Recombinant Pichia Pastoris Research Articles

Oral Vaccination With Recombinant Pichia pastoris Expressing Iridovirus Major Capsid Protein Elicits Protective Immunity in Largemouth Bass (Micropterus salmoides).

Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.

Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with the recombinant Corynebacterium glutamicum producing high-level proline

The application of antibacterial lipopeptides is limited by high cost and low yield. Herein, the exogenous L-proline significantly improved lipopeptide production by Bacillus amyloliquefaciens HM618. A recombinant Corynebacterium glutamicum producing high levels of proline using genetically modifying proB and putA was used to establish consortium, to improve lipopeptide production of strain HM618. Compared to a pure culture, the levels of iturin A, fengycin, and surfactin in consortium reached 67.75, 39.32, and 37.25 mg L-1, respectively, an increase of 3.19-, 2.05-, and 1.63-fold over that produced by co-cultures of B. amyloliquefaciens and recombinant C. glutamicum with normal medium. Commercial amylase and recombinant Pichia pastoris with a heterologous amylase gene were used to hydrolyze kitchen waste. A three-strain consortium with recombinant P. pastoris and C. glutamicum increased the lipopeptide production of strain HM618 in medium containing KW. This work provides new strategies to improve lipopeptide production by B. amyloliquefaciens.

High Production of Trametes cinnabarina Laccase (lac1) by Suspended and Immobilized Cells of Recombinant Pichia pastoris from Crude Glycerol

High Production of Trametes cinnabarina Laccase (lac1) by Suspended and Immobilized Cells of Recombinant Pichia pastoris from Crude Glycerol

Anaerobically Digesting Hazardous Waste Pichia pastoris Associated with Butyric Acid Cleaner Production.

Recombinant Pichia pastoris semisolid hazardous waste treatment is difficult and traditional solid waste treatment is not applicable. However, P. pastoris wastes have features of high density and enriched proteins/polysaccharides, which could supply nitrogen/carbon sources for butyric acid production. The waste P. pastoris was first treated using NaOH to form a waste yeast suspension, and then the suspension was mixed with glucose to obtain a starting medium containing 5.6 g DCW/L (dry cell weight) yeast to initiate butyrate fermentation. The suspension was intermediately supplemented to bring the total waste yeast concentration to 26.3 g DCW/L while continuously feeding the concentrated glucose solution. With the proposed strategy, butyrate concentration reached high levels of 51.0–54.0 g/L using Clostridium tyrobutyricum as the strain. Amino acids/oligosaccharides/SO42– in the suspension, raw material costs, complicated pretreatment process, and butyric acid cleaner production could be effectively utilized, reduced, eliminated, and realized. However, the apparent waste P. pastoris reduction rate was only 49% per batch, thus a “tanks in-series type’s repeated waste treating system” model was developed to theoretically explore the possibility of increasing the waste yeast reduction rate R. The simulation results indicated that when setting the treatment unit numbers at 4, waste solid concentration could decrease from 26.3 to 3.37 g DCW/L and the hazardous waste yeast reduction rate R would increase from 49 to 97%.

Dynamic Feeding for Pichia pastoris.

The knowledge of certain strain-specific parameters of recombinant Pichia pastoris strains is required to be able to set up a feeding regime for fed-batch cultivations. These parameters are commonly determined either by time-consuming and labor-intensive continuous cultivations or by several, consecutive fed-batch cultivations. Here, we describe a fast method based on batch experiments with substrate pulses to extract certain strain characteristic parameters, which are required to set up a dynamic feeding strategy for P. pastoris strains based on the specific substrate uptake rate. We further describe in detail the course of actions, which have to be taken to obtain the desired dynamics during feeding.

Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles.

Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.

Impacts of Magnetic Immobilization on the Growth and Metabolic Status of Recombinant Pichia pastoris.

Downstream processing is an expensive step for industrial production of recombinant proteins. Cell immobilization is known as one of the ideal solutions in regard to process intensification. In recent years, magnetic immobilization was introduced as a new technique for cell immobilization. This technique was successfully employed to harvest many bacterial and eukaryotic cells. But there are no data about the influence of magnetic immobilization on the eukaryotic inducted recombinant cells. In this study, impacts of magnetic immobilization on the growth and metabolic status of induced recombinant Pichia pastoris as a valuable eukaryotic model cells were investigated. Results based on colony-forming unit, OD600, and trypan blue assay indicated that magnetic immobilization had no adverse effect on the growth and viability of P. pastoris cells. Also, about 20-40% increase in metabolic activity was recorded in immobilized cells that were decorated with 0.5-2mg/mL nanoparticles. Total protein and carbohydrate of the cells were also measured as main indicatives for cell function and no significant changes were observed in the immobilized cells. Current data show magnetic immobilization as a biocompatible technique for application in eukaryotic expression systems. Results can be considered for further developments in P. pastoris-based expression systems.

PSX-B-12 Сreation of a pichia pastoris strain: A producer of recombinant peptidase M9 from Aeromonas salmonicida

Abstract Proteases are produced by a wide variety of microorganisms and have a wide range of applications. Peptidase M9 from Aeromonas salmonicida is a 915 amino acid residue protein. Earlier, we cloned the 864 bp C end of the gene. (col2 gene) encoding the actual peptidase domain of 238 aa in the vector plasmid pPic9K. It was demonstrated that the recombinant Pichia pastoris strain, created on the basis of this plasmid, has a specific enzymatic activity against collagen. The aim of this work was to further optimize the expression of the col2 gene of the M9 peptidase domain from Aeromonas salmonicida in Pichia pastoris, isolation, purification, and characterization of the protein.First, the col2 gene was optimized based on the use of P. pastoris codons. Secondly, a study of collagenase activity with 4 different signaling sequences was carried out. It was shown that the strain for which the collagenase gene had the P. pastoris alpha peptide as a signal sequence was shown to have the maximum activity. One of the approaches leading to an increase in the yield of the target protein is the cloning of the gene encoding the chaperone proteins. We have created strains containing, in addition to the collagenase gene, also the pdi and hac chaperone genes. It was shown that the collagenase activity in the strain containing the pdi chaperone gene was 2–3 times higher than in the strain containing only the col2 gene. So, the Pichia pastoris strain was created - the producer of the recombinant peptidase M9 from Aeromonas salmonicida, the target protein was isolated and characterized.Research conducted as part of the state assignment of Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences 0437-2019-0001.

Purification of Recombinant Mouse C-Reactive Protein fromPichia PastorisGS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography andP-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

Shrimp protected from a virus by feed containing yeast with a surface-displayed viral binding protein

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

Highly efficient porous magnetic polydopamine/copper phosphate with three-dimensional hierarchical nanoflower morphology as a selective platform for recombinant proteins separation

The separation and purification of recombinant pharmaceutical proteins is a fundamental and challenging step in the biotechnology industry. Hierarchical nanostructures with unique features and high stability can be used as efficient adsorbents. In this study, hierarchical magnetic polydopamine-copper phosphate nanoflowers (Cu-PDA MNFs) were synthesized as high-performance magnetic adsorbents in a simple and low-cost method based on green chemistry. The prepared hybrid Cu-PDA MNFs revealed great performance for separating pure recombinant human growth hormone (rhGH) and the rhGH acquired from recombinant Pichia pastoris yeast fermentation. The analysis confirmed that Cu-PDA MNFs exhibited a high adsorption capacity of 257.4 mg rhGH g−1 Cu-PDA MNFs and a fast adsorption rate for approaching the adsorption equilibrium within less than 30 min with a recovery efficiency of 74% of rhGH from the real sample. In addition, recycling tests demonstrated the stability and recyclability of Cu-PDA MNFs for at least six cycles with almost constant adsorption capacity and no toxicity. Based on these results, Cu-PDA MNFs could be considered as a promising candidate for separation and purification of rhGH. This work presents a new approach to using organic-inorganic nanoflowers as the hierarchical nanostructure for purification of pharmaceutical proteins with high performance.

Lipase-active heterogeneous biocatalysts for enzymatic synthesis of short-chain aroma esters

The lipase-active heterogeneous biocatalysts were prepared by adsorptive immobilization of Thermomyces lanuginosus lipase (designated as rPichia/lip) produced by authoring recombinant Pichia pastoris strain. Biocatalysts of LipoCarb type were prepared by spontaneous adsorption of rPichia/lip on the authoring macroporous carbon aerogel. Biocatalysts of LipoSil type were prepared by forced adsorption of rPichia/lip on mesoporous silica. The prepared biocatalysts were studied carefully in the esterification of short-chain fatty acids (butyric C4, C5–C7) and aliphatic alcohols (ethanol C2, C3–C5, C8) in order to produce valuable fruity-smelling esters. Biocatalytic properties, such as enzymatic activity, substrates specificity, and operational stability, were found to depend on chemical nature of both the adsorbents and organic solvents, primarily on their polarity. When comparing the activity in the synthesis of n-butyl heptanoate, it was found that the LipoCarb biocatalysts demonstrated activity an order of magnitude higher than LipoSil type. The Michaelis-Menten kinetic constants differed by a factor of 30, e.g. 1.1·102 s−1 and 3.7 s−1, respectively. The operational stability of the lipase-active biocatalysts was sufficiently high; rPichia/lip adsorbed on carbon aerogel retained till 96% of the initial activity after 29 reaction cycles, whereas rPichia/lip adsorbed on silica completely retained the activity after 38 reaction cycles of esterification under optimal reaction conditions selected. The prepared biocatalysts of LipoCarb and LipoSil types were promising for the Green enzymatic synthesis of fragrance and aroma esters under very mild ambient conditions (20±2 °C, 1 bar).

Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate

This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.

Enhancing the expression of recombinant small laccase in Pichia pastoris by a double promoter system and application in antibiotics degradation.

Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5mg/L ciprofloxacin (CIP) and 99% of 100mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.

Pilot-Scale Production of Peroxygenase from Agrocybe aegerita.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Covalent Immobilization of Candida antarctica Lipase B on Functionalized Hollow Mesoporous Silica Nanoparticles

AbstractCandida antarctica lipase B (CALB) produced by recombinant Pichia pastoris was covalently immobilized to a carboxyl‐functionalized hollow mesoporous silica sphere (HMS) synthesized by sol‐gel precipitation. The HMS was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffractometer (XRD), and Brunauer‐Emmett‐Teller (BET). The immobilized CALB was characterized through Fourier transform infrared spectra (FT‐IR) and elemental analysis to verify the binding of enzyme. The free and immobilized CALB were tested in tributyrin hydrolysis. Results revealed that CALB loading capacity of HMS‐COOH was about 28.6 mg/g support and the corresponding enzyme activity was up to about 2700 U/g support at optimal pH and temperature, which was 6.5 and 30 °C respectively. Moreover, compared with free CALB, the immobilized CALB presented more resistant towards the reaction pH and temperature changes. Furthermore, the immobilized CALB retained 60 % residual activity after eight batches of recycling and exhibited better thermal stability and storage stability, which was promising in the potential industrial applications.

Production of bio-xylitol from d-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

BackgroundXylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from d-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from d-xylose.ResultsPichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform d-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.ConclusionsWe demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform d-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of d-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

Implementing Chemostat Fermentation of Pichia Pastoris Producing Recombinant HBsAg to Optimize Cell Density Affected by Methanol Rate

High cell density fed-batch fermentation is the main strategy for recombinant hepatitis B surface antigen (rHBsAg) production. In this study, we employed short-term continuous fermentation to optimize the cell density of recombinant Pichia pastoris (P. pastoris). After reaching the maximum specified broth volume of 5 L in the fed-batch fermentation process, the operation mode was altered into the continuous mode with a dilution rate of 0.009 1/h. We used various values of methanol inflow to examine its impact as a limiting nutrient on cell density. After reaching the steady-state point, the continuous fermentation was stopped. The process's performance was evaluated based on titer, yield, productivity, and ease of process control. According to the results, the optimal methanol inflow in the pilot-scale fermentation process was 39.9 ml/h as the cell density increased from 363 g/l wet cell weight (WCW) in the fed-batch stage to 450 g/l WCW. We could successfully scale up the fermentation process with the biomass concentration of 450 g/l without having any major issues such as excessive heat dissipation or insufficient oxygen supply. This approach is a simple method for enhancing rHBsAg production efficiency in P. pastoris without requiring any new and complex facility.

Combined strategies to improve the expression of acidic mammalian chitinase in Pichia pastoris for the production of N, N'-diacetylchitobiose

Acidic mammalian chitinase (AMCase) could hydrolyze chitin into (GlcNAc)2 at pH 2.0. Therefore, it was essential to develop an engineered strain with a high-yield AMCase. Herein, multiple strategies including codon optimization, multiple expression cassettes insertion, co-expression of helper proteins, were developed to construct a recombinant Pichia pastoris with the high-level secretion of AMCase. The recombinant strain harboring four copies of codon-optimized AMCase-encoding gene ChiD and co-expression of HAC1 presented the highest chitinase activity of 570 U/L in high density fermentation, which was 934 folds higher than that in E. coli reported before. Then, the recombinant AMCase was applied to hydrolyze colloidal chitin with a conversion rate of 90 % at pH 2.0, which could save half of the water consumption during the preparation of colloidal chitin. Finally, we also demonstrated that the hydrolysate of AMCase could promote seed germination of wheat and rice, improving the growth of roots and seedling.

Heterogeneous biocatalytical esterification by recombinant Thermomyces lanuginosus lipase immobilized on macroporous carbon aerogel

Heterogeneous biocatalysts prepared by immobilizing recombinant lipases are promising for implementation in organic synthesis of various esters used as fragrances, emollients, emulsifiers, non-ionic surfactants, plasticizers, etc. For this research, the Thermomyces lanuginosus lipase produced by the authoring recombinant Pichia pastoris strain was adsorbed on the authoring macroporous carbon aerogel. The prepared biocatalysts were studied in the periodic processes of enzymatic esterification carried out in nonconventional anhydrous media of organic solvents at ambient conditions (22 ± 2 °C, 1 bar). Biocatalytical activities depended strongly on both the selected pair of lipase substrates (acid and alcohol) involved in reaction and the organic solvent used for esterification. The reaction rates of fatty C7 acid esterification with double and triple bond C3 alcohols, such as allyl and propargyl alcohols, were found to be 2–4 times lower than with aliphatic propanol. Isomerism of an acid (not alcohol) molecule greatly affected the esterification rates. It was observed that isomers of C4 and C5 acids (iso-butyric and iso-valeric) practically did not react with butanol. The esterification rates strongly depended on the polarity of the organic solvent characterized by the parameter logP. With an increase in the solvent polarity from non-polar hexane to polar acetone, the esterification rate decreased by two orders of magnitude. The prepared lipase-active biocatalysts had a sufficiently high operational stability under the chosen optimal reaction conditions; their stationary activity (300 ± 100 U·g–1) did not change in periodic reaction cycles during several tens of hours.