Combination of algae and yeast fermentation for an integrated process to produce single cell oils.

Economic and ecological reasons cause the industry to develop new innovative bio-based processes for the production of oil as renewable feedstock. Petroleum resources are expected to be depleted in the near future. Plant oils as sole substituent are highly criticized because of the competitive utilization of the agricultural area for food and energy feedstock production. Microbial lipids of oleaginous microorganisms are therefore a suitable alternative. To decrease production costs of microbial lipids and gain spatial independence from industrial sites of CO2 emission, a combination of heterotrophic and phototrophic cultivation with integrated CO2 recycling was investigated in this study. A feasibility study on a semi-pilot scale was conducted and showed that the cultivation of the oleaginous yeast Cryptococcus curvatus on a 1.2-L scale was sufficient to supply a culture of the oleaginous microalgae Phaeodactylum tricornutum in a 21-L bubble column reactor with CO2 while single cell oils were produced in both processes due to a nutrient limitation.

Stereoselective hydrolysis of aryl-substituted dihydropyrimidines by hydantoinases.

In this study, we investigated the possibility of using a modified hydantoinase process for the production of optically pure ß-amino acids. Two aryl-substituted dihydropyrimidines D,L-6-phenyl-5,6-dihydrouracil (PheDU) and para-chloro-D,L-6-phenyl-5,6-dihydrouracil (pClPheDU) were synthesized. Hydrolysis of these novel substrates to the corresponding N-carbamoyl-ß-amino acids by three recombinant D-hydantoinases and several bacterial strains was tested. All applied recombinant D-hydantoinases and eight bacterial isolates catalyzed the conversion of PheDU to N-carbamoyl-ß-phenylalanine (NCßPhe). Some of these biocatalysts showed an enantioselectivity for either the D- or the L-PheDU enantiomer. The second dihydropyrimidinase substrate pClPheDU was hydrolyzed by all three recombinant D-hydantoinases and six of the wild-type strains. To our knowledge, this is the first dihydropyrimidinase activity reported with this aryl-substituted dihydropyrimidine. For selected biocatalysts, hydantoinase activity towards aryl-substituted hydantoins was demonstrated as well. However, none of the bacterial strains tested so far exhibited any carbamoylase activity towards NCßPhe.

Kinetic analysis and modeling of the liquid-liquid conversion of emulsified di-rhamnolipids by Naringinase from Penicillium decumbens.

The enzymatic conversion of an aggregate-forming substrate was kinetically analyzed and a model was applied for the prediction of reaction-time courses. An L-rhamnose molecule from a di-rhamnolipid is cleaved by Naringinase from Penicillium decumbens leading to a mono-rhamnolipid. Optimal reaction rates were found when both, substrate and product build large co-aggregates in a slightly acidic aqueous phase. On the other hand, reaction rates were independent of initial di-rhamnolipid concentration and this was interpreted by assuming that the reaction occurs in the aqueous phase according to Michaelis-Menten kinetics in combination with competitive L-rhamnose inhibition. Rhamnolipids were therefore assumed to be highly concentrated in aggregates, a second liquid phase, whereas diffusive rhamnolipid transport from and to the aqueous phase occurs due to the enzymatic reaction. Furthermore, ideal surfactant mixing between di- and mono-rhamnolipid was assumed for interpretation of the negative effect of the last on the reaction rate. A model was created that describes the system accordingly. The comparison of the experimental data, were in excellent agreement with the predicted values. The findings of this study may beneficially be adapted for any bioconversion involving aggregate-forming substrate and/or product being catalyzed by hydrophilic enzymes.

Deactivation kinetics and response surface analysis of the stability of alpha-L-rhamnosidase from Penicillium decumbens.

The stability of the mixed enzyme preparation Naringinase from Penicillium decumbens was studied in dependence of the temperature, the pH value, and the enzyme concentration by means of response surface methodology. Deactivation kinetics by formation of an intermediate state was proposed for fitting deactivation data. Empirical models could then be constructed for prediction of deactivation rate constants, specific activity of intermediate state, and half-life values under different incubation conditions. From this study, it can be concluded that (1) Naringinase is most stable in the pH range of 4.5-5.0, being quite sensitive to lower pHs (<3.5) and (2) the glyco-enzyme is a rather thermo-stable enzyme preserving its initial activity for long times when incubated at its optimal pH up to temperatures of 65 degrees C. Enriched alpha-L-rhamnosidase after column treatment and ultrafiltration presented similar deactivation kinetics pattern and half-life values as the unpurified enzyme. Thus, any influence of low molecular weight substances on its deactivation is most probably negligible. The intermediate state of the enzyme may correspond to unfolding and self-digestion of its carbohydrate portion, lowering its activity relative to the initial state. The digestion- and unfolding-grade of this intermediate state may also be controlled by the pH and temperature of incubation.

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion.

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.

Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747.

The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96-98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids.

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.

High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter.

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

Development of an Escherichia coli whole cell biocatalyst for the production of L-amino acids.

A whole cell biocatalyst for the enzymatic production of L-amino acids from hydantoins was created by coexpressing the genes encoding the L-hydantoinase, the L-N-carbamoylase and the hydantoin racemase from Arthrobacter aurescens in Escherichia coli. In order to construct a well balanced reaction system the enzymatic activity in the cells was varied by using vectors with different copy numbers for expression of the genes. Derivatives of pSC101, pACYC184 and pBR322 were employed for the various constructions and in one construct the hydantoinase gene was integrated into the E. coli chromosome. All constructs carried the E. coli rhamnose promoter system enabling gene expression control by transcriptional regulation. The productivity for L-tryptophan from the corresponding hydantoin was more than 6-fold higher than achieved with Arthrobacter aurescens.

Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation.

Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.

Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids.

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization.

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.

Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins.

Two enzymes, hydantoinase (HyuH) and L-N-carbamoylase (HyuC), are required for the biocatalytic production of natural and unnatural, optically pure L-amino acids starting from D,L-5-monosubstituted hydantoins using the so called 'hydantoinase-method'. For the preparation of immobilized enzymes, which omit several drawbacks of whole cell biocatalysts, purified or at least enriched HyuH and HyuC have to be provided. In order to simplify existing purification protocols several genetically modified derivatives of HyuH and HyuC from Arthrobacter aurescens DSM 3747 have been cloned and expressed in E. coli. A fusion protein consisting of maltose-binding protein (MalE) and HyuH resulted in an enhanced solubility of the hydantoinase, which easily forms inclusion bodies. On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose resin) and the complex purification protocol of the wild-type enzyme could therefore be simplified and shortened significantly. Interestingly, the specific activity of the MalE-HyuH fusion protein was as high as the wild-type enzyme despite that the molecular mass was doubled. A second modification of HyuH carrying a histidine-tag was efficiently bound to a metal affinity matrix but inactivated completely during elution from the column at either low pH or in the presence of imidazole. In the case of HyuC, an aspartate-tag has been added to the biocatalyst to allow an integrated purification-immobilization procedure since this enzyme is immobilized efficiently only via its carboxylic groups. The diminished isoelectric point of the Asp-tagged HyuC resulted in a simplified purification procedure. Compared to the wild-type enzyme expressed in E. coli HyuC-Asp6 was shifted off the elution range of the contaminating proteins and higher purification factors were obtained even in the capturing step. In contrast to HyuH, it was possible to purify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity using immobilized metal affinity chromatography. Therefore, the existing three step purification protocol was reduced to one chromatographic step and the yield of this relatively unstable protein enhanced remarkably.

Production of sophorolipids from whey. II. Product composition, surface active properties, cytotoxicity and stability against hydrolases by enzymatic treatment.

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1',4"-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFTmin 39 mN m-1, CMC 130 mg l-1), water solubilities (2-3 g l-1) and low cytotoxicities (LC50 300-700 mg l-1). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m-1, CMC 10 mg l-1), less water soluble (max. 70 mg l-1) and showed stronger cytotoxic effects (LC50 15 mg l-1). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect.

Microbial hydantoinases--industrial enzymes from the origin of life?

Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5'-monosubstituted hydantoins and are therefore classified in the EC nomenclature as cyclic amidases (EC 3.5.2.). In the EC nomenclature, four different hydantoin-cleaving enzymes are described: dihydropyrimidinase (3.5.2.2), allantoinase (EC 3.5.2.5), carboxymethylhydantoinase (EC 3.5.2.4), and N-methylhydantoinase (EC 3.5.2.14). Beside these, other hydantoinases with known metabolic functions, such as imidase and carboxyethylhydantoinase and enzymes with unknown metabolic function, are described in the literature and have not yet been classified. An important question is whether the distinct hydantoinases, which are frequently classified as L-, D-, and non-selective hydantoinases depending on their substrate specificity and stereoselectivity, are related to each other. In order to investigate the evolutionary relationship, amino acid sequence data can be used for a phylogenetic analysis. Although most of these enzymes only share limited sequence homology (identity < 15%) and therefore are only distantly related, it can be shown (i) that most of them are members of a broad set of amidases with similarities to ureases and build a protein superfamily, whereas ATP-dependent hydantoinases are not related, (ii) that the urease-related amidases have evolved divergently from a common ancestor and (iii) that they share a metal-binding motif consisting of conserved histidine residues. The difference in enantioselectivity used for the classification of hydantoinases on the basis of their biotechnological value does not reflect their evolutionary relationship, which is to a more diverse group of enzymes than was assumed earlier. This protein superfamily probably has its origin in the prebiotic conditions of the primitive earth.

Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli.

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates.

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1:1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola.

Molecular evolution of hydantoinases.

The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively. A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5). Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution. We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria. Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.