Xanthomonas campestris expansin-like X domain is a structurally disordered beta-sheet macromolecule capable of synergistically enhancing enzymatic efficiency of cellulose hydrolysis.

OBJECTIVES: To biochemically characterize an expansin-like X protein domain from Xanthomonas campestris (XcEXLX1) and to study its synergy with cellulases in cellulose depolymerization. RESULTS: The protein was purified using a combination of ion exchange and size exclusion chromatography rendering about 30 mg pure protein/l culture medium. Circular dichroism spectroscopy and small-angle X-ray scattering studies of XcEXLX1 reveal that it is a strongly disordered ß-sheet protein. Its low resolution envelope fits nicely the crystallographic structure of the homologous protein EXLX1 from Bacillus subtillis. Furthermore, we demonstrate that XcEXLX1 shows a synergistic, pH-dependent effect when combined with a commercial enzymatic preparation (Accellerase 1500), enhancing its hydrolytic activity on a cellulosic substrate. The strongest effect was observed in acid pHs with an increase in sugar release of up to 36 %. CONCLUSION: The synergistic effect arising from the action of the expansin-like protein was considerable in the presence of significantly larger amounts of the commercial enzymatic cocktail then previously observed (0.35 FPU of Accellerase 1500/g substrate).

Marine extremophiles: a source of hydrolases for biotechnological applications.

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

Enzymatic hydrolysis of steryl glucosides, major contaminants of vegetable oil-derived biodiesel.

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae).

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 µM); CAL2 (17 kDa, pHo 5.5, km 11 µM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.

Initial characterization of a recombinant kynureninase from Trypanosoma cruzi identified from an EST database.

Kynureninase has been described in bacteria, fungi and animals as an enzyme involved in the catabolic degradation pathway of l-tryptophan. This pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the hydrolytic cleavage of l-kynurenine and 3-hydroxy-l-kynurenine to yield l-alanine and either anthranilic or 3-hydroxyanthranilic acid, respectively. We identified a putative kynureninase gene from a Trypanosoma cruzi project aiming at the structural and functional characterization of more than 100 proteins differentially expressed during metacyclogenesis. This gene encodes a protein similar in size and sequence to kynureninases from other sources. This open reading frame was cloned and the recombinant enzyme was overexpressed. Recombinant T. cruzi kynureninase was purified to homogeneity and its identity was confirmed by mass spectrometry. The apparent molecular mass of the native T. cruzi kynureninase was estimated by gel filtration, suggesting that the protein is a homodimer. Circular dichroism spectrum indicated a mixture of alpha-helix and beta-sheet structure, expected for an aminotransferase fold. l-kynurenine, preferentially hydrolyzed by prokaryotic inducible kynureninases, and 3-hydroxy-l-kynurenine, the preferred substrate in fungi and vertebrates, are both catabolized equally well by T. cruzi kynureninase. Further experimental assays will be performed to fully understand the importance of this enzyme for T. cruzi metabolism.

Hydrolases in the hypopharyngeal glands of workers of Scaptotrigona postica and Apis mellifera (Hymenoptera, Apinae).

Hydrolytic enzymes from hypopharyngeal gland extracts of newly emerged, nurse and foraging workers of two eusocial bees, Scaptotrigona postica, a native Brazilian stingless bee, and the Africanized honey bee (Apis mellifera) in Brazil, were compared. The hypopharyngeal gland is rich in enzymes in both species. Fifteen different enzymes were found in the extracts, with only a few quantitative differences between the species. Some of the enzymes present in the extracts may have intracellular functions, while others seem to be digestive enzymes. Scaptotrigona postica, had lower beta-glucosidase and higher lipase esterase activities than A. mellifera. The differences may be due to different feeding habits and behavioral peculiarities of the two species.

Hydrolases in the hypopharyngeal glands of workers of Scaptotrigona postica and Apis mellifera (Hymenoptera, Apinae)

Hydrolytic enzymes from hypopharyngeal gland extracts of newly emerged, nurse and foraging workers of two eusocial bees, Scaptotrigona postica, a native Brazilian stingless bee, and the Africanized honey bee (Apis mellifera) in Brazil, were compared. The hypopharyngeal gland is rich in enzymes in both species. Fifteen different enzymes were found in the extracts, with only a few quantitative differences between the species. Some of the enzymes present in the extracts may have intracellular functions, while others seem to be digestive enzymes. Scaptotrigona postica, had lower beta-glucosidase and higher lipase esterase activities than A. mellifera. The differences may be due to different feeding habits and behavioral peculiarities of the two species.

Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59.

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV-A59) contained autoantibodies (autoAb) that bound to a 40-kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross-reacted with a 40-kDa protein from human, rat and sheep liver, but not with liver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and the occurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV-infection. The 40-kDa protein was purified from mouse liver extracts by ion-exchange chromatography, gel filtration and SDS-PAGE. Because the N-terminal was blocked, we digested the protein in-gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.

The biochemical basis for L-canavanine tolerance by the tobacco budworm Heliothis virescens (Noctuidae).

The tobacco budworm, Heliothis virescens (Noctuidae), a destructive insect pest, is remarkably resistant to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an arginine antimetabolite that is a potent insecticide for nonadapted species. H. virescens employs a constitutive enzyme of the larval gut, known trivially as canavanine hydrolase (CH), to catalyze an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. As such, it represents a new type of hydrolase, one acting on oxygen-nitrogen bonds (EC 3.13.1.1). This enzyme has been isolated from the excised gut of H. virescens and purified to homogeneity; it exhibits an apparent Km value for L-canavanine of 1.1 mM and a turnover number of 21.1 micromol x min(-1)x micromol(-1). This enzyme has a mass of 285 kDa and is composed of two subunits with a mass of 50 kDa or 47.5 kDa. CH has a high degree of specificity for L-canavanine as it cannot function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homoserine, are not metabolized significantly by CH.

Hydrolytic activity of an onchocercoma.

The onchocercoma or nodule produced by the nematode Onchocerca volvulus (Filarioidea) in the skin of patients suffering from onchocerciasis has not been examined by histochemical techniques. In this work we have used histochemical techniques to evaluate 5 hydrolytic enzymes, namely phosphatases, esterases and beta-glucuronidase. The results show increased enzymatic activity at the sites of major metabolic activity and within reactive cells including macrophages (mc) and giant cells (gc) of the onchocercoma.

Hydrolytic activity of an onchocercoma

The onchocercoma or nodule produced by the nematode Onchocerca volvulus (Filarioidea) in the skin of patients suffering from onchocerciasis has not been examined by histochemical techniques. In this work we have used histochemical techniques to evaluate 5 hydrolytic enzymes, namely phosphatases, esterases and beta-glucuronidase. The results show increased enzymatic activity at the sites of major metabolic activity and within reactive cells including macrophages (mc) and giant cells (gc) of the onchocercoma