The resistance of cellulases and xylanases to proteolytic inactivation

The sensitivity of a range of cellulases and xylanases to proteolytic inactivation was investigated. The xylanases, all the Clostridium thermocellum cellulases and cellulase E from Pseudomonas fluorescens subsp. cellulosa exhibited no decrease in catalytic activity during a 3-h incubation with proteinases of the small intestine. Under these conditions, the control Escherichia coli enzymes analysed had half-lives of 4.3–13.5 min. The addition of substrate significantly decreased the sensitivity of proteinase-labile enzymes to inactivation. The significance of these data in relation to the use of cellulases and xylanases for improving animal nutrition is discussed.     

Potentially Probiotic Limosilactobacillus fermentum Fruit-Derived Strains Alleviate Cardiometabolic Disorders and Gut Microbiota Impairment in Male Rats Fed a High-Fat Diet

Probiotics and Antimicrobial Proteins - High-fat diet (HFD) consumption is a risk factor for dyslipidemias, insulin resistance, and arterial hypertension linked with gut dysbiosis. Probiotic...     

Role of Pectinolytic Enzymes Identified in Clostridium thermocellum Cellulosome

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence {"type":"entrez-protein","attrs":{"text":"ABN54148.1","term_id":"125715656","term_text":"ABN54148.1"}}ABN54148.1 and {"type":"entrez-protein","attrs":{"text":"ABN53381.1","term_id":"125714889","term_text":"ABN53381.1"}}ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.     

Acanthamoeba polyphaga Mimivirus Prevents Amoebal Encystment-Mediating Serine Proteinase Expression and Circumvents Cell Encystment

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.     

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature''s most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (K_a ∼10¹² m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.     

Seasonal versatility in the feeding ecology of a group of titis (Callicebus coimbrai) in the northern Brazilian Atlantic Forest

The feeding behavior of a group of titis (Callicebus coimbrai) was monitored over an annual cycle at a site in northeastern Brazil. Behavioral data were collected in scan samples (1-min scan at 5-min intervals), and complementary data on fruit availability and new leaf cover were collected. Feeding time accounted for 28.9% of daily activity. Fruit was the principal item of the diet (61.2% of records) and the primary category in all months except September, when it was surpassed by leaves. Young leaves were the second most important category (20.0%). The consumption of seeds and insects was prominent in November and December. Fifty-two plant species were exploited, and the Elaeocarpaceae, Myrtaceae, Sapotaceae, and Passifloraceae provided the vast majority (86.0%) of plant feeding records. The phenological record did not provide a good measure of fruit availability, but a strong correlation (r(s) =0.902, P<0.0001, n=12) was found between the consumption of leaves and the exploitation of lianas each month. Lianas accounted for 28.2% of plant feeding records, and predominated between August and December. This suggests that lianas may represent a key factor in the ability of the species to tolerate the intense habitat fragmentation found throughout its geographic range.     

Stable and high-level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.     

EARLY RESPONSIVE to DEHYDRATION 15, a new transcription factor that integrates stress signaling pathways

The Early Responsive to Dehydration (ERD) genes are defined as those genes that are rapidly activated during drought stress. The encoded proteins show a great structural and functional diversity, with a particular class of proteins acting as connectors of stress response pathways. Recent studies have shown that ERD15 proteins from different species of plants operate in cross-talk among different response pathways. In this mini-review, we show the recent progress on the functional role of this diverse family of proteins and demonstrate that a soybean ERD15 homolog can act as a connector in stress response pathways that trigger a programmed cell death signal.