relate the present microbiota in the food and its properties remains limited. In this review, reports on the applications of omics in the study of traditional fermented foods and beverages are reviewed to propose new ways to analyze the fermentation phenomena.

Regulation of the cutinases expressed by Aspergillus nidulans and evaluation of their role in cutin degradation.

Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.

Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.

Poly(lactic acid) Degradation by Recombinant Cutinases from Aspergillus nidulans.

Poly(lactic-acid) (PLA) is a biodegradable polymer widely used as a packaging material. Its monomer, lactic acid, and its derivatives have been used in the food, cosmetic, and chemical industries. The accumulation of PLA residues leads to the development of green degrading methodologies, such as enzymatic degradation. This work evaluates the potential use of three cutinolytic enzymes codified in the Aspergillus nidulans genome to achieve this goal. The results are compared with those obtained with proteinase K from Tritirachium album, which has been reported as a PLA-hydrolyzing enzyme. The results show that all three cutinases act on the polymer, but ANCUT 1 releases the highest amount of lactic acid (25.86 mM). Different reaction conditions assayed later led to double the released lactic acid. A decrease in weight (45.96%) was also observed. The enzyme showed activity both on poly L lactic acid and on poly D lactic acid. Therefore, this cutinase offers the potential to rapidly degrade these package residues, and preliminary data show that this is feasible.

Biotechnological Aspects and Mathematical Modeling of the Biodegradation of Plastics under Controlled Conditions.

The strong environmental impact caused by plastic pollution has led research to address studies from different perspectives. The mathematical modeling of the biodegradation kinetics of solid materials is a major challenge since there are many influential variables in the process and there is interdependence of microorganisms with internal and external factors. In addition, as solid substrates that are highly hydrophobic, mass transfer limitations condition degradation rates. Some mathematical models have been postulated in order to understand the biodegradation of plastics in natural environments such as oceans. However, if tangible and optimizable solutions are to be found, it is necessary to study the biodegradation process under controlled conditions, such as using bioreactors and composting systems. This review summarizes the biochemical fundamentals of the main plastics (both petrochemical and biological origins) involved in biodegradation processes and combines them with the main mathematical equations and models proposed to date. The different biodegradation studies of plastics under controlled conditions are addressed, analyzing the influencing factors, assumptions, model developments, and correlations with laboratory-scale results. It is hoped that this review will provide a comprehensive overview of the process and will serve as a reference for future studies, combining practical experimental work and bioprocess modeling systems.

Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: changes in k(cat) and thermal dependence.

Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k(cat) and a remarkable increase in its stability. While the increase in k(cat) is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k(cat), K(M) and k(cat)/K(M) were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.

Molecular characterization of StcI esterase from Aspergillus nidulans.

Aspergillus nidulans produces StcI esterase, which is involved in the biosynthesis of sterigmatocystin, a precursor of aflatoxins. Previous reports of this esterase in A. nidulans suggest that it is composed of 286 amino acid residues with a theoretical molecular mass of 31 kDa. Various conditions were evaluated to determine the optimal expression conditions for StcI; the highest level was observed when A. nidulans was cultured in solid oat media. Various esterases were expressed differentially according to the culture media used. However, specific antibodies designed to detect StcI reacted with a protein with an unexpected molecular mass of 35 kDa in cell extracts from all expression conditions. Analysis of the gene sequence and already reported expressed sequence tags indicated the presence of an additional 29-amino-acid N-terminal region of StcI, which is not a signal peptide and which has not been previously reported. We also detected the presence of this additional N-terminal region using reverse-transcriptase polymerase chain reaction. The complete protein (NStcI) was cloned and successfully expressed in Pichia pastoris.

The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction.

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and ß-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

Data concerning secondary structure and alpha-glucans-binding capacity of the LaCBM26.

Carbohydrate-binding modules (CBMs) are auxiliary domains into glycoside-hydrolases that allow the interaction between the insoluble substrate and the solubilized enzyme, through hydrophobic, CH-π interactions and hydrogen bonds. Here, we present the data article related to the interaction of one LaCBM26 and some mutated proteins with soluble α-glucans determined by enzyme-linked carbohydrate-binding assay, isothermal titration calorimetry (ITC), and affinity gel electrophoresis (AGE). The data of the behavior of proteins in presence and absence of substrate analyzed by circular dichroism CD and thermofluor are also presented. These results are complementary to the research article "The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction" (Armenta et al., 2019).

Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein.

Highly hydrophobic protein Z19 zein shows a tendency towards oligomerization. The role of temperature and pH on the oligomerization process was studied monitoring the secondary structure content and the appearance of aggregates by Circular Dichroism Spectroscopy (CD) and Dinamic Light Scattering (DLS). Z19 zein suffers irreversible thermal denaturalization, as demonstrated by far-UV CD measurements. DLS data indicate that this denaturalization is accompanied by oligomerization processes which are strongly dependent on temperature. The aggregates that appear when the sample is heated maintain a certain amount of their native structure. Oligomers, showing high stability to temperature changes and other denaturing conditions with molecular weights of 45, 66 kDa and higher, were detected by SDS-PAGE. The secondary structure strongly depends on pH. Thus, at pH above pI (6.8), all the protein structure is in alpha helix. The formation of disulfide bonds plays an important role in the aggregation process, since most of the sulfhydryls in the protein (97.52%) form disulfide bonds and only 2.47% of them are free and superficially exposed. The sensitivity towards thermal denaturalization is also affected by pH rises.

Effect of alkaline deamidation on the structure, surface hydrophobicity, and emulsifying properties of the Z19 alpha-zein.

Different deamidation conditions for the Z19 alpha-zein were studied in order to find the best conditions for the development of the emulsifying properties. Alkaline deamidation was chosen, and the effects on the peptide bond cleavage, secondary structure, emulsifying properties, and surface hydrophobicity were studied. The Z19 alpha-zein was deamidated by using 0.5 N NaOH containing 70% ethanol at 70 degrees C for 12 h. A deamidation degree (DD) of 60.6 +/- 0.5%, and a degree of hydrolysis (DH) of 5 +/- 0.5% were achieved. Analysis by far-UV circular dichroism showed that the denaturation was mainly promoted by the high temperature used during the incubation. The adequate balance between the DD and the DH results in an effective emulsifying property improvement for the Z19 alpha-zein. Thus, after the deamidation treatment, the surface hydrophobicity decreased from 9.5 x 104 +/- 6.8 x 103 to 46 x 104 +/- 2.1 x 103, and the emulsion stability increased from 18 +/- 0.7% to 80 +/- 4.7% since the oil globules stabilized by the modified protein were smaller (57.7 +/- 5.73 nm) and more resistant to coalescence than those present in the native protein emulsions (1488 +/- 3.92 nm).

ANCUT2, a Thermo-alkaline Cutinase from Aspergillus nidulans and Its Potential Applications.

Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.

Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.

Characterization of a 19 kDa alpha-zein of high purity.

A highly pure alpha-zein was extracted from corn flour using ethanol (95%). Subsequently, ion-exchange chromatography was performed, using SP-Sepharose that yielded a highly homogeneous protein. This protein migrated as a single band in 20% SDS-PAGE and in pH gradient gels, showing an isoelectric point of 6.8. Mass spectrometry (MALDI-TOF-MS) showed a single peak with a molecular mass of 24 535 Da. It was identified as Z19, when comparing the sequence obtained in an automatic Edman sequencer with the Swissprot database using BLAST. The molar extinction coefficient, determined by dry weight in 70% methanol, was 12 415.49 M(-1) cm(-1) at 280 nm. Light scattering showed its presence in a monodispersed state of 44-66 kDa aggregates in methanol (70%). Circular dichroism spectra allowed the estimation of an alpha-helix content that was lower than the one found for a mixture of two alpha-zeins but with a higher content of beta sheets.

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus.

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.

Evaluation of strategies to improve the production of alkaline protease PrtA from Aspergillus nidulans.

Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.

Immobilization and Biochemical Properties of the Enantioselective Recombinant NStcI Esterase of Aspergillus nidulans.

The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1 h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72 h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, a w of 0.07, and 120 h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures.