Increased production of β-cyclodextrin using an aqueous two-phase system

Summary -Cyclodextrin(-CD) was produced by cyclodextrin glycosyltransferase(CGTase) in aqueous two-phase system. -CD production from soluble starch was catalyzed by CGTase in dextran-rich bottom phase, and the -CD produced was transferred to PEG-rich top phase in aqueous two-phase system, composed of 7% (w/w) polyethylene glycol(Mr 20,000) and 10% (w/w) dextran(Mr 38,900). Partition coefficients of -CD and CGTase were 1.5 and 0.25, respectively. The total productivity of -CD in aqueous two-phase system was about 3 times of that in dextran phase.     

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.     

α-Amylase production by immobilized whole cells of Bacillus subtilis

Summary Whole cells of Bacillus subtilis were immobilized in polyacrylamide gel prepared from 5% total acrylamide (85% acrylamide and 15% N,N-methylenebisacrylamide). Production of -amylase by the immobilized whole cells was attempted in a batch system. -Amylase produced by the immobilized whole cells was about three times larger than that produced by washed cells at optimum conditions. The reusability of the immobilized whole cells and washed cells was examined. The activity of -amylase production by washed cells decreased with increasing use cycles. On the other hand, the activity of the immobilized cells increased gradually, and it reched a steady state after seven cycles. -Amylase was produced from a simple reaction medium containing 1% meat extract and 0.05% yeast extract by the immobilized whole cells. The rate of -amylase production by the immobilized whole cells was the same as in submerged cultivation using starch bouillon medium. Growth of B. subtilis in polyacrylamide gel was observed by electron microscopy.     

Continuous production of α-peptide using immobilized recombinant yeast cells: A model for continuous production of foreign peptide by recombinant yeast

α-Peptide, a portion of Escherichia coli β-galactosidase, was cloned downstream of the yeast α-factor promoter and the signal peptide by one of the authors. In this study, we utilized recombinant yeast cells, transformed the α-peptide secretion vector and attempted continuous production of α-peptide as a model of foreign peptide production. The continuous production of α-peptide was performed by using immobilized recombinant yeast cells on a column reactor, after characterizing the secretion, using minimal and complex medium. Utilizing minimal medium, with a productivity of 100 000 U h⁻¹ l⁻¹, α-peptide was continuously produced for more than 200 h. We then attempted to improve the productivity of α-peptide by alternating minimal and complex medium. Utilizing this medium changing method, 1.4 times higher α-peptide was produced during 150 h of operation compared with that achieved only by feeding minimal medium.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Mechanism for reversible inactivation of immobilized β-galactosidase from Bacillus circulans during continuous production of galacto-oligosaccharides

Summary The specific activity-dependent stability of the immobilized -galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans during the continuous production of galactooligosaccharides from lactose was studied. This was done by measuring the elution pattern of saccharides from the various immobilized Merckogel (controlled pore silica gel) columns and the amount of saccharides remaining in the gel. It was suggested that oligosaccharides produced were trapped inside the three dimensional enzyme aggregate with the immobilized enzyme having a specific activity of 240 units/g of wet gel, causing gradual inactivation, while the immobilized enzyme with 15 units/g of wet gel was stable since the oligosaccharides were not accumulated.Free -galactosidase-2 was stable during continuous reaction in a membrane reactor.     

Glucose production using immobilized mycelial-associated β-glucosidase of Trichoderma E58

Summary The immobilization of the mycelial-associated -glucosidase of Trichoderma E-58 has been carried out by encapsulating, in calcium alginate beads, the fungal mycelium obtained durinq liquid culture. The activity of this immobilized -glucosidase was found to vary with culture age and to be more thermally stable than the extracellular -glucosidase produced by this organism. The activity of the immobilized enzyme was successfully demonstrated in both static and shake-flask batch reaction mixtures at 50°C using both cellobiose and salicin as substrates.     

Toward a cost-effective method for α-arbutin production by using immobilized hydroquinone as a glucosyl acceptor

α-Arbutin is a glycosylated hydroquinone (HQ) which has inhibitory function against tyrosinase. The aim of present study is to develop an efficient and inexpensive method for the production of α-arbutin by using Xanthomonas maltophilia BT-112 as a biocatalyst. HQ, substrate for the bioconversion as glucosyl acceptor, was immobilized on different macroporous resins to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. The research results indicated that H107 resin offered the best adsorption capacity for HQ than other resins. When immobilized hydroquinone (IMHQ) was applied, the maximal HQ tolerance of cells and yield of α-arbutin were 254 mM and 64.7 g L^?1 respectively. The α-arbutin productivity (0.90 g L^?1 h^?1) was 526% higher than that of the control (free HQ) in 72 h reaction and the fermentation broth maintained its full activity for almost three consecutive batch reactions. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the results in this work provide a cost-effective method for the production of α-arbutin.     

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.     

Polyploids as a “model system” for the study of heterosis

Heterosis research over the past century has focused primarily on diploid plants and animals. This is despite the fact that most heterotic organisms contain polyploid events in their recent and/or ancient past and various important crop species are heterotic polyploids. We present an argument for the study of heterosis within polyploid systems and give examples of how their study can improve current hypotheses and generate new ones. Polyploid systems allow experiments not possible in diploids but the insights gained must be incorporated into models to explain heterosis at all levels.     

Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris–sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.     

Yeast mitochondrial biogenesis: a model system for humans?

Recently, our knowledge of yeast mitochondrial biogenesis has considerably progressed. This concerns the import machinery that guides preproteins synthesized on the cytoplasmic ribosomes through the mitochondrial outer and inner membranes, as well as the inner membrane insertion machinery of mitochondrially encoded polypeptides, or the proteins participating in the assembly and quality control of the respiratory complexes and ATP synthase. More recently, two new fields have emerged, biosynthesis of the iron-sulfur clusters and dynamics of the mitochondrion. Many of the newly discovered yeast proteins have homologues in human mitochondria. Thus, Saccharomyces cerevisiae has proven a particularly suitable simple organism for approaching the molecular bases of a growing number of human mitochondrial diseases caused by mutations in nuclear genes identified by positional cloning.     

High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

Background  

Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable.     

Fast screening method for benzophenanthridine alkaloids in plant cell cultures of California poppy using a dansyl-glycine-β-cyclodextrin molecular recognition system

The association constant K for the host-guest complex comprised of sanguinarine/dansyl-glycine--cyclodextrin was found to be 5.75 × 105 M. The sensitivity factor(), which is defined as the ratio of the change of the fluorescence intensity after complex formation to the intensity of free, uncomplexed dansyl-glycine--cyclodextrin, showed an almost linear dependence on analyte concentration up to 50M sanguinarine. This molecular recognition system can be exploited to rapidly estimate the total concentration of benzophenanthridine alkaloids in suspension cultures of Eschscholtzia californica. © Rapid Science Ltd. 1998     

Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae

This work studied the hydrolysis of lactose using β-galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 °C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 °C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Steroid hydroxylation with free and immobilized cells of Penicillium raistrickii in the presence of β-cyclodextrin

Free and Ca-alginate-immobilized cells of Penicillium raistrickii i 477 were used for 15-hydroxylation of 13-ethyl-gon-4-en-3,17-dione. The product formation in the presence of -cyclodetrin, in comparison with reactions carried out in the presence of methanol. Application of -cyclodextrin led to increasing solubility of the steroid substrate. The fungus was able to utilize -cyclodextrin as a carbon source. Correspondence to: H.-P. Schmauder     

Comparative study of methodologies for obtaining β-glucosidase immobilized on dextran-modified silica

In this study, two procedures for the immobilization of β-glucosidase on silica are compared. The first approach comprises a preliminary stabilization of β-glucosidase by coupling with dextran dialdehyde and subsequent immobilization of the obtained β-glucosidase dextran dialdehyde with aminopropylsilica. In the second approach, β-glucosidase is immobilized on silica modified with a dextran-dialdehyde coating. Enzyme immobilized via coupling with dextran dialdehyde and subsequent attachment with aminopropylsilica show a remarkably enhanced thermostability. Enzyme immobilized by the alternative approach demonstrated an inferior thermoresistance. The difference in behavior of the immobilized enzyme obtained via these two methods can be explained considering the number of links between the enzyme and carrier. Enzyme immobilized on dextran dialdehydecoated silica is fixed via a limited number of links. On the other hand, with soluble β-glucosidase-dextran conjugates, the enzyme configuration is already stabilized via a high number of links with the dextran backbone. It is clear from this study that the sequence of reactions in immobilizing enzymes on silica support via a dextran-dialdehyde linker has a significant effect on the final properties.     

Optimization of β-mannanase production by Bacillus subtilis US191 using economical agricultural substrates

The Bacillus subtilis US191 strain producing highly thermostable β-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular β-mannanase production by this strain was 1.48 U ml⁻¹. To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett–Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on β-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box–Behnken design. Under optimized conditions (1 g L⁻¹ LBG, 8 g L⁻¹ soymeal, temperature of 30°C and inoculum size of 10¹⁰ CFU ml⁻¹), a 2.59-fold enhancement in β-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L⁻¹) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a β-mannanase production level of 8.75 U ml⁻¹ in presence of 0.25 g L⁻¹ LBG, 5 g L⁻¹ of coffee residue powder, 5 g L⁻¹ of date seeds powder, and 5 g L⁻¹ of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in β-mannanase production by B. subtilis US191 was achieved.