Screening for suitable solvents as substrate carriers for the microbial side-chain cleavage of sitosterol using microtitre plates

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to whole cell multistep biotransformations and to organic solvent screening/non-conventional medium bioconversion processes, which is an issue of major relevance when bioconversion processes are addressed. The present study aims to fill in this gap by using 24-well microtitre plates as platforms for the screening of suitable organic solvents as substrate carriers for effective biocatalysis. The side-chain cleavage of sitosterol with resting cells of Mycobacterium sp. NRRL B-3805 was used as model system. Series of miscible and immiscible alcohols with primary, secondary and tertiary structure were tested as carriers of the hydrophobic substrate, thus ruling out the effect of functionality on biocatalytic activity.Results suggest that microtitre plates may be used for solvent selection in complex bioconversion systems. The highest bioconversion yields were observed when methanol and ethanol were used as substrate carriers. An empirical correlation could be established between overall catalytic activity and physicochemical properties of the solvents.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

Anchoring high-throughput screening methods to scale-up bioproduction of siderophores

The use of high throughput strategies is of acknowledged relevance since the rational use of small-scale reactors, coupled with suitable analytic tools, is contributing to the acceleration of process development in several areas of biotechnology. These small-scale reactors are available in different working volumes and configurations, being useful in a wide array of applications, from cell screening to process optimization.The present work was focused on the development of a high-throughput strategy, combining microtiter plates and analytic methodologies, to screen an in-house library of environmental bacteria in order to identify good siderophore producers. From a library of roughly 500 marine microorganisms, it was possible to ultimately obtain 11 bacterial strains with high production capabilities. Two of them had not been previously identified as siderophore producers. The bioprocess was scaled-up from microtiter plates to a 5 L stirred tank reactor, while maintaining the overall volumetric productivity, using the k_La similarity as scale-up criterion.This novel approach is a suitable alternative to traditional screening tools.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Δ′-Dehydrogenation of steroids byArthrobacter simplex immobilized in calcium polygalacturonate beads

Summary Arthrobacter simplex ATCC 6946 (viable cells) was immobilized in a calcium polygalacturonate gel. The trapped cells were used for repeated batchwise bioconversion of steroids. Reichstein's compound S and hydrocortisone were dehydrogenated introducing a double bond between C₁ and C₂ of ring A. The products 1-dehydro S and prednisolone, respectively, were identified by high pressure liquid chromatography. Steroid dehydrogenase activity increased in the system when an artificial electron acceptor, such as menadione (vitamin K₃) was present in the reaction mixture. An airlift-type reactor was used to bioconvert up to 90% of substrate in 15 min, under optimal conditions. The gel entrapped cell preparations were used for repeated batch bioconversion during 30 days; 69 batch bioconversions for Reichstein's compound S were performed during 15 days of operation of the reactor. The operational stability of the process and the feasibility of repeated batch bioconversions was shown to be comparable to similar processes.     

Microwell engineering characterization for mammalian cell culture process development

Experimentation in shaken microplate formats offers a potential platform technology for the rapid evaluation and optimization of cell culture conditions. Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas–liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridoma cells producing IgG1, 24‐well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted k_La values varied between 1.3 and 29 h^?1; liquid‐phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m^?3. CFD simulations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridoma cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm, 2,000 µL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24‐well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and comparable to currently used shake flask systems while offering at least a 30‐fold decrease in scale of operation and material requirements. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic suspension culture conditions. Biotechnol. Bioeng. 2010; 105: 260–275. © 2009 Wiley Periodicals, Inc.     

Improved scale-up strategies of bioreactors

Effective scale-up is essential for successful bioprocessing. While it is desirable to keep as many operating parameters constant as possible during the scale-up, the number of constant parameters realizable is limited by the degrees of freedom in designing the large-scale operation. Scale-up of aerobic fermentations is often carried out on the basis of a constant oxygen transfer coefficient, k _L a, to ensure the same oxygen supply rate to support normal growth and metabolism of the desired high cell populations. In this paper, it is proposed to replace the scale-up criterion of constant k _L by a more direct and meaningful criterion of equal oxygen transfer rate at a predetermined value of dissolved oxygen concentration. This can be achieved by using different oxygen partial pressures in the influent gas streams for different scales of operation. One more degree of freedom, i.e., gas-phase oxygen partial pressure, is thus added to the process of scale-up. Accordingly, one more operating factor can be maintained constant during scale-up. It can be used to regulate the power consumption in large-scale fermentors for economical considerations or to describe the fluid mixing more precisely. Examples are given to show that the results of optimization achieved in the bench-scale study can be translated to the production-scale fermentor more successfully with only a small change in the gas-phase oxygen partial pressure employed in the bench-scale operation.List of Symbols a m²/m³ Specific gas/liquid interfacial area - C _L mole/m³ Dissolved oxygen concentration in bulk liquid phase - C ^* mole/m³ Equilibrium oxygen concentration at gas/liquid interface - D _i m Impeller diameter - D _T m Bioreactor diameter - H _L mole/m³ · atm Henry's-law constant - k _L m/s Liquid-phase mass transfer coefficient - N 1/s Impeller agitation speed - N _i Number of impellers - OTR mole/s · m³ Oxygen transfer rate per unit volume of the medium - P _g kW Power input in aerated fermentation - P _o kW Power input in non-gassed fermentation - p _g atm Gas-phase oxygen partial pressure - Q m³/s Volumetric gas flow rate - Re _i Impeller Reynolds number - T _Q Joule Torque applied to the mixer shaft - V m³ Liquid volume - v _s m/s Superficial gas velocity - kg/m · s Liquid viscosity - kg/m³ Liquid density     

Mixing and oxygen transfer characteristics of a microplate bioreactor with surface-attached microposts

Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, process development may be carried out in small volume systems, with the caveat that such systems be predictive for process scale-up. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, k_La, has been identified as a critical parameter for predictive process scale-up. Here, we describe the development of a 96-well microplate with integrated Redbud Posts to provide mixing and enhanced k_La. Mixing in the microplate is characterized by observation of dyes and analyzed using the relative mixing index (RMI). The k_La is measured via dynamic gassing out method. Actuating Redbud Posts are shown to increase rate of planar homogeneity (2 min) verse diffusion alone (120 min) and increase oxygenation, with increasing stirrer speed (3500-9000 rpm) and decreasing fill volume (150-350 μL) leading to an increase in k_La (4-88 h⁻¹). Significant increase in Chinese Hamster Ovary growth in Redbud Labs vessel (580,000 cells mL^-1) versus the control (420,000 cells mL^-1); t(12.814) = 8.3678, p ≤ .001), and CD4⁺ Naïve cell growth in the microbioreactor indicates the potential for this technology in early stage bioprocess development and optimization.     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Optimization of media composition for improving conversion of isoeugenol into vanillin with Pseudomonas sp. strain KOB10 using the Taguchi method

Abstract

The popular demand for natural food additives has resulted in a number of processes for producing natural vanillin. Although there are chemical procedures and plant sources for vanillin production, microbial bioconversions are being sought as a suitable ‘natural’ alternative. The present paper describes the conversion of isoeugenol to vanillin by a novel bacterial strain isolated from soil. The strain was identified as Pseudomonas sp. strain KOB10 based on morphological and physiochemical characteristics and its 16S rDNA gene sequence. We optimized medium composition for vanillin production using a Taguchi experimental design. Eight factors, i.e. isoeugenol, glycerol, tryptone, K₂HPO₄, KH₂PO₄, Cu²⁺, Mg²⁺ and Ca²⁺ concentrations, were selected and experiments based on an orthogonal array layout of L₁₈ (2² × 3⁶) were performed. Analysis of the experimental data using the Taguchi method indicated that Cu²⁺ and glycerol concentrations had the highest impact on isoeugenol conversion into vanillin at a substrate concentration of 0.9 g L^?1. Under the optimized conditions, growing cells of Pseudomonas sp. strain KOB10 produced 0.153 g vanillin L^?1 from 0.9 g isoeugenol L^?1, with a molar yield of 18.3% after incubation for 48 h. To improve the vanillin yield, the effect of other bioconversion parameters including time of isoeugenol addition, initial isoeugenol concentration and conversion time was studied; the results showed a maximum concentration of 3.14 g vanillin L^?1 after a total incubation time of 88 h with 15 g isoeugenol L^?1, which corresponded to a molar yield of 22.5%. Further standardization and optimization for vanillin production was challenging.     

Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Bioconversion of naturally occurring precursors and related synthetic compounds using plant cell cultures.

The nearly unlimited enzymatic potential of cultured plant cells can basically be employed for bioconversion purposes. Plant enzymes are able to catalyze regio- and stereospecific reactions and can therefore be applied to the production of compounds of pharmaceutical interest. Naturally occurring as well as related synthetic compounds may be used as precursors. A review of the current status of such bioconversions is given. It includes the performance of bioconversions by freely suspended and immobilized plant cells or enzyme preparations. In addition, the kinetic aspects of immobilized plant cells are discussed. Special attention is paid to the bioconversion of poorly or water insoluble precursors. Finally, a model scheme for the development of a commercially available drug, produced by bioconversion, and perspectives are discussed.     

Reproducible preparation of spheroids of pancreatic hormone positive cells from human iPS cells: An in vitro study

BackgroundTransplantation of islets of Langerhans is regarded as a promising therapy for type 1 diabetes. A large number of β-cells are required for the treatment of human type 1 diabetes. Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have been considered as new sources for cell replacement therapy.MethodsCell aggregates were prepared from human iPS cells using agarose microwell plates and differentiated into pancreatic endocrine cells by changing the culture media with different additives.ResultsAfter 20 days of culture, approximately 30% of cells in aggregates were positive for C-peptide. After another 14 days in culture, the cells gained an ability to alter C-peptide release in response to changes in the glucose concentration.ConclusionsUniform aggregates of human iPSCs were easily prepared on agarose microwell plates and efficiently differentiated into the pancreatic endocrine lineage. Thus, aggregate culture is a suitable method for preparing islet-like aggregates from human iPSCs.General significanceOur results indicate that the microwell plate is suitable for scaling up the preparation of pancreatic endocrine cells from human iPS cells in a robotic system.     

Effect of inoculation strategies,substrate to biomass ratio and nitrogen sources on the bioconversion of wood sterols by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

4-Androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) are the main precursors in the production of steroidal drugs from phytosterols. To carry out the bioconversion, different inoculation strategies have been proposed. We compared the use of whole fermented broth and of free resting cells of two mutant strains of Mycobacterium sp. (DSMZ2966 and DSMZ2967) in shake flasks. Also the effect of the nitrogen source (ammonium sulfate, ammonium chloride and ammonium nitrate) and the sterol to biomass ratio at high substrate concentrations (19.2 g/l and 48.1 g/l) was evaluated. We found that the bioconversion with free resting cells (cell pellets) is more efficient than that with whole fermented broth, increasing both AD and ADD production. The use of ammonium nitrate in the culture medium and low substrate to biomass ratios (close to 1.0) increased the production yield. We also found that the bioconversion can be run at high substrate concentration under non-sterile conditions.     

Glass microspargers as effective frit spargers in single use bioreactors

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO₂ profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and k_La studies. Mass transfer (k_La) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard “P40” according to ISO 4793-80, which corresponds to a range of 16–40 μm.     

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Abstract

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (k_La) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by Mut^S P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2?kW m^?3. Specific growth rate, and then dextranase production, increased as k_La and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2?L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut⁺ strain. After a scale-up process, at constant k_La, the specific growth rate was kept at 30?L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30?L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.     

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k_cat/K_m) for d-xylose and l-sorbose. P2OxB1 was provided with a C-terminal His₆-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k_cat/K_m for d-glucose (5.3-fold), methyl--d-glucoside (2.0-fold), d-galactose (4.8-fold), d-xylose (59.9-fold), and l-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of l-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.This revised version was published online in February 2005 with corrections to Table 2.     

Vergleich von Messungen des kLa-Wertes nach der stationären und der dynamischen Methode bei der Fermentation von L-Lysin und Turimycin

The calculation and scale-up of fermentation processes need k_La as one of the most important engineering data. There are two methods to determine k_La depending on power input, aeration rate and the properties of the fermentation broth: static with a balance between air supply and exit, dynamic gassing out with following the changes of dissolved oxygen concentration during periods of air off and a following air on. Within early intervals of fermentation time the data from both methods agree well, while for later time intervals the dynamic method always gives much lower values for k_La than static. The only explanations for this phenomenon are quick changes in the oxygen metabolism or an enzymatic storage of oxygen. For both gassing out and saturation period it is possible to calculate the same absolute amounts of this additional oxygen.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. I. Derivatives of L-phenylalanine

Summary Resting cells of a mutant ofArthrobacter sp. (DSM 3747) were used for the bioconversion of D,L-5-benzylhydantoin and related compounds to the corresponding L-amino acids. After optimization of the reaction conditions in shake flask experiments, bioconversions were performed in a preparative scale in a 2-l-bioreactor under nitrogen atmosphere. Specific productivities of 0.4 (p-NO₂-L-phenylalanine) up to 3.9 mM amino acid x g cell dry mass^–1 x h^–1 (p-Cl-L-phenylalanine) were obtained. D,L-5-p-COOH-Benzylhydantoin, D,L-5-phenylhydantoin and D,L-5-p-OH-phenylhydantoin were not accepted as substrates.