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

Funcionalización enzimática de la celulosa bacteriana para su aprovechamiento y valorización

[spa] Recientemente, el interés en la celulosa se ha desplazado hacia materiales de la nanoescala que incluyen las nanofibrillas de celulosa (NFC), los nanocristales de celulosa (NCC) y la celulosa bacteriana (CB). La CB es un exopolisacárido sintetizado por algunas bacterias. Su composición química es la misma que la de la celulosa vegetal, pero su conformación y sus propiedades fisicoquímicas son diferentes. La CB presenta un mayor grado de pureza, tiene índices de cristalinidad más elevados y mayor capacidad de retención de agua. Además, presenta una gran elasticidad, buena resistencia mecánica y es biocompatible. Todas estas propiedades le confieren una gran aplicabilidad en campos muy diversos. La CB se produce en forma de una red tridimensional de nanofibras que genera una estructura con una elevada área superficial con potencial para la adhesión y retención de moléculas. El objetivo de esta tesis, la funcionalización de la CB dentro de un marco de tecnologías amigables con el medio ambiente, se ha abordado desde dos vertientes: la inmovilización de enzimas y la generación de NCC, todo ello reflejado en un compendio de cinco artículos. Previamente al estudio de la idoneidad de diferentes matrices de CB como soporte para la inmovilización de enzimas lipasas, se procedió a la mejora genética de la lipasa LipJ de Bacillus cereus JR3. Esta cepa exhibía una actividad lipasa hipertermófila en su sobrenadante, una característica de gran interés de cara a una aplicación industrial. Sin embargo, LipJ, a pesar de su similitud de secuencia con otras lipasas termófilas bacterianas, no era la responsable de esta actividad. La meta del primer estudio fue, pues, revertir este comportamiento mesófilo a termófilo a través de una mutagénesis dirigida en los dominios de su centro catalítico y en el péptido señal y a través de la creación de una librería de degeneración NNK en la posición H110, implicada en la activación dependiente de la temperatura de lipasas termófilas. Las diferentes variantes obtenidas con ambas estrategias mostraron un cambio de tendencia en la especificidad de sustrato, pero sin rasgos de termofília. Estas circunstancias, unidas a la baja actividad de la lipasa original, condujo a su descarte como enzima a inmovilizar sobre la CB. La dificultad en discriminar entre la actividad debida a LipJ o a la actividad intrínseca de la cepa hospedadora, Escherichia coli, inspiró el objetivo del segundo estudio de la tesis. Esta actividad basal, aunque conocida por la comunidad científica, apenas se encuentra documentada, y es la causa de interferencias en la caracterización de lipasas. Por estas razones, se procedió a la caracterización de esta actividad en las cepas más comunes de E.coli empleadas en la clonación y expresión heteróloga de lipasas. Como se describe en el tercer artículo, se empleó una lipasa comercial para estandarizar el proceso de inmovilización por adsorción física sobre dos matrices de CB: CB en suspensión acuosa (BCS, por sus siglas en inglés Bacterial Cellulose Suspension) y papel de CB (BCP por sus siglas en inglés Bacterial Cellulose Paper). Los nanocomposites de Lipasa/CB obtenidos presentaron excelentes propiedades operacionales. Los nanocomposites Lipasa/BCP mostraron una gran estabilidad térmica, reusabilidad y durabilidad, además de mantenerse activos después de ser almacenados durante más de un mes a temperatura ambiente, por lo que podrían ser potenciales candidatos en la elaboración de papeles bioactivos de dispositivos simples, portátiles y desechables. Siguiendo la misma metodología, en la siguiente investigación se generó un papel de CB funcional con actividad antimicrobiana y antioxidante, mediante la inmovilización de la lisozima. A temperatura ambiente, la enzima inmovilizada mostró una mayor estabilidad que la lisozima libre, aparte de conservar la totalidad de su actividad durante casi tres meses. Debido a la naturaleza intrínseca de sus componentes, el papel Lisozima/BCP es biodegradable y biocompatible, lo que lo convierte en candidato ideal para el diseño de nuevos materiales de envasado en la industria alimentaria. Finalmente, en el último artículo se describe la producción de NCC de CB (NCCB), a partir de un proceso más sencillo y respetuoso con el medioambiente que la tradicional hidrólisis ácida con ácido sulfúrico o ácido clorhídrico. Mediante un tratamiento con monooxigenasas líticas de polisacáridos (LPMOs), responsables de aportar cargas negativas y proporcionar una mayor estabilidad, y una digestión con glucosil hidrolasas, se obtuvieron unos NCCB de entre 80 nm y 2 µm de longitud y 9 nm de ancho. Sus propiedades permitieron su uso como agentes de recubrimiento sobre soportes celulósicos de origen vegetal, aportándoles propiedades barrera al agua y al aceite, además de mejorar sus cualidades mecánicas.[eng] Recently, interest in cellulose has shifted to nanoscale materials including cellulose nanofibrils (CNF), cellulose nanocrystals (CNC) and bacterial cellulose (BC). BC is an exopolysaccharide synthesized by some bacteria, with the same chemical structure than that of vegetable cellulose, but with different conformation and physicochemical properties. BC has a higher degree of purity, a higher crystallinity index and a greater water retention capacity. In addition, it has great elasticity and good mechanical resistance and is biocompatible. According to all these features, BC is a promising biomaterial that can meet the needs of different fields. Due to its three-dimensional structure of nanofibers, BC have a high superficial area, a feature that makes BC a suitable material to entrap different types of molecules. The main goal of this thesis, the functionalization of BC in a framework of more environmentally friendly methodologies, has been approached by enzyme immobilization and the obtention of CNC in a compendium of five research articles. Prior to studying the suitability of different bacterial cellulose matrices lipase enzyme carriers, LipJ, a previously cloned lipase from Bacillus cereus JR3 was genetically improved. This stain deserved great interest for industrial applications because of its remarkably high tolerance to the extreme temperatures of its lipolytic system. However, even though its sequence has a markedly similarity to thermophilic lipases, LipJ showed the highest activity levels at 30°C, with no signs of being a thermophilic lipase. In the first study, with the objective of reversing its mesophilic activity into thermophilic trough a point directed mutation essay and through the construction of a NNK degenerancy library in the H110 position, a putative position involved in temperature dependent activation of thermophilic lipases. The obtained variants showed a a general shift in specificity towards longer chain-length substrates, but without thermophilic traits. These circumstances, in addition to the low activity of native LipJ lipase, led to reject this enzyme for immobilization onto BC. This difficulty in discriminating between the activity due to LipJ or the intrinsic activity of the host strain, Escherichia coli, aimed the purpose of the second study of the thesis. Even if this E.coli’s basal lipolytic activity is widely known in the scientific community, it has been barely explored. For these reasons, this activity was deeply characterized in the most common strains of E.coli used in the cloning and heterologous expression of lipases. Then, as it is described in the third article, a commercial lipase was used to standardize the immobilization process on two BC matrices: BC in aqueous suspension (BCS) and BC paper (BCP). Once the Lipase BC by physical adsorption, it was found that neither the morphology nor the crystallinity were affected. The specific activity was measured under different conditions and the operational properties were evaluated. The Lipase / BCP nanocomposite showed great thermal stability, reusability and durability. Besides, it remained active after being storaged at room temperature for more than a month, which indicated that it could be a key element in the development of bioactive papers for simple, portable and disposable devices. Following the same methodology, the next research was focused on the production of a functional BC paper with antimicrobial and antioxidant activities through the immobilization of lysozyme. At room temperature, the immobilized enzyme showed greater stability than free lysozyme, apart from conserving all of its activity for almost three months. Due to the intrinsic nature of its components, Lysozyme- BCP is biodegradable and biocompatible, which makes it a great candidate for the design of new packaging materials in the food industry. Finally, in the last article, NCC from BC (BCNC) were produced by an easier and more environmentally friendly process than traditional harsh acid hydrolysis. The combination of a lytic polysaccharide monooxygenase, who provided BC with negative charges and lead to a better stability, and a mixture of glycosyl hydrolases, BCNC with a length ranging from 80 nm to 2 µm and a width of 9 nm were obtained. Their properties allowed their use as a coating agent in two different pre-existing cellulosic materials, providing them with different degrees of barrier and mechanical properties

Data - Bacterial cellulose matrices to develop enzymatically active paper-based nanocomposites

Data - Bacterial cellulose matrices to develop enzymatically active paper-based nanocomposite

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 lysozyme was 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 lysozyme were similar. Moreover, immobilized enzyme showed a broader working temperature and higher thermal stability. The composites maintained its activity for at least 80 days without 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 packagingPostprint (author's final draft

Bacterial cellulose : a smart biomaterial with diverse applications

Natural biomaterials have benefited the human civilisation for millennia. However, in recent years, designing of natural materials for a wide range of applications have become a focus of attention, spearheaded by sustainability. With advances in materials science, new ways of manufacturing, processing, and functionalising biomaterials for structural specificity has become feasible. Our review is focused on bacterial cellulose (BC), an exceptionally versatile natural biomaterial. BC is a unique nanofibrillar biomaterial extruded by microscopic single- cell bacterial factories utilising the chemical energy harvested from renewable substrates. BC is extracellular and is intrinsically pure, unlike other biopolymers that require extraction and purification. BC fibres are 100 times thinner than plant-derived cellulose and exist in a highly porous three-dimensional network that is highly biocompatible. Macro fibres fabricated from BC nanofibrils are stronger and stiffer, have high tensile strength values and can be used as substitutes for fossil fuel-derived synthetic fibres. The increased surface area to volume ratio allows stronger interactions with the components of composites that are derived from BC. The reactive hydroxyl groups on BC allows various chemical modifications for the development of functionalised BC with a plethora of ‘smart’ applications. In this review we consolidate the current knowledge on the production and properties of BC and BC composites, and highlight the very recent advancements in bulk applications, including food, paper, packaging, superabsorbent polymers and the bio-concrete industries. The process simplicity of BC production has the potential for large scale low-cost applications in bioremediation. Furthermore, the emerging high value applications of BC will be in electrochemical energy storage devices as a battery separator, and in transparent display technologies will be explored. Finally, the extensive biomedical applications of BC are discussed including, wound healing, controlled drug delivery, cancer treatment, cell culture and artificial blood vessels. In a further development on this, additive manufacturing considers enhancing the capabilities for manufacturing complex scaffolds for biomedical applications. An outlook on the future directions of BC in these and other innovative areas is presented