All good things come in threes!

The CARBAFIN team of UGent reported a successful application of engineered enzymes for the production of cellotriose! Cellotriose belongs to the group of cellodextrins, compounds that have the potential to act as prebiotics. Currently, cellodextrins are synthesized from cellulose by the chemical procedures that result in a complex mixture of oligosaccharides.

The idea within CARBAFIN is to produce cellotriose, believed to be the most potent component in the mixture, by an enzymatic bottom-up approach that would enable its defined synthesis. Cellotriose is composed of 3 glucose-units. Two bacterial strains (Cellulomonas uda and Clostridium cellulosi) provide the most promising enzyme candidates, which can be applied in a one-pot reaction. In the first step, enzyme number 1 (cellobiose phosphorylase, CBP) from Cellulomonas uda assembles two elements to produce cellobiose. Then, enzyme number 2 (cellodextrin phosphorylase, CDP) from Clostridium cellulosi elongates cellobiose by other glucose units to produce the three-piece compound, cellotriose but also larger cello-oligosaccharides.

In the recent paper, a special focus is given to the CBP enzyme and its improved variant that can synthesize cellotriose on its own. It represents a crucial step in enabling further research and commercial exploitation of cellotriose.

Read the full research article here!

New CARBAFIN paper about Scale-up of enzymatic cellodextrin production

Cellodextrins are functional carbohydrates that are expected to be promising water-soluble dietary fibres. They can act as a prebiotic ingredient and, therefore, have a positive effect on health: Prebiotics are stimulating the immune system, regulating the gastrointestinal function or preventing from carcinogenesis for example. An important criterium for prebiotic components is that they are not digested by humans. Cello-oligosaccharides (COS), also known as cellodextrin meet this requirement and are, therefore, one of the main target products of the CARBAFIN project.

The recent publication explains the enzymatic production of COS at larger scale in order to bring the technology that has been proven previously, to the next level and to open the door towards industrial application. The CARBAFIN researchers from acib and TU Graz together with researchers from BOKU demonstrate the soluble COS production to approximately 100 gram per litre in a 20 mL reaction volume. The enzymatic reaction turned out to be very efficient and to provide a product with a very satisfying yield and purity from simple and expedient substrates. The stimulation of growth among probiotic bacteria was also shown.

Read more!

 

Biotechnology Journal – new paper accepted: Cellobiose production in whole cells

Cellobiose, the simplest cellodextrin, is one of CARBAFIN’s target products, derived from sugar (glucose) molecules. The biocatalytic production process is divided into two steps:

  • Enzyme No. 1 (sucrose phosphorylase) produces an activated glucose component from sucrose, namely glucose-1-phosphate.
  • Enzyme No. 2 (cellobiose phosphorylase) uses the glucose-1-phosphate from the prior reaction and glucose for producing cellobiose.

For a cascade reaction like this, a combined process within one cell would be beneficial. But convincing the cell to do so, comes along with a huge effort of research. The CARBAFIN team was successful in designing an appropriate tool (plasmid) that allows the co-production of the two enzymes in perfect relation to each other. The obtained process and derived knowledge can be easily transferred to other cascadic reactions like this and, therefore, is of  broad interest across the whole field of biocatalysis.

Read more

Reactive extraction: an efficient method for fructose recovery

In the recently published Green Chemistry Article, the CARBAFIN team of acib GmbH describes the efficient separation of fructose during enzymatic production of Glucosylglycerol.

The sucrose-based reaction comes along with a significant fructose fraction as a side product. For commercial use, fructose needs to be separated from the main product (glucosylglycerol). The idea of the CARBAFIN project is to generate value of this “waste” part in order to strengthen the project derived glycosylation technology. Therefore, a high efficiency and purity after separation efforts would be highly desirable!

The team developed a protocol for reactive extraction that delivered a recovery of 90% glucosylglycerol and 83% fructose at a purity degree of 90%!

 

Review: Downstream Processing Technologies in the Biocatalytic Production of Oligosaccharides

We are proud to present our recent review about oligosaccharide production and its downstream processing. Oligosaccharides are an important ingredient for not only food and feed but also cosmetic products. The biocatalytic production processes offer a variety of advantages compared to chemical synthesis, such as an improved sustainability, environmental-friendliness and a lower energy effort. However, a successful production process requires an efficient downstream processing in terms of yield and costs. The review gives a good overview about actual unit operations and their corresponding separation principles. Our authors shed a light on membrane technologies (eg nanofiltration), chromatographic methods or solvent extractions, for example. Furthermore, they discuss the combination of different approaches. Access the full article from the Journal “Biotechnology Advances” here: https://doi.org/10.1016/j.biotechadv.2020.107568

New data about co-immobilized enzymes for production of cello-oligosaccharides

The paper “Three-enzyme phosphorylase cascade immobilized on solid support for biocatalytic synthesis of cello-oligosaccharides” was published in the journal ChemCatChem in December 2019. Now, all data are open access as well!

What is it about?

The synthesis of cello-oligosaccharides is a multistep process, which consists of several enzymatic reactions. For the sake of efficiency, biotechnologists would prefer to string these steps together (enzyme cascades), but this is challenging. Enzymes need to be recycled in order to keep their functionality. The recycling process is easier when enzymes are immobilized, but this process is well developed only for single enzymes so far. The actual CARBAFIN paper presents a controlled co-immobilization of three enzymes (glycoside phosphorylases) for the conversion of sucrose and glucose into soluble cello-oligosaccharide chains. The system is highly active AND recylable!

This is a very important step forward, since multienzyme cascades play an important role in synthetic carbohydrate chemistry.


Picture credits: Pixabay (Symbol for immobilized enzymatic cascades)

New Publication about Glucosylglycerol production and Nanofiltration

Glucosylglycerol (GG) is one of the main CARBAFIN targets that is an attractive compound for commercial applications in the cosmetics as well as the food industry. The enzymatic GG-production comes along with a high excess of glycerol, when using the enzyme sucrose phosphorylase. For the final product, glycerol needs to be removed and our CARBAFIN team found a good solution: nanofiltration. The researchers screened membranes at different varied pressure and temperature. The most suitable separation was achieved with a specific polyamide membrane and after 6 filtration cycles glycerol could be completely removed.

Find out more about the details!

The article was published in Separation and Purification Technology, Vol. 241 (2020).

CARBAFIN partner AVA Biochem teams-up with Michelin group

AVA Biochem had an interesting press release on 31st January 2020 about their cooperation with Michelin group:

[AVA Biochem has developed, patented and piloted a fully water-based
process for the conversion of industrial sugars into the 100% bio-based molecule 5-HMF. This platform chemical is ideally positioned to replace petroleum-sourced chemicals in various mass-market applications, due to its versatility, non-toxicity and bio-sourcing. Applications include biopolymers (such as yarns, films, bottles and other packaging) as well as resins and adhesives, where 5-HMF replaces highly toxic formaldehyde. With the common goal to further implement 5-HMF in various materials and chemical applications, AVA Biochem has entered into a Joint Development Agreement with the Michelin Group.

This collaboration aims to establish the world’s first commercial-scale production plant of 5-HMF, and to ultimately bring novel product applications onto the market based on this versatile chemical. These applications are all targeted to improve the performance, economics, sustainability (low carbon footprint) and safety (non-toxicity) of a variety of both consumer goods and industrial
products.

“We are proud to be working together with one of the most innovative players in their industry. The collaboration with Michelin represents a powerful opportunity to develop eco-friendly industrial products. It is an important step forward in AVA Biochem’s ambition to become a preferred partner and key provider of sustainable chemicals solutions and worldwide licenses to industrial and consumer goods groups”, says Peter Achermann, Chairman & Co-Founder of AVA Biochem.

Christophe Rahier (Michelin Senior vice president, Strategy and new businesses High Tech Materials) says: “This collaboration demonstrates the ability of AVA Biochem to master their new process. We will contribute to its know-how in the industrialization and evaluation of the performance of these products in different applications. This collaboration also illustrates Michelin’s growing interest in “green chemistry”, in line with our sustainable development strategy
and our vision of tomorrow’s mobility.”]

In CARBAFIN, AVA Biochem is currently testing the chemical-technical feasibility of using the fructose streams from cellodextrin or glucosylglycerol synthesis for the 5-HMF production. Therefore, they have installed a CARBAFIN mini reaction plant. We are curious about the results, stay tuned!

New Publication on Cellodextrin Production

The CARBAFIN team is proud to present another peer-reviewed publication about improved cellodextrin production processes! Cellodextrins are oligosaccharides that have promising applications as nutrial ingredients as long as they are soluble enough. The solubility mainly depends on the degree of polymerization, which is an important parameter for the biotechnological production.

The recent publication proposes a cascade approach by using three enzymes (glycoside phosphorylase cascade) for the conversion of sucrose and glucose into short-chain (soluble) cellodextrins. Our CARBAFIN team identified the three major factors that are crucial for an efficient reaction: the ratio of enzyme activity, the ratio of sucrose and glucose (substrate) and the phosphate concentration used. The article describes a balanced approach for product solubility control and yield and gives some important hints about upstream and downstream processing.

Preview to the Biotechnology Journal at Wiley Online Library

For reading the full article, please follow the link. The article was published in the Biotechnology Journal (Nov. 2019).