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Cellulose for enzymes immobilization

Nanobiocatalysis, a common approach used in nanobiotechnology can be defined as a process based on incorporation of enzymes onto nanostructured material.

Enzymes are remarkable biocatalysts and have been used in the industrial biotechnology because of their interesting characteristics, such as green chemistry, substrate and product specificity, and ease of preparation.

The catalytic activity of enzymes implies that they accelerate reactions by decreasing the activation energy.

In biocatalyst fields, major objectives are:

to stabilize and to recover the enzymes, since they are too costly in the current market. Achieving good stability, enzyme separation, recovery, and life-time cycle rate are successful keys in enzymatic production and commercialization.

Native enzymes are commonly applied as industrial biocatalysts due to their higher enzyme activity.

Unfortunately, native enzymes often lack long-term stability in operational conditions and pose difficulties for recovery and reuse.

For example, capillary gel electrophoresis can be used for separation between enzyme and product, but it requires high energy consumption, is expensive, and is not applicable (difficult and time consuming) for large-scale operation.

The performance of enzyme immobilization strongly depends on the properties of support, such as material type, composition, structure, and mechanical properties.

Better support properties provide a good mechanical strength, which can contribute to stability and reusability. The use of nanosized supports in enzyme immobilization is not only to enhance the stability and reusability of immobilized enzyme but also to overcome lower immobilized enzyme activity due to the presence of high surface area per volume ratio.

In order words, high surface area of nanosized support provides a high number of functional group on the surface support. Thus, CNF have more chance of interacting with enzyme molecules.

A unique behavior of nanoscale support would distinguish them from traditional immobilized systems. Development of nanoscale biocatalyst system would show a benefit for both enzyme and nanosized support, since the size of enzyme molecule is already in the nanoorder.


Course creator: Sergiu Coseri

Nanomaterials-nano

Nanotechnology is science, engineering, and technology conducted at the nanoscale (about 1 to 100 nanometers)

Nano can refer to technologies, materials, particles, objects – we are focusing on nanomaterials as these are already being used in workplaces more widely

A sheet of paper is about 100,000 nanometers thick, a human hair is around 80,000- 100,000 nanometers wide

What are they?

•Nano = 10-9 or one billionth in size
•Materials with dimensions and tolerances in the range of 100 nm to 0.1 nm
•Metals, ceramics, polymeric materials, or composite materials
•One nanometer spans 3-5 atoms lined up in a row
•Human hair is five orders of magnitude larger than nanomaterials

Course creator: Sergiu Coseri

Cellulose, to depolymerize or not to?

Oxidation of the primary OHgroups in cellulose is a pivotal reaction both at lab and industrial scale, leading to the
value-added products, i.e. oxidized cellulosewhich have tremendous applications in medicine, pharmacy and hitech
industry. Moreover, the introduction of carboxyl moieties creates prerequisites for further cellulose
functionalization through covalent attachment or electrostatic interactions, being an essential achievement designed
to boost the area of cellulose-based nanomaterials fabrication. Variousmethods for the cellulose oxidation
have been developed in the course of time, aiming the selective conversion of theOHgroups. These methods use:
nitrogen dioxide in chloroform, alkali metal nitrites and nitrates, strong acids alone or in combination with permanganates
or sodium nitrite, ozone, and sodium periodate or lead (IV) tetraacetate. In the case of the last two
reagents, cellulose dialdehydes derivatives are formed, which are further oxidized by sodium chlorite or hydrogen
peroxide to formdicarboxyl groups. A major improvement in the cellulose oxidationwas represented by the
introduction of the stable nitroxyl radicals, such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). However, a
major impediment for the researchers working in this area is related with the severe depolymerisation occurred
during the TEMPO-mediated conversion of\\CH2\\OH into COOH groups. On the other hand, the cellulose
depolymerisation represent the key step, in the general effort of searching for alternative strategies to develop
newrenewable, carbon-neutral energy sources. In this connection, exploiting the biomass feed stocks to produce
biofuel and other low molecular organic compounds, involves a high amount of research to improve the overall
reaction conditions, limit the energy consumption, and to use benign reagents. This course is therefore focused on
the parallelism between these two apparently antagonist processes involving cellulose, building a necessary
bridge between them, thinking how the reported drawbacks of the TEMPO-mediated oxidation of cellulose are
heading towards to the biomass valorisation, presenting why the apparently undesired side reactions could be
turned into beneficial processes if they are correlated with the existing achievements of particular significance
in the field of cellulose conversion into small organic compounds, aiming the general goal of pursuing for alternatives
to replace the petroleum-based products in human life.

Course creator: Sergiu Coseri

N-hydroxyphthalimide (NHPI) powerful catalyst for the cellulose oxidation

Catalytic aerobic oxidation of organic substrates is of fundamental importance in the industrial synthesis of a large variety of oxy-functionalized compounds from both economic and environmental points of view.

Among all >NO● radicals, N-hydroxyphthalimide (NHPI), has emerged as a powerful and popular catalyst for organic oxidation reactions. NHPI is thought to catalyze oxidation through initial generation of the phthalimide-N-oxyl (PINO) radical by abstraction of the O-H hydrogen in NHPI. The PINO radical then abstracts a hydrogen atom from a target substrate, thus reverting NHPI and a carbon-centered radical. This carbon radical reacts with O2 to yield a peroxy radical, and the peroxy radical abstracts the O-H hydrogen from NHPI, forming a stable hydroperoxide, while also regenerating the PINO radical.

However, the PINO radical was for the first time reported as early as 1964 by Lemaire and Rassat, using EPR spectroscopy for the NHPI and lead tetraacetate reaction in benzene.

 

Since there, a large variety of methods to generate PINO radical have been developed and reported. These methods to generate PINO radical, can be divided into three categories of catalytic systems:

i)Biocatalytic systems for the generation of PINO radical;
ii)Electrocatalytic systems for the generation of PINO radical;
iii)Chemocatalytic systems for the generation of PINO radical;


Course creator: Sergiu Coseri

Applications of TEMPO-oxidized polysaccharides

Human skin plays an essential role in preventing water loss in the body as it provides the outermost
layer of protection from the external environment. In skincare applications, bio-polymers, synthetic
polymers, and organic polymers are used to control formulation viscosities, transfer moisture to the
skin, increase the stability of the formulation and active ingredient, and protect the skin by forming a
coating on its surface [1]. In particular, bio-polymers, such as cellulose, chitosan, and polysaccharide,
are known to be skin-friendly substances as they are biocompatible and biodegradable. Among these,
cellulose is the most abundant bio-polymer in plants and microorganisms and possesses a number
of unique properties that, depending on its origin and the extraction process, allow it to be used in
various applications.
Recently, cellulose nanofibers (CNFs) have attracted wide interest due to their nanoscopic
size, ease of preparation, low cost, tunable surface properties, and enhanced mechanical properties,
which makes them well suited for use as drug carriers, tissue regenerating sca olds, water purifying
membranes, electrodes, supercapacitors, fluorescent probes, and flexible electronics [2]. In the
field of skincare, CNFs have attracted attention as a new potential bio-material with thixotropic
properties that allow it to be used for emulsion stabilization, water retention, and rheology modification
applications [3].Bacterial cellulose (BC), which is referred to as bio-cellulose in the skincare field, is a bio-based
polymer that is synthesized directly from microorganisms, such as Acetobacter xylinum (A. xylinum).
BC nanofibers (BCNFs) have a number of advantages over plant-derived cellulose, including a
high physical strength, water absorption and retention properties, and a uniform fiber network
structure [4–7]. In contrast, a higher yield of CNFs can be obtained through the oxidation of cellulose mediated
via 2,2,6,6–tetramethyl–1–piperidine–N–oxy radical (TEMPO), which is an oxidation catalyst capable
of replacing alcoholic groups of cellulose with aldehyde, ketone, and carboxy groups under mild
conditions at room temperature and normal pressure. TEMPO-oxidized cellulose can be dispersed in
the aqueous phase by the repulsion force caused by the anionic charge on the surface of the carboxyl
groups in the modified cellulose [10,11]. As the resulting material has the advantage of maintaining its
physical fiber structure when dispersed in water, its use has been studied in a variety of fields, such as
papermaking, membrane filters, heavy metal removal, and cell transfer.

References
1. Gross, R.A.; Kalra, B. Biodegradable Polymers for the Environment. Science 2002, 297, 803–807. [CrossRef]
[PubMed]
2. Menon, M.P.; Selvakumar, R.; Kumar, P.S.; Ramakrishna, S. Extraction and modification of cellulose nanofibers
derived from biomass for environmental application. RSC Adv. 2017, 7, 42750–42773. [CrossRef]
3. Halib, N.; Perrone, F.; Cemazar, M.; Dapas, B.; Farra, R.; Abrami, M.; Chiarappa, G.; Forte, G.; Zanconati, F.;
Pozzato, G.; et al. Potential Applications of Nanocellulose-Containing Materials in the Biomedical Field.
Materials 2017, 10, 977. [CrossRef] [PubMed]
4. Tahara, N.; Tabuchi, M.; Watanabe, K.; Yano, H.; Morinaga, Y.; Yoshinaga, F. Degree of Polymerization
of Cellulose from Acetobacter xylinum BPR2001 Decreased by Cellulase Produced by the Strain.
Biosci. Biotechnol. Biochem. 1997, 61, 1862–1865. [CrossRef] [PubMed]
5. Naritomi, T.; Kouda, T.; Yano, H.; Yoshinaga, F. E ect of lactate on bacterial cellulose production from
fructose in continuous culture. J. Ferment. Bioeng. 1998, 85, 89–95. [CrossRef]
6. Lee, J.W.; Deng, F.; Yeomans,W.G.; Allen, A.L.; Gross, R.A.; Kaplan, D.L. Direct Incorporation of Glucosamine
and N-Acetylglucosamine into Exopolymers by Gluconacetobacter xylinus (5Acetobacter xylinum) ATCC
10245: Production of Chitosan-Cellulose and Chitin-Cellulose Exopolymers. Appl. Environ. Microbiol. 2001,
67, 3970–3975. [CrossRef] [PubMed]
7. Svensson, A.; Nicklasson, E.; Harrah, T.; Panilaitis, B.; Kaplan, D.L.; Brittberg, M.; Gatenholm, P. Bacterial
cellulose as a potential sca old for tissue engineering of cartilage. Biomaterials 2005, 26, 419–431. [CrossRef]
[PubMed]
8. Chen,W.; Li, Q.; Cao, J.; Liu, Y.; Li, J.; Zhang, J.; Luo, S.; Yu, H. Revealing the structures of cellulose nanofiber
bundles obtained by mechanical nanofibrillation via TEM observation. Carbohydr. Polym. 2015, 117, 950–956.
[CrossRef]
9. Kondo, T.; Kose, R.; Naito, H.; Kasai, W. Aqueous counter collision using paired water jets as a novel means
of preparing bio-nanofibers. Carbohydr. Polymer. 2014, 112, 284–290. [CrossRef]
10. Spaic, M.; Small, D.P.; Cook, J.R.; Wan, W. Characterization of Anionic and Cationic Functionalized Bacterial
Cellulose Nanofibres for Controlled Release Applications. Cellulose 2014, 21, 1529–1540. [CrossRef]
11. Jun, S.-H.; Lee, S.-H.; Kim, S.; Park, S.-G.; Lee, C.-K.; Kang, N.-K. Physical properties of TEMPO-oxidized
bacterial cellulose nanofibers on the skin surface. Cellulose 2017, 24, 5267–5274.

Course creator: Sergiu Coseri

TEMPO-oxidation_Generalities

2,2,6,6-Tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation is a unique reaction to native and regenerated celluloses, and has advantages in terms of position-selective reaction at room temperature under aqueous conditions. When the TEMPO/NaBr/NaClO oxidation is applied to native celluloses in water at pH 10 under suitable conditions, the C6-primary hydroxy groups present on crystalline cellulose microfibril surfaces are mostly converted to sodium C6-carboxylate groups. Anionic sodium glucuronosyl units are densely, regularly, and position-selectively formed on crystalline cellulose microfibril surfaces, while maintaining the original cellulose morphology, cellulose I crystal structure, crystallinity, and crystal width. When TEMPO-oxidized celluloses (TOCs) prepared from, for example, wood cellulose have sodium C6-carboxylate contents >1 mmol/g, transparent highly viscous gels consisting of TEMPO-oxidized cellulose nanofibrils (TOCNs) with homogeneous widths of ≈3 nm and lengths >0.5 μm, dispersed at the individual nanofiber level, are obtained by gentle mechanical disintegration of TOCs in water. Alternative systems are as follows: TEMPO/NaClO/NaClO2 system, TEMPO electro-mediated oxidation, etc. TOCNs are promising new plant-based renewable nanofibers applicable to high-tech material fields.

Course creator: Sergiu Coseri

Cellulose, chemical modification, reactions

This course is devoted to the chemical modification of cellulose. Chemical modification of cellulose is of crucial importance, because the properties of the native cellulose can be modified, new moieties are introduced in the cellulose backbone, which confer new properties for a wide range of applications.

Course creator: Sergiu Coseri

Cellulose_Introduction_Importance

A general introduction on cellulose structure and properties.

Course creator: Sergiu Coseri