Pilar Diaz

A multidomain xylanase from a Bacillus sp. with a region homologous to thermostabilizing domains of thermophilic enzymes.

The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120,567 Da. A comparison of the deduced amino acid sequence of xylanase C with known beta-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology to family 10 xylanases. A domain homologous to family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology to thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar to that of enzymes from mesophilic micro-organisms. Maximum activity was found at 45 degrees C, and the enzyme was only stable at 55 degrees C lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous to thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 degrees C instead of 45 degrees C, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 degrees C.

Use of methylumbeliferyl-derivative substrates for lipase activity characterization

Lipases and esterases have been recognized as very useful biocatalysts because of their wide-ranging versatility in industrial applications, their stability, low cost, and non-requirement for added cofactors. The physical properties of lipidic substrates, typically water insoluble, have determined a great difficulty in studying lipolytic enzymes. A method for fast and simple detection of lipolytic activity, based on the use of 4-methylumbelliferone (MUF)-derivative substrates was developed. The system has been used for the detection of lipase activity either from microbial colonies, cell culture suspensions, or from proteins separated on SDS-polyacrylamide or isoelectric focusing gels. The use of MUF-derivative substrates has also been extended to the quantitative determination of lipolytic activity from a variety of assays including optimum pH and temperature determination, growth dependency, kinetics or stability studies, or residual activity quantification after treatment with potential inhibitors. The method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.

An unusual pectate lyase from a Bacillus sp. with high activity on pectin: cloning and characterization.

The gene pelA encoding a pectate lyase from the strain Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1214 bp DNA fragment containing pelA gene was determined, revealing an ORF of 666 nucleotides that encoded a protein of 23233 Da. The deduced amino acid sequence of the encoded enzyme showed homology to pectate lyases A, B, C and D from Fusarium solani, Pel-3 and PelB from Erwinia carotovora and Pell from Erwinia chrysanthemi. Homology was also found to the protein deduced from the Bacillus subtilis yvpA gene, the function of which is unknown. The heterologous expressed enzyme depolymerized polygalacturonate and pectins of methyl esterification degree from 22 to 89%, and exhibited similar activity on polygalacturonate and on 89% esterified citrus pectin. Optimum temperature and pH for enzymic activity were 50 degrees C and pH 10, respectively. Ca2+ was required for activity on pectic substrates, while the enzyme was strongly inhibited by Ba2+.

Pectinolytic Systems of Two Aerobic Sporogenous Bacterial Strains with High Activity on Pectin

Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS–polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca2+ for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca2+ requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.

estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases.

Screening of a gene library from Paenibacillus sp. BP-23 generated in Escherichia coli led to identification of a clone that directed the production of lipolytic activity. From the sequencing data, we found an open reading frame encoding a protein of 485 amino acids with an estimated molecular mass of 53 kDa and a pI of 5.1. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-XI-S-X2-G included in most serine-esterases and lipases. The cloned protein showed high homology with enzymes belonging to the bacterial subclass of type B carboxylesterases. The enzyme had a significant preference for esters of short-chain fatty acids and showed the kinetics behaviour of a true esterase. Maximum activity was found at pH 7.5 and 37 degrees C, although the enzyme was active in the pH range 6.0- 9.0 and at temperatures up to 45 degrees C. As expected for a serine-esterase, activity was inhibited by phenylmethylsulphonyl fluoride.

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

Abstract The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect.

Effect of cellulase-assisted refining on the properties of dried and never-dried eucalyptus pulp

The effect of two different cellulases on the hornification phenomenon,in which drainability (Schopper–Riegler method) and mechanical propertiesdiminish when pulps are dried, was studied. The enzyme applications testedincluded a commercial enzyme named ComC (Pergalase A40 from CIBA) and alaboratory enzyme from Paenibacillus sp. strain BP-23namedCelB. Industrial never-dried Eucalyptus globulus bleachedkraft pulp was split in two halves and one of them was dried at ambientcontrolled conditions. We compared enzyme effects on both pulps (wet pulp anddried pulp) before and after PFI mill refining. Enzyme applications increaseddrainability (Schopper–Riegler method) and water retention value (WRV) ofnever-dried bleached pulp, although this did not imply an enhancement of themechanical properties of paper. Cellulase treatment of dried pulps, bycontrast,gave rise to increased drainability and WRV and also to improved mechanicalproperties. The changes caused by drying became less significant after enzymeapplication. Handsheets from CelB-treated dried pulps showed an improvement oftensile and burst indexes while tear decreased. The effect produced by CelB canbe considered a biorefining step. In fact, by means of enzyme treatment withCelB the properties of paper manufactured from dried pulp equalled theproperties attained from wet fibres, with the exception of tear index. Changeswere also found in surface fibre morphology, such as flakes and peeling due tocellulase treatment. The surface modification of fibres with cellulases givesrise to better bonding properties and a closer structure of paper. The finalconclusion is that treatment with cellulases could compensate the hornificationeffect and lead to an important saving of refining energy. The novel enzyme,CelB, was the most effective in improving paper properties and counterbalancingthe hornification effect caused by drying.

Characterization of a Family GH5 Xylanase with Activity on Neutral Oligosaccharides and Evaluation as a Pulp Bleaching Aid

ABSTRACT A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization.

Aims:  To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry.

Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

ABSTRACT Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a Km of 14.72 mg/ml and a k cat value of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8 barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.