Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5′-CTGGGGTCTGGGG-3′ CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite (R) 545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI. 3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I. 3, requiring a refolding step, was poorly immobilized on all supports tested ( best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Paenibacillus barcinonensis xylanase 10C containing the CBM22-1-CBM22-2 tandem

A construct containing the CBM22-1-CBM22-2 tandem forming the N-terminal domain of Paenibacillus barcinonensis xylanase 10C (Xyn10C) has been purified and crystallized. A xylan-binding function and an affinity for mixed [beta]-1,3/[beta]-1,4 glucans have previously been demonstrated for some members of the CBM22 family. The sequence of the tandem is homologous to the N-terminal domains found in several thermophilic enzymes. Crystals of this tandem were grown by the streak-seeding method after a long optimization strategy. The structure has been determined by molecular replacement to a resolution of 2.43 Å and refinement is under way. This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine-tuning substrate affinit

Crystallization and preliminary X-ray diffraction analysis of Xyn30D from Paenibacillus barcinonensis

Xyn30D, a new member of a recently identified group of xylanases, has been purified and crystallized. Xyn30D is a bimodular enzyme composed of an N-terminal catalytic domain belonging to glycoside hydrolase family 30 (GH30) and a C-terminal family 35 carbohydrate-binding domain (CBM35) able to bind xylans and glucuronic acid. Xyn30D shares the characteristic endo mode of action described for GH30 xylanases, with the hydrolysis of the [beta]-(1,4) bonds of xylan being directed by [alpha]-1,2-linked glucuronate moieties, which have to be placed at the -2 subsite of the xylanase active site. Crystals of the complete enzyme were obtained and a full data set to 2.3 Å resolution was collected using a synchrotron X-ray source. This represents the first bimodular enzyme with the domain architecture GH30-CBM35. This study will contribute to the understanding of the role that the different xylanases play in the depolymerization of glucuronoxylan

A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil

A new xylanolytic strain, Paenibacillus favisporus CC02-N2, was isolated from sugarcane plantation fi elds in Brazil. The strain had a xylan-degrading system with multiple enzymes, one of which, xylanase Xyn30A, was identifi ed and characterized. The enzyme is a single-domain xylanase belonging to family 30 of the glycosyl hydrolases (GH30). Xyn30A shows high activity on glucuronoxylans, with a Vmax of 267.2 U mg–1, a Km of 4.0 mg/ml, and a kcat of 13,333 min–1 on beechwood xylan, but it does not hydrolyze arabinoxylans. The three- dimensional structure of Xyn30A consists of a common (β/α)8 barrel linked to a side-chain-associated β-structure, similar to previously characterized GH30 xylanases. The hydrolysis products from glucuronoxylan were methylglucuronic-acid-substituted xylooligomers (acidic xylooligosaccharides). The enzyme bound to insoluble xylan but not to crystalline cellulose. Our results suggest a specifi c role for Xyn30A in xylan biodegradation in natural habitats. The enzyme is a good candidate for the production of tailored xylooligosaccharides for use in the food industry and in the biotechnological transformation of biomass. [Int Microbiol 2014; 17(3):175-184]Keywords: Paenibacillus favisporus · xylanase · glycosyl hydrolases GH3

A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil

A new xylanolytic strain, Paenibacillus favisporus CC02-N2, was isolated from sugarcane plantation fi elds in Brazil. The strain had a xylan-degrading system with multiple enzymes, one of which, xylanase Xyn30A, was identifi ed and characterized. The enzyme is a single-domain xylanase belonging to family 30 of the glycosyl hydrolases (GH30). Xyn30A shows high activity on glucuronoxylans, with a Vmax of 267.2 U mg1, a Km of 4.0 mg/ml, and a kcat of 13,333 min1 on beechwood xylan, but it does not hydrolyze arabinoxylans. The three- dimensional structure of Xyn30A consists of a common (β/α)8 barrel linked to a side-chain-associated β-structure, similar to previously characterized GH30 xylanases. The hydrolysis products from glucuronoxylan were methylglucuronic-acid-substituted xylooligomers (acidic xylooligosaccharides). The enzyme bound to insoluble xylan but not to crystalline cellulose. Our results suggest a specifi c role for Xyn30A in xylan biodegradation in natural habitats. The enzyme is a good candidate for the production of tailored xylooligosaccharides for use in the food industry and in the biotechnological transformation of biomass. [Int Microbiol 2014; 17(3):175-184

Development of an antimicrobial bioactive paper made from bacterial cellulose

Bacterial cellulose (BC) has emerged as an attractive adsorptive material for antimicrobial agents due to its fine network structure, its large surface area, and its high porosity. In the present study, BC paper was first produced and then lysozymewas immobilized onto it by physical adsorption, obtaining a composite of lysozyme-BC paper. The morphology and the crystalline structure of the composite were similar to that of BC paper as examined by scanning electron microscopy and X-ray diffraction, respectively. Regarding operational properties, specific activities of immobilized and free lysozymewere similar. Moreover, immobilized enzyme showed a broaderworking temperature and higher thermal stability. The composites maintained its activity for at least 80 dayswithout any special storage. Lysozyme-BC paper displayed antimicrobial activity against Gram-positive and Gram-negative bacteria, inhibiting their growth by 82% and 68%, respectively. Additionally, the presence of lysozyme increased the antioxidant activity of BC paper by 30%. The results indicated that BC is a suitable material to produce bioactive paper as it provides a biocompatible environment without compromising the activity of the immobilized protein. BC paper with antimicrobial and antioxidant properties may have application in the field of active packaging

Bacterial cellulose matrices to develop enzymatically active paper

This work studies the suitability of bacterial cellulose (BC) matrices to prepare enzymatically active nanocomposites, in a framework of more environmentally friendly methodologies. After BC production and purification, two kind of matrices were obtained: BC in aqueous suspension and BC paper. A lipase was immobilised onto the BC matrices by physical adsorption, obtaining Lipase/BC nanocomposites. Neither morphology nor crystallinity, measured by scanning electron microscopy (SEM) and X-Ray diffractometry (XRD) respectively, of the BC were affected by the binding of the protein. The activity of Lipase/BC suspension and Lipase/BC paper was tested under different conditions, and the operational properties of the enzyme were evaluated. A shift towards higher temperatures, a broader pH activity range, and slight differences in the substrate preference were observed in the immobilised lipase, compared with the free enzyme. Specific activity was higher for Lipase/BC suspension (4.2 U/mg) than for Lipase/BC paper (1.7 U/mg) nanocomposites. However, Lipase/BC paper nanocomposites showed improved thermal stability, reusability, and durability. Enzyme immobilised onto BC paper retained 60% of its activity after 48 h at 60 ºC. It maintained 100% of the original activity after being recycled 10 times at pH 7 at 60 ºC and it remained active after being stored for more than a month at room temperature. The results suggested that lipase/BC nanocomposites are promising biomaterials for the development of green biotechnological devices with potential application in industrials bioprocesses of detergents and food industry and biomedicine. Lipase/BC paper nanocomposite might be a key component of bioactive paper for developing simple, handheld, and disposable devices

Study of non-covalent interactions on dendriplex formation: Influence of hydrophobic, electrostatic and hydrogen bonds interactions

The interaction of a double stranded small interference RNA (siRNA Nef) with cationic carbosilane dendrimers of generations 1-3 with two different ammonium functions at the periphery ([-NMe2R](+), R=Me, (CH2)(2)OH) has been studied by experimental techniques (zeta potential, electrophoresis, single molecule pulling experiments) and molecular dynamic calculations. These studies state the presence of different forces on dendriplex formation, depending on generation and type of ammonium group. Whilst for higher dendrimers electrostatic forces mainly drive the stability of dendriplexes, first generation compounds can penetrate into siRNA strands due to the establishment of hydrophobic interactions. Finally, in the particular case of first generation dendrimer [G(1)O(3)(NMe2(CH2)(2)OH))(6)](6+); the presence of hydroxyl groups reinforces dendriplex stability by hydrogen bonds formation. However, since these small dendrimers do not cover the RNA, only higher generation derivatives protect RNA from degradation.University of Alcalá; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN); Instituto de Investigación Sanitaria Gregorio Marañón; Universitat de Barcelon

Bacterial Cellulose-Chitosan paper with antimicrobial and antioxidant activities

The production of paper-based bacterial cellulose-chitosan (BC-Ch) nanocomposites was accomplished following two different approaches. In the first, BC paper sheets were produced and then immersed in an aqueous solution of chitosan (BC-ChI); in the second, BC pulp was impregnated with chitosan prior to the production of paper sheets (BC-ChM). BC-Ch nanocomposites were investigated in terms of physical characteristics, antimicrobial and antioxidant properties, and the ability to inhibit the formation of biofilms on their surface. The two types of BC-Ch nanocomposites maintained the hydrophobic character, the air barrier properties, and the high crystallinity of the BC paper. However, BC-ChI showed a surface with a denser fiber network and with smaller pores than those of BC-ChM. Only 5% of the chitosan leached from the BC-Ch nanocomposites after 96 h of incubation in an aqueous medium, indicating that it was well retained by the BC paper matrix. BC-Ch nanocomposites displayed antimicrobial activity, inhibiting growth of and having a killing effect against bacteria Staphylococcus aureus and Pseudomonas aeruginosa and yeast Candida albicans. Moreover, BC-Ch papers showed activity against the formation of a biofilm on their surface. The incorporation of chitosan increased the antioxidant activity of the BC paper. Paper-based BC-Ch nanocomposites combined the physical properties of BC paper and the antimicrobial, antibiofilm, and antioxidant activities of chitosan