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Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition.

Hildebrand A, Kasuga T, Fan Z - PLoS ONE (2015)

Bottom Line: By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase.The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate.This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA, 95616, United States of America.

ABSTRACT
We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5∆ace-1∆cre-1∆ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

No MeSH data available.


Related in: MedlinePlus

The effect of potassium phosphate buffer concentration and the conversion of cellobiose to CBA via CDH-ABTS-laccase mediated conversion with the F5Δace-1Δcre-1ΔndvB strain in 1x Vogel’s medium and 20 g/L Avicel.The values shown are the means of biological duplicates with the error bars representing the standard deviations.
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pone.0123006.g003: The effect of potassium phosphate buffer concentration and the conversion of cellobiose to CBA via CDH-ABTS-laccase mediated conversion with the F5Δace-1Δcre-1ΔndvB strain in 1x Vogel’s medium and 20 g/L Avicel.The values shown are the means of biological duplicates with the error bars representing the standard deviations.

Mentions: When F5Δace-1Δcre-1ΔndvB is grown on 20 g/L Avicel, approximately 20 mM of cellobiose is produced along with 10 mM CBA. If laccase and ABTS are added to convert cellobiose to CBA, 30 mM of CBA is produced [19]. Because of the high level of CBA production, the pH rapidly drops from pH 6 to pH 4. Buffering the fermentation medium was evaluated to determine the effect of this pH drop on the conversion of cellobiose to CBA. Three different concentrations of potassium phosphate buffer were evaluated to control the rate of pH drop and compared to the unbuffered Vogel’s medium (Fig 3). In the case of the unbuffered Vogel’s medium, the pH drops to 4 over the course of the fermentation. The addition of sodium phosphate buffer results in a slower decrease in pH and higher final pH as the buffer concentration increases. 200 mM potassium phosphate held the pH at 6 until the last day of fermentation where it dropped to 5.5. Although pH varied across the conditions tested, cellobiose and CBA production were not significantly affected. As a result, unbuffered Vogel’s medium was used for the remainder of the fermentation experiments in this study.


Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition.

Hildebrand A, Kasuga T, Fan Z - PLoS ONE (2015)

The effect of potassium phosphate buffer concentration and the conversion of cellobiose to CBA via CDH-ABTS-laccase mediated conversion with the F5Δace-1Δcre-1ΔndvB strain in 1x Vogel’s medium and 20 g/L Avicel.The values shown are the means of biological duplicates with the error bars representing the standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388835&req=5

pone.0123006.g003: The effect of potassium phosphate buffer concentration and the conversion of cellobiose to CBA via CDH-ABTS-laccase mediated conversion with the F5Δace-1Δcre-1ΔndvB strain in 1x Vogel’s medium and 20 g/L Avicel.The values shown are the means of biological duplicates with the error bars representing the standard deviations.
Mentions: When F5Δace-1Δcre-1ΔndvB is grown on 20 g/L Avicel, approximately 20 mM of cellobiose is produced along with 10 mM CBA. If laccase and ABTS are added to convert cellobiose to CBA, 30 mM of CBA is produced [19]. Because of the high level of CBA production, the pH rapidly drops from pH 6 to pH 4. Buffering the fermentation medium was evaluated to determine the effect of this pH drop on the conversion of cellobiose to CBA. Three different concentrations of potassium phosphate buffer were evaluated to control the rate of pH drop and compared to the unbuffered Vogel’s medium (Fig 3). In the case of the unbuffered Vogel’s medium, the pH drops to 4 over the course of the fermentation. The addition of sodium phosphate buffer results in a slower decrease in pH and higher final pH as the buffer concentration increases. 200 mM potassium phosphate held the pH at 6 until the last day of fermentation where it dropped to 5.5. Although pH varied across the conditions tested, cellobiose and CBA production were not significantly affected. As a result, unbuffered Vogel’s medium was used for the remainder of the fermentation experiments in this study.

Bottom Line: By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase.The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate.This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA, 95616, United States of America.

ABSTRACT
We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5∆ace-1∆cre-1∆ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

No MeSH data available.


Related in: MedlinePlus