Rolf Wichmann

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 104 [PEG‐10,000‐NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L‐leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of α‐ketoisocaproate (2‐oxo‐4‐methylpentanoic acid) to L‐leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L‐leucine. The kinetic constants Vmax and km of the enzymes are all in the same range regardless of whether native NAD(H) or PEG‐10,000‐NAD(H) is used as coenzyme. L‐leucine was produced continuously out of α‐ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space–time yield was 324 mmol/L day (or 42.5 g/L day).

Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

BackgroundRhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.ResultsHere, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. Whats more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.ConclusionsA functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

Enzyme-assisted process for DAG synthesis in edible oils.

This study deals with the enzymatic synthesis of diacylglycerols in rapeseed oil by the esterification of free fatty acids and monoacylglycerols. As enzymatic reactions are influenced by many factors, a statistical design of experiments was conducted to investigate the enrichment of diacylglycerols, systematically. Simultaneously, the investigated method contributes to the refining process, as the amount of free fatty acids could be reduced significantly from 2% to 0.3%. Utilizing an immobilized lipase from Rhizomucormiehei, a maximum diacylglycerol content of 23% was obtained, after optimization. By washing with iso-propanol and hexane the immobilised lipase could be reused in 14 consecutive batches. In addition, glycerol was proven to be an alternative to MAG as acyl-group acceptor. However, the diacylglycerol enrichment was not accomplished in the same yields as for monoacylglycerols. Summarizing, the present study revealed the potential of an enzymatic diacylglycerol synthesis in edible oils as a suitable alternative to conventional processes also enabling the reduction of free fatty acids in crude oils.

Improvement in the bioreactor specific productivity by coupling continuous reactor with repeated fed-batch reactor for acetone-butanol-ethanol production.

In comparison to the different fermentation modes for the production of acetone, butanol and ethanol (ABE) researched to date, the continuous fermentation is the most economically favored. Continuous fermentation with two or more reactor cascade is reported to be the most efficient as it results in a more stable solvent production process. In this work, it is shown that a continuous (first-stage) reactor coupled to a repeated fed-batch (second stage) is superior to batch and fed-batch fermentations, including two-stage continuous fermentation. This is due to the efficient catalyst use, reported through the specific product rate and rapid glucose consumption rate. High solvents are produced at 19.4 g(ABE) l⁻¹, with volumetric productivities of 0.92 g(butanol) l⁻¹ h⁻¹ and 1.47 g(ABE) l ⁻¹ h⁻¹. The bioreactor specific productivities of 0.62 and 0.39 g g⁻¹(cdw) h⁻¹ obtained show a high catalyst activity. This new process mode has not been reported before in the development of ABE fermentation and it shows great potential and superiority to the existing fermentation methods.

Influence of substrate or product inhibition on the performance of enzyme reactors

For the design of an enzyme reactor a detailed knowledge of the kinetic parameters of the catalyst under operational conditions is essential. For technical applications high initial substrate concentrations and high degrees of conversions are desirable, in order to save reactor volume and energy in recovery processes. Most of the kinetic data available in the literature have been derived from dilute solutions under initial rate conditions. These data cannot be extrapolated with confidence for technically interesting concentrations because substrate as well as product-inhibition may occur, which would not be observed in dilute solutions and by initial rate measurements. Because of this difficulty effective and fast methods to obtain significant data for technical applications have been developed based on-line rate determinations. Such extensive treatment has proved necessary for the following enzymes: alanine dehydrogenase, formate dehydrogenase and alpha-glucosidase, indicating that we are dealing with a general phenomenon.

Continuous microbial production of L-leucine with cell retention

SummaryContinuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting fromα-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.

Foam adsorption as an ex situ capture step for surfactants produced by fermentation

In this report, a method for a simultaneous production and separation of a microbially synthesized rhamnolipid biosurfactant is presented. During the aerobic cultivation of flagella-free Pseudomonas putida EM383 in a 3.1L stirred tank reactor on glucose as a sole carbon source, rhamnolipids are produced and excreted into the fermentation liquid. Here, a strategy for biosurfactant capture from rhamnolipid enriched fermentation foam using hydrophobic-hydrophobic interaction was investigated. Five adsorbents were tested independently for the application of this capture technique and the best performing adsorbent was tested in a fermentation process. Cell-containing foam was allowed to flow out of the fermentor through the off-gas line and an adsorption packed bed. Foam was observed to collapse instantly, while the resultant liquid flow-through, which was largely devoid of the target biosurfactant, eluted towards the outlet channel of the packed bed column and was subsequently pumped back into the fermentor. After 48h of simultaneous fermentation and ex situ adsorption of rhamnolipids from the foam, 90% out of 5.5g of total rhamnolipids produced were found in ethanol eluate of the adsorbent material, indicating the suitability of this material for ex situ rhamnolipid capture from fermentation processes.

Characterisation of 2,5-diketo-D-gluconic acid reductase from Corynebacterium sp.

Summary2,5-diketo-D-gluconic acid reductase, that converts 2,5-diketo-D-gluconic acid into 2-keto-L-gulonic acid (the direct precursor of vitamin C) was extracted and purified from Corynebacterium sp.. The enzyme was characterised in terms of kinetic parameters, molecular weight and isoelectric point. Enzyme stability at different operating temperatures was investigated, as well.