Immobilized yeast membranes as biocatalysts for sucrose inversion

Yeast membranes were obtained by autolysis of various strains with relatively high invertase activity. Heterogeneous biocatalysts for sucrose inversion were made of the yeast membranes and granulated carbon-containing supports made of common natural materials: expanded clay aggregate (ECA), sapropel, and lignin. The properties of these biocatalysts were studied. It was shown that the biocatalyst activity and stability of the immobilized yeast membranes increased with reference to the initial ECA, independent of the structure of the carbon layer synthesized on the support surface. Heterogeneous biocatalysts prepared by adsorption of yeast membranes on sapropel had the greatest activity and stability, whereas lignin-based biocatalysts were relatively unstable.     

Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis

Heterogeneous biocatalysts of starch saccharification based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in the enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase immobilized by adsorption on the surface of carriers covered with a layer of catalytic filamentous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.     

Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis

Heterogeneous biocatalysts of starch conversion based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase that was immobilized by adsorption on the surface of carriers covered with a layer of catalytic fibrous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas the biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.     

Immobilization of cells with nitrilase activity from a thermophilic bacterial strain

Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Heterologous coexpression of the benzoate-para-hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts

Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large-scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self-sufficient P450s for their potential as whole-cell biocatalysts. Most P450s are, however, not self-sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450-reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450-CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad-range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole-cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450-based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole-cell biocatalyst was emphasized, supporting earlier findings.     

Immobilization of biocatalysts with poly(vinyl alcohol) supports.

Two polymer materials, poly(vinyl alcohol) (PVA) superfine fibers and photocrosslinkable PVA bearing styrylpyridinium groups, have been developed to immobilize biocatalysts. The former has a large surface consisting of relatively large-size pores and the fibers can immobilize a large amount of biocatalyst on their surface by ionic interaction. The latter entraps many kinds of biocatalysts by cyclodimerization caused by visible light irradiation. The biocatalysts on/in these supports maintain high activity and thermal stability. These materials can easily be formed into various shapes suitable for various applications. A new bioreactor system was constructed for evaluating a variety of biocatalysts and supports.     

Application of immobilized cells ofThermoanaerobium brockii for stereoselective reductions of oxo-acid esters

Summary Cells ofThermoanaerobium brockii were immobilized by entrapment methods as easy-to-handle biocatalyst for stereoselective reductions of oxo-acid esters. Different matrix materials were tested: agarose, k-carrageenan, alginate, polyacrylamide and polyurethanes. The two latter matrices allowed useful lifetimes of the immobilized biocatalysts of more than 2 months at thermophilic operation temperatures (around 65°C). Permeabilization of cells did not improve the catalytic activity. Immobilization of the cells did not enhance the thermostability. Only after a considerable period of operation could the immobilized biocatalysts be fed with medium lacking the complex substrates yeast extract and tryptone. Compared with freely suspended cells, reaction rates were lower. The immobilized system proved to be a relatively stable easy-to-handle biocatalyst, however, the freely suspended cells were superior with respect to flexibility of application and reaction velocity.     

Effects of organic solvents on activity and stability of lipases produced by thermotolerant fungi in solid-state fermentation

Dried solid-state fermented solids (biocatalysts) produced by seven thermotolerant fungal strains were tested for lipase activity and stability in organic solvents. Two strains of Rhizopus sp. (19 and 43a) produced biocatalysts (L-19 and L-43a) that showed high lipase activities (74 and 72 U/g of dry matter, respectively) comparable to Lipozyme® RM IM (118 U/g DM). The use of the dipole moment of the organic solvents along with their classification based on the functional groups (non-polar, protic polar, aprotic polar) allowed the establishment of four different relative activity profiles for the seven biocatalysts evaluated. Compared to a biocatalyst not exposed to the organic solvent (100% relative activity), all biocatalysts showed a high relative activity (greater than 90%) in aprotic polar solvents (acetonitrile, acetone and ethyl acetate), whereas in protic polar solvents (ethanol and i-propanol) activity was reduced (lower than 40%). In addition, the incubation of biocatalysts L-19 and L-43a in i-amyl alcohol increased lipase activity in the synthesis of ethyl oleate 3.36 and 1.46 times, respectively. L-19 activity also increased after incubation in toluene (2.0 times), i-propanol (1.5 times) and acetonitrile (1.3 times) at temperatures from 30 to 50 °C. The results suggest that these biocatalysts can be used for a broad range of lipase reactions.     

Catalytic properties of lipase adsorbed on nanocarbon-containing mesoporous silica in esterification and transesterification reactions

Nanocarbon-containing mesoporous silica covered with a varying amounts of nanostructured carbon of different morphologies were used as supports to immobilize Thermomyces lanuginosus lipase. The catalytic properties of the prepared biocatalysts were studied in both the transesterification of vegetable (linseed) oil in the presence of ethyl acetate and the esterification of the fatty acid (capric C10:0) in the presence of secondary (isopropyl or isoamyl) alcohols. The physico-chemical characteristics, such as the amount of adsorbed lipase, its specific activity, and the dependence of the activity and stability of the prepared biocatalysts on the support type were evaluated. The Michaelis-Menten kinetics was studied in the esterification of capric acid with isoamyl alcohol. The prepared biocatalysts were shown to retain up to 90% activity for >1000 h in the synthesis of isoamyl caprate. The half-time of the biocatalysts inactivation in the transesterification of linseed oil was found to be more than 700 h at 40°C.     

An automated method for measuring the operational stability of biocatalysts with carbonic anhydrase activity

The operational stability of an enzyme can be quantified by its half-life, or the length of time after which 50% of its original activity has degraded. Ideally, continuous methods for measuring half-lives are preferred but they can be expensive and relatively low throughput. Batch methods, while simple, cannot be used for all enzymes. For example, batch reactions can be difficult when there is a gas phase reactant or when there is significant product or substrate inhibition. Here we describe a repeated-batch method for measuring the half-life of carbonic anhydrase (CA)-based biocatalysts by automated periodic switching between a forward and reverse reaction. This method is inexpensive and can be multiplexed for high-throughput analysis of enzyme variants. Several purified CA enzymes as well as whole-cell biocatalysts with engineered CA activity were evaluated with this method. The results indicate a significant increase in operational stability is achieved upon immobilization of CA in the cellular periplasm of Escherichia coli.     

非水酶学和非水相生物催化研究进展

近年来工业生物技术飞速发展,酶学和生物催化领域也取得突破性进展,特别在酶在非水相中活性及稳定性研究,耐溶剂生物催化剂的筛选、构建、修饰和改造,生物相容性和环境相容性好的绿色介质等方面取得了较大的进展。最近的研究热点和未来几年的研究方向主要为:基于基因组信息的耐溶剂酶的虚拟筛选和构建;基于自然界筛选新酶基因的耐溶剂酶重构和改造;离子液体等环境友好的绿色介质系统等几个方面。     

Functional Exoenzymes as Indicators of Metabolically Active Bacteria in 124,000-Year-Old Sapropel Layers of the Eastern Mediterranean Sea

Hydrolytic exoenzymes as indicators of metabolically active bacteria were investigated in four consecutive sapropel layers collected from bathyal sediments of the eastern Mediterranean Sea. For comparison, the organic carbon-poor layers between the sapropels, sediment from the anoxic Urania basin, and sediments of intertidal mud flats of the German Wadden Sea were also analyzed. The sapropel layers contained up to 1.5 · 10⁸ bacterial cells cm⁻³, whereas cell numbers in the intermediate layers were lower by a factor of 10. In sapropels, the determination of exoenzyme activity with fluorescently labeled substrate analogues was impaired by the strong adsorption of up to 97% of the enzymatically liberated fluorophores (4-methylumbelliferone [MUF] and 7-amino-4-methylcoumarin [MCA]) to the sediment particles. Because all established methods for the extraction of adsorbed fluorophores proved to be inadequate for sapropel sediments, we introduce a correction method which is based on the measurement of equilibrium adsorption isotherms for both compounds. Using this new approach, high activities of aminopeptidase and alkaline phosphatase were detected even in a 124,000-year-old sapropel layer, whereas the activity of β-glucosidase was low in all layers. So far, it had been assumed that the organic matter which constitutes the sapropels is highly refractory. The high potential activities of bacterial exoenzymes indicate that bacteria in Mediterranean sapropels are metabolically active and utilize part of the subfossil kerogen. Since a high adsorption capacity was determined not only for the low-molecular-weight compounds MUF and MCA but also for DNA, the extraordinarily strong adsorption of structurally different substrates to the sapropel matrix appears to be the major reason for the long-term preservation of biodegradable carbon in this environment.     

Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.

     

Enhanced biodegradation of phenol by magnetically immobilized <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis>

Aromatic compounds are abundant in aqueous environments due to natural resources or different manufacturer’s wastewaters. In this study, phenol degradation by the yeast, Trichosporon cutaneum ADH8 was compared in three forms namely: free cells, nonmagnetic immobilized cells (non-MICs), and magnetically immobilized cells (MICs). In addition, three different common immobilization supports (alginate, agar, and polyurethane foams) were used for cell stabilization in both non-MICs and MICs and the efficiency of phenol degradation using free yeast cells, non-MICs, and MICs for ten consecutive cycles were studied. In this study, MICs on alginate beads by 12 g/l Fe₂O₃ magnetic nanoparticles had the best efficiency in phenol degradation (82.49%) and this amount in the seventh cycle of degradation increased to 95.65% which was the highest degradation level. Then, the effect of magnetic and nonmagnetic immobilization on increasing the stability of the cells to alkaline, acidic, and saline conditions was investigated. Based on the results, MICs and non-MICs retained their capability of phenol degradation in high salinity (15 g/l) and acidity (pH 5) conditions which indicating the high stability of immobilized cells to those conditions. These results support the effectiveness of magnetic immobilized biocatalysts and propose a promising method for improving the performance of biocatalysts and its reuse ability in the degradation of phenol and other toxic compounds. Moreover, increasing the resistance of biocatalysts to extreme conditions significantly reduces costs of the bioremediation process.     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Immobilization of a recombinant strain producing glucose isomerase inside SiO<Subscript>2</Subscript>-xerogel and properties of prepared biocatalysts

An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

Production of S‐lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S‐lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S‐lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4°C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70–80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S‐lactoylglutathione production rates of these permeabilized cells were 1.5–2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 54–60, 1999.