Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases.

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases' biodiversity and structure-function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C2 to C16). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C12 and C16, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in kcat values with increasing substrate chain length, whereas KM values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low KM values, resulting in high catalytic efficiencies toward pNP-C16. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

Characterization of an immunogenic cellulase secreted by Cryptococcus pathogens.

Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

Combination of two ß-galactosidases during the synthesis of galactooligosaccharides may enhance yield and structural diversity.

In this study, the combination of two ß-galactosidases to synthesize prebiotic galactooligosaccharides (GOS) was evaluated in terms of total GOS yield as well as GOS structures (chain length). Two different combinations of either Aspergillus oryzae and Cryptococcus laurentii or Aspergillus oryzae and Kluyveromyces lactis were tested to examine the influence of enzyme origin. Neither consecutive nor simultaneous synthesis with A. oryzae and C. laurentii led to an increased GOS yield. However, with the latter, synthesis of higher GOS (≥3 monomer units) was enhanced from 38.5% to 40% with special emphasis of tetra- and pentasaccharides, which increased from 6.7% to 12.8% and from 0.4% to 3.3%, respectively. Additionally, due to the different preferences of the two ß-galactosidases in terms of types of glycosidic linkages, the structural diversity of the final GOS product could be increased. Using K. lactis following the synthesis with A. oryzae increased the yield of total GOS from 24.6% to 33.1%, which was mainly due to the formation of GOS disaccharides. On the other hand, applying A. oryzae as the second enzyme led to a degradation of di- and trisaccharides, and thus total GOS yield was diminished, although the yield of tetrasaccharides could be enhanced. In conclusion, with both studied enzyme combinations it was possible to increase the percentage of higher GOS and reduce the residual lactose content of the final mixture, which is beneficial for subsequent purification processes. Thus, using more than one ß-galactosidase during the synthesis of GOS represents an interesting research area, which should be explored in more detail in the future.

Extensive hydrolysis of raw rice starch by a chimeric α-amylase engineered with α-amylase (AmyP) and a starch-binding domain from Cryptococcus sp. S-2.

Recombinant chimeric α-amylase (AmyP-Cr) was constructed by a catalytic core of α-amylase (AmyP) from a marine metagenomic library and a starch-binding domain (SBDCr) of α-amylase from Cryptococcus sp. S-2. The molecular fusion did not alter optimum pH, optimum temperature, hydrolysis products, and an ability of preferential and rapid degradation towards raw rice starch, but catalytic efficiency and thermostability were remarkably improved compared with those of the wild-type AmyP. AmyP-Cr achieved the final hydrolysis degree of 61.7 ± 1.2% for 10% raw rice starch and 47.3 ± 0.8% for 15% raw rice starch after 4 h at 40 °C with 1.0 U per mg of raw starch. The catalytic efficiency was very high, with 3.6-4.0 times higher than that of AmyP. The enhanced catalytic efficiency was attributed to the better thermostability and the higher adsorption and disruption to raw rice starch caused by SBDCr. The properties of AmyP-Cr open a new way in terms of a new design of raw rice starch processing.

Expression of a chitin deacetylase gene, up-regulated in Cryptococcus laurentii strain RY1, under nitrogen limitation.

This study reports the identification of a chitin deacetylase gene in Cryptococcus laurentii strain RY1 over-expressing under nitrogen limitation by differential display. The up-regulation took place in robustly growing cells rather than in starving quiescent autophagic cells. Quantitative Real Time-PCR, enzyme activity in cell lysate and cell wall analysis corroborated the up-regulation of chitin deacetylase under nitrogen limitation. These results suggest chitin deacetylase might play a significant role in nitrogen limiting growth of Cryptococcus laurentii strain RY1.

[Component composition of Cryptococcus albidus and Eupenicillium erubescens alpha-L-rhamnosidases].

The component composition of Cryptococcus albidus and Eupenicillium erubescers alpha-L-rhamnosidases have studied. It was shown that enzymes have a monomeric structure. Enzyme preparations of C. albidus and E. erubescens have similar qualitative but differ in quantitative amino acid composition. alpha-L-rhamnosidase of C. albidus characterised by high amount of histidine, proline, cysteine, methionine in compared with alpha-L-rhamnosidase of E. erubescens. alpha-L-Rhamnosidase of E. erubescens, in contrast to the alpha-L-rhamnnosidase of C. albidus, contained higher levels of lysine, arginine, threonine, alanine, isoleucine, leucine, tyrosine, phenylalanine. It is shown that purified preparations of alpha-L-rhamnosidase C. albidus and E. erubescens contained 5 and 1% carbohydrates respectively. Enzyme preparations differ in quantitative monosaccharide composition, which represented by rhanmose, xylose, mannose, galactose and glucose. Furthermore, alpha-L-rhannosidase C. albidus contained fuicose, whereas alpha-L-rhamnosidase E. erubescens--ribose and arabinose. A significant percentage of hydrophobic amino acids, which is 31 and 34% of the total content, and the presence of the carbohydrate component are essential in stabilization of enzymes molecule.

[Influence of coordination compounds of germanium (IV) and stannum (IV) on activity of some microbial enzymes with glycolytic and proteolytic action].

Influence of coordinative compounds of germanium (IV) and stanum (IV) (complexes of germanium (IV) with nicotinamide (Nad) [GeCl2(Nad)4]Cl2 (1) and complexes of stanum (IV) with 2-hydroxybenzoilhydrazone 4-dimetylaminobenzaldehide (2-OH-HBdb) [SnCl4(2-OH-Bdb-H)] (2), 3-hydroxy-2-naphtoilhydrazone 2-hydroxynaphtaldehide (3-OH-H2Lnf) [SnCl3(3-OH-HLnf)] (3) and izonicotinoilhydrazone 2-hydroxyibenzaldehide [SnCl3 (Is·H)] (4) on activity of peptidases 1 and 2 Bacillus thuringiensis, α-L-rhamnosidase Cryptococcus albidus, Eupenicillium erubescens and α-amylase Aspergillus flavus var. oryzae. Results testify that all studied compounds differ on their influence on activity of the enzymes tested: significantly don't change elastolytic activity of peptidases 1 and 2 B. thuringiensis, completely inhibit A. flavus var. oryzae amylase, activate or oppress of α-L-rhamnosidase C. albidus and E. erubescens. Considerable differences in compounds (3, 4) on activity observed in case of the last. It's possible that peculiarity of influence (1) in compare with (2-4) is connected with existence of different central atoms of complexants: germanium (IV) (1) and stanum (IV) (2-4). A certain analogy in oppression of C. albidus α-L-rhamnosidase by compounds (1) and (4) can explain with presence of a pyridinic ring at molecules of their ligands. The less activsty displayed compound (2) with coordinative knot {SnCl4ON}. Nature of compounds (3, 4) activity was absolutely different: essential increase of activity of C. albidus α-L-rhamnosidase and full oppression of E. erubescens α-L-rhamnosidase by compound (3), while the action of compound (4) was feed back. Taking into account identical coordination knot {SnCl3O2N} the major role in this case play change of a hydrazide fragment in molecules of their ligands.

Assessment of Cryptococcus albidus for biopulping of eucalyptus.

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei.

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 °C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.

Antifungal susceptibility, enzymatic activity, PCR-fingerprinting and ITS sequencing of environmental Cryptococcus laurentii isolates from Uberaba, Minas Gerais, Brazil.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.

Characterization of laccase activity produced by Cryptococcus albidus.

This study deals with the characterization of laccase enzyme activity produced by Cryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a number of chemicals. These chemicals can interfere with the proper functioning of the enzymes used for bioremediation. Thus, it is important to study the effect of such interfering solvents, detergents, metal chelators, and other chemicals on enzyme activity before industrial applications. Laccase showed maximum activity at pH 2.5 and temperature 20-30°C when ABTS was used as a substrate. The enzyme followed Michaelis-Menten kinetics: K(m) was 0.8158 mM and V(max) was 1527.74 U/mg. Laccase showed good thermostability with a half-life of 81 min at 25°C, 77 min at 35°C, 64 min at 45°C, 36 min at 55°C, and 21 min at 65°C. There was no effect of sodium dodceyl sulfate (SDS) (0.1-1.0%) and EDTA (0.1-0.5%) on laccase activity. Sodium azide and 2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lower concentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, the activity was completely inhibited at a concentration of 50% or above of acetone, methanol, 1,4-dioxan, and acetonitrile.

[Purification and physico-chemical properties of Cryptococcus albidus 1001 alpha-L-rhamnosidase].

A scheme of isolation and purification of the enzyme with alpha-L-rhamnosidase activity from culture liquid Cryptococcus albidus 1001 has been developed. It included fractionation by ammonium sulfate and chromatography on TSK-gels Toyopearl HW-60, Fractogel TSK DEAE-650-s and Sepharose 6B. The enzyme was purified 42 times with the yield of 0.7 %. The enzyme preparation did not contain any glycosidase (except of beta-D-glucosidase) and proteolytic activity. Molecular mass of the alpha-L-rhamnosidase preparation by the data of Sepharose 6B gel-filtration was 50 kDa. The enzyme preparation was stable during 48 hours at 20 degrees C, its pH and thermal optimum were 4.0-5.0 and 60 degrees C, correspondingly.

[Investigation of functional groups of Cryptococcus albidus alpha-L-rhamnosidase].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino)propyl]-3-ethylcarbodimide methiodide, EDTA, o-phenantroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the alpha-L-rhamnosidase activity of Cryptococcus albidus has been investigated. The essential role of Ag+ which inhibits the alpha-L-rhamnosidase activity by 72.5% was shown. Rhamnose at 1-5 mM protect the enzyme from the negative effect of Ag(+). It was expected that carboxyl group of C-terminal aminoacid and imidazole group of histidine would participate in the catalytic action of alpha-L-rhamnosidase on the basis of inhibition and kinetic analysis.

Salt-tolerant and thermostable glutaminases of cryptococcus species form a new glutaminase family.

Genes encoding salt-tolerant and thermostable glutaminases were isolated from Cryptococcus species. The glutaminase gene, CngahA, from C. nodaensis NISL-3771 was 2,052 bp in length and encoded a 684-amino acid protein. The gene, CagahA, from C. albidus ATCC20293 was 2,100 bp in length and encoded a 700-amino acid protein. These glutaminases showed 44% identity. By searches on public databases, we found that these glutaminases are not similar to any other characterized glutaminases, but are similar to certain hypothetical proteins. On searching the conserved domain with the basic local alignment search tool (BLAST), it was found that they have the amidase domain and are members of the amidase signature superfamily. They were expressed in Saccharomyces cerevisiae, and their activity was detected on the cell surface. This study revealed that they are a new type of glutaminase with the amidase signature sequence, and that they form a new glutaminase family.

[Optimization of cultivation conditions of Cryptococcus albidus--producers of alpha-L-rhamnosidase].

The influence of content of cultivation media and fermentation conditions on synthesis of alpha-L-rhamnosidase by Cryptococcus albidus 1001 and 1003 were studied. It was shown that L-rhamnose and NaNO, are the optimum sources of carbon and nitrogen, respectively. The temperature of 25 degrees C, pH 6.0, growing in 50 ml of the medium at rotation rate 220 rev/min during 6 days are optimum parameters for both strains.

[Optimization of cultivation conditions of alpha-L-rhamnosidases producers-- representatives of different taxonomic groups of microorganisms].

Influence ofsome technological parameters of cultivation ofproducers Cryptococcus albidus, Eupenicillinum erubescens, Bacillus sp. on the process of synthesis of extracellular enzyme alpha-L-rhamnosidase has been studied. The authors have determined optimal sources of carbon (0.2-0.3% rhamnose) and nitrogen (0.2% sodium nitrate for C. albidus and E. erubescens and ammonium sulphate for Bacillus sp.) (the ratio 1:2), cultivation temperature (28 degrees C, 25 degrees C, 42 degrees C, respectively) for maximum synthesis of alpha-L-rhamnosidase. Use of the medium with initial pH value from 4 to 8 was most efficient for all the studied strains. The maximum level of alpha-L-rhamnosidase activity of E. erubescens and Bacillus sp. was established at the value of sulphite number of 0.44, while for C. albidus--it was 0.56. Maximum alpha-L-rhamnosidase activity of C. albidus, E. erubescens, Bacillus sp. is achieved at 4, 8 days and 27 hours of cultivation, respectively. The cultures being grown in selected conditions, the alpha-L-rhamnosidase synthesis has increased by 30, 50 and 20%, respectively.

Esterification of Polymeric Carbohydrate Through Congener Cutinase-Like Biocatalyst.

Cutinase-like enzymes (CLEs) are bi-functional hydrolases, which share the conserved catalytic site of lipase and consensus pentapeptide sequence of cutinase. Here, we have genetically replaced the canonical amino acids (CAA) by their non-canonical fluorinated surrogates to biosynthesize a novel class of congener biocatalyst for esterification of polymeric carbohydrate with long-chain fatty acid. It is a new enzyme-engineering approach used to manipulate industrially relevant biocatalyst through genetic incorporation of new functionally encoded non-canonical amino acids (NCAA). Global fluorination of CLE improved its catalytic, functional, and structural stability. Molecular docking studies confirmed that the fluorinated CLE (FCLE) had developed a binding affinity towards different fatty acids compared with the parent CLE. Importantly, FCLE could catalyze starch oleate synthesis in 24 h with a degree of substitution of 0.3 ± 0.001. Biophysical and microscopic analysis substantiated the efficient synthesis of the ester by FCLE. Our data represent the first step in the generation of an industrially relevant fluorous multifunctional enzyme for facile synthesis of high fatty acid starch esters.

Regulation of virulence factors, carbon utilization and virulence by SNF1 in Cryptococcus neoformans JEC21 and divergent actions of SNF1 between cryptococcal strains.

We describe here the functions of a Snf1/AMPK homolog in the human pathogenic yeast Cryptococcus neoformans, strain JEC21. We found that JEC21 SNF1 is a key regulator for the biosynthesis of the major virulence factors, stress resistance and alternative carbon source utilization. Disruption of JEC21 SNF1 results in defects of laccase activity and capsule production, sensitivity to cation stress. Especially, we found that JEC21 SNF1 is essential for growth at elevated temperature and for thermotolerance. To our knowledge, a role for Snf1 proteins in thermotolerance has not been reported. Furthermore, we observed a functional divergence between JEC21 SNF1 and its equivalent from serotype A strain H99. A high temperature is needed for H99 SNF1 to function in stress response and carbon source preference, but not for the JEC21 SNF1. Our results confirmed a critical role of JEC21 SNF1 in regulation of stress response and virulence. Revelation of divergent actions of SNF1 may help to understand the evolution of cryptococcal pathogenesis and provides insights into the strain-associated biosynthesis of virulence factors.

Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.

Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.

Enzymatic characterisation of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii and other environmental Cryptococcus spp.

This study compared the enzymatic activity of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii, environmental isolates of C. neoformans and non-neoformans Cryptococcus. Most of the cryptococcal isolates investigated in this study exhibited proteinase and phospholipase activities. Laccase activity was detected from all the C. neoformans and C. gattii isolates, but not from the non-neoformans Cryptococcus isolates. There was no significant difference in the proteinase, phospholipase and laccase activities of C. neoformans and C. gattii. However, significant difference in the enzymatic activities of beta-glucuronidase, alpha-glucosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase between C. neoformans and C. gattii isolates was observed in this study. Environmental isolates of C. neoformans exhibited similar enzymatic profiles as the clinical isolates of C. neoformans, except for lower proteinase and laccase activities.