Production and purification of a novel thermostable phytase by Pichia pastoris FPHY34

A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L⁻¹ and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (Y_X/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL⁻¹ at a methanol feed rate of 3.0 mL L⁻¹ h⁻¹ after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg⁻¹ and 3281 U mg⁻¹ in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

Amperometric detection of lignin-degrading peroxidase activities from Phanerochaete chrysosporium

An amperometric enzyme sensor for rapid and simultaneous detection of the lignin-degrading peroxidase activities secreted by Phanerochaete chrysosporium was developed, using H₂O₂, hydroquinone and veratryl alcohol as substrates. In the amperometric measurement, samples of culture filtrate with different lignin-degrading peroxidase activities measured by spectrophotometry were placed into electrochemical cells. The slope of the current increase (Δ_current/Δ_time) upon the addition of H₂O₂ into the culture filtrate solution containing hydroquinone was used as the index for total activity of lignin peroxidase and manganese peroxidase. Then a specific detection of lignin peroxidase was achieved by the addition of veratryl alcohol, which led to current decrease due to the redox competition between veratryl alcohol and hydroquinone. A good linear correlation was found between the electrochemical response and lignin peroxidase activity, manganese peroxidase activity in the range of 8.14–29.79 U l⁻¹ and 0.085–1.37 U l⁻¹, respectively. A regression model was established describing the relationship. The amperometric sensor described here is more rapid, sensitive and precise than conventional spectrophotometric assays, free from interference of turbidity and UV–vis-light-absorbing substances. In this paper, it was also applied in the detection of lignin-degrading peroxidases in compost bioremediation using P. chrysosporium, showing considerable advantages.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli–Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m⁻³ at the initial glucose concentration of C_Go=20 kg m⁻³ and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm⁻³ at C_Go=20 kg m⁻³. Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g⁻¹ cm⁻³) and r-B. licheniformis (130 U g⁻¹ cm⁻³). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasmid, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm⁻³) and r-B. sphaericus (820 U cm⁻³) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

Lipolytic activity in submerged cultures of Issatchenkia orientalis

The ability of the yeast Issatchenkia orientalis CECT 10688 to secrete lipolytic activity in submerged culture was investigated. The yeast was grown in a complex medium supplemented with a fixed concentration of several lipidic compounds (triglycerides, fatty acids). Maximum enzyme activity around 70–80 U cm⁻³ was produced in cultures supplemented with tributyrin. An optimum tributyrin concentration of 10 g dm⁻³ was selected. Several surfactants were added to the cultures, but no significant increase in activity was detected. Finally, the effect of the type of carbon source on lipolytic enzyme production was studied. The best results were obtained with glucose and fructose (60–80 U cm⁻³), while rather low enzyme activity was found in cultures grown on lactose and maltose (about 20 U cm⁻³).     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Physiological and morphological study of encapsulated Saccharomyces cerevisiae

The impact of encapsulation on the anaerobic growth pattern of S. cerevisiae CBS 8066 in a defined synthetic medium over 20 consecutive batch cultivations was investigated. In this period, the ethanol yield increased from 0.43 to 0.46 g/g, while the biomass and glycerol yields decreased by 58 and 23%, respectively. The growth rate of the encapsulated cells in the first batch was 0.13 h⁻¹, but decreased gradually to 0.01 h⁻¹ within the 20 sequential batch cultivations. Total RNA content of these yeast cells decreased by 39% from 90.3 to 55 mg/g, while the total protein content decreased by 24% from 460 to 350 mg/g. On the other hand, the stored carbohydrates, that is, glycogen and trehalose content, increased by factors of 4.5 and 4 within 20 batch cultivations, respectively. Higher biomass concentrations inside capsules led to a lower glucose diffusion rate through the membrane, and volumetric mass transfer coefficient for glucose was drastically decreased from 6.28 to 1.24 (cm³/min) by continuing the experiments. Most of the encapsulated yeast existed in the form of single and non-budding cells after long-term application.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l⁻¹ and a total anthocyanin production of 3.87 g l⁻¹ by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l⁻¹, the maximum cell density of 38.3 g dry cells l⁻¹ was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l⁻¹, the highest pigment production of>5.8 g l⁻¹ was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l⁻¹. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l⁻¹ and inoculum size of 15 g wet cells l⁻¹, respectively.     

Critical analysis of application of generalized distance function for optimization of important variables for esterase synthesis by Saccharomyces cerevisiae

The generalized distance function approach is employed to obtain a suitable near optimal conditions of variables. The optimal values of variables (medium constituents, microbiological parameters, and process parameters) have been evaluated separately using single responses (either specific esterase activity or cell mass) as per central-composite-design and multi-responses following generalized distance function approach. The optimal conditions (medium composition (g l⁻¹): dextrose, 13.43; peptone, 7.285; yeast extract, 2.55; and malt extract, 1.695; microbiological parameters: slant age, 39.9 h; inoculum age, 9.6 h; and number of cells, 1.49 × 10⁸ numbers ml⁻¹; process conditions: temperature, 29.9 °C; and pH, 6.2) obtained by generalized distance approach can be considered as a ‘near optimal’ solution of interactive multi-response systems of intracellular esterase synthesis by Saccharomyces cerevisiae.     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.