Related Experiment Video
Updated: Nov 9, 2025
09:27
Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability
Published on: April 22, 2016
17.7K
Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation
Lisa Bretschneider1, Ingeborg Heuschkel1, Afaq Ahmed1
1Department of Solar Materials, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany.
Biotechnology and Bioengineering
|April 12, 2021
Summary
This study engineered Pseudomonas taiwanensis VLB120 to express cyclohexanone monooxygenase (CHMO) for biocatalysis. Biofilms showed potential for process stability, while suspended cells offered promising kinetics for cyclohexanone conversion.
Area of Science:
- Biocatalysis and Enzyme Engineering
- Microbial Biotechnology
- Green Chemistry
Background:
- Cyclohexanone monooxygenase (CHMO) is a Baeyer-Villiger monooxygenase crucial for converting cyclic ketones to lactones.
- Efficient biocatalysts are sought for sustainable chemical synthesis.
- Recombinant expression of biocatalysts in microbial hosts is a key strategy.
Purpose of the Study:
- To express and characterize an Acidovorax-derived CHMO in Pseudomonas taiwanensis VLB120.
- To compare the kinetic performance of isolated CHMO, suspended whole cells, and biofilms for cyclohexanone conversion.
- To evaluate the potential of different biocatalyst formats for industrial applications.
Main Methods:
- Gene expression of Acidovorax-derived CHMO in Pseudomonas taiwanensis VLB120.
- Purification and in vitro characterization of the recombinant CHMO enzyme.
- Kinetic analysis (KS, kcat, KI) of cyclohexanone conversion using isolated enzyme, suspended cells, and biofilms.
- Assessment of NADPH regeneration in whole-cell biocatalysts.
Main Results:
- The recombinant CHMO exhibited a broad substrate spectrum and high conversion efficiency.
- Biofilms displayed less favorable KS and kcat but a higher KI for cyclohexanone compared to isolated CHMO.
- Suspended cells showed improved KS, kcat, and KI values, coupled with efficient NADPH regeneration.
- Mass transfer limitations and heterogeneity were identified as challenges for biofilm performance.
Conclusions:
- Recombinant CHMO in P. taiwanensis VLB120 is a viable biocatalyst for cyclic ketone conversion.
- Suspended whole cells present a promising format due to favorable kinetics and efficient cofactor regeneration.
- Further engineering of biofilms is needed to overcome limitations and harness their process stability potential.
Related Concept Videos
01:22Reactions of Aldehydes and Ketones: Baeyer–Villiger Oxidation
4.6K
Baeyer–Villiger oxidation converts aldehydes to carboxylic acids and ketones to esters. The reaction uses peroxy acids or peracids and is often catalyzed by acid. The reaction is named after its pioneers, Adolf von Baeyer and Victor Villiger. The reaction is achieved by a wide range of peracids such as m-chloroperoxybenzoic acid (mCPBA), perbenzoic acid (C6H5COOOH), peracetic acid (CH3COOOH), hydrogen peroxide (H2O2), and tert-butyl hydroperoxide (t-BuOOH).
The carbonyl center is activated by...
The carbonyl center is activated by...
4.6K
01:28Reduction of Benzene to Cyclohexane: Catalytic Hydrogenation
5.2K
Unlike the easy catalytic hydrogenation of an alkene double bond, hydrogenation of a benzene double bond under similar reaction conditions does not take place easily. For example, in the reduction of stilbene, the benzene ring remains unaffected while the alkene bond gets reduced. Hydrogenation of an alkene double bond is exothermic and a favorable process. In contrast, to hydrogenate the first unsaturated bond of benzene, an energy input is needed; that is, the process is endothermic. This is...
5.2K
01:12Cycloaddition Reactions: MO Requirements for Photochemical Activation
2.3K
Some cycloaddition reactions are activated by heat, while others are initiated by light. For example, a [2 + 2] cycloaddition between two ethylene molecules occurs only in the presence of light. It is photochemically allowed but thermally forbidden.
2.3K
03:08Hydroboration-Oxidation of Alkenes
9.8K
In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.
9.8K
02:36Regioselectivity and Stereochemistry of Hydroboration
8.8K
A significant aspect of hydroboration–oxidation is the regio- and stereochemical outcome of the reaction.
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
8.8K
02:21Oxidation of Alkenes: Syn Dihydroxylation with Potassium Permanganate
14.6K
Alkenes can be dihydroxylated using potassium permanganate. The method encompasses the reaction of an alkene with a cold, dilute solution of potassium permanganate under basic conditions to form a cis-diol along with a brown precipitate of manganese dioxide.
14.6K