Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml^?1) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml^?1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l^?1 inulin, an enzyme concentration of 40 U g^?1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.     

Production of thermo-alkali-stable xylanase by a novel polyextremophilic Bacillus halodurans TSEV1 in cane molasses medium and its applicability in making whole wheat bread

A high titre of thermo-alkali-stable xylanase was attained in cane molasses medium. When the culture variables for endoxylanase production were optimized [cane molasses 7 %, soluble alkaline extract of wheat bran (SAE-WB) 37 % and ammonium chloride 0.30 %], a 4.5-fold enhancement in xylanase production (69 U ml^?1) was achieved as compared to that in the unoptimized medium (15 U ml^?1). The enzyme titre attained in shake flasks could be sustained in a 7-l laboratory bioreactor. An activity band corresponding to 40 kDa was visualized on SDS-PAGE zymogram analysis. The enzyme has broad range of pH and temperature for activity with optima at 9.0 and 80 °C, and stable between pH 4.0 and 11.0 with 85 % retention of activity. It has T _1/2 of 40 and 15 min at 70 and 80 °C. The enzyme is halotolerant since it displays activity in the presence of salt up to 15 %, and remains 100 % active in the absence of salt. The supplementation of whole wheat dough with xylanase improves antistaling property, reducing sugar content, bread volume with prebiotic xylooligosaccharides in bread. This is the first report on xylanase production in cane molasses medium with SAE-WB as the inducer and its applicability in whole wheat bread making that improves human health.     

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml^?1 Rf, and 10.6 U ml^?1 enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l^?1 h^?1 after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l^?1 h^?1 after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml^?1 APPT from 2.29 mg ml^?1 PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l^?1 h^?1, and 0.62 mg ml^?1 APPT from 1.35 mg ml^?1 PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l^?1 h^?1. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.     

Lipase production by Serratia marcescens strain SN5gR isolated from the scat of lion-tailed macaque (Macaca silenus) in Silent Valley National Park, a biodiversity hotspot in India

An extracellular lipase-producing bacterium was isolated from a fecal sample of lion-tailed macaque (Macaca silenus), an endangered Old World monkey that is endemic to the Western Ghats of South India. Morphological, biochemical and molecular analyses identified the bacterium as Serratia marcescens. Production of lipase was investigated in shake-flask culture. Optimum tributyrin concentration of 1.5 % was found to be the most suitable triglyceride to increase lipase production (13.3 U ml^?1). The next best lipid source observed was olive oil (11.94 U ml^?1), followed by castor oil, coconut oil and palm oil. Analyzing the effect of different carbon sources on lipase production revealed that 2 % glucose yielded higher lipase production than the other tested carbon sources. Investigations on suitable nitrogen source for lipase production revealed that 2 % meat extract yielded higher lipase production. The most suitable trace element for maximum lipase production was zinc sulfate, followed by magnesium sulfate and copper sulfate. Partial characterization of the crude lipase revealed that pH 7.0 and a temperature of 40 °C gave optimal lipase activity. Enzymatic activity of the crude sample was retained over a wide temperature range (20–75 °C), and 70 % of enzyme activity was retained at 60 °C. Testing the effect of various organic solvents on lipase activity revealed that hexadecane increased lipase activity by 85 % over the control.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

Optimization of culture medium for microsclerotia production by Nomuraea rileyi and analysis of their viability for use as a mycoinsecticide

Microsclerotia (MS) formation was successfully induced in Nomuraea rileyi (Ascomycetes: Hypocreales) in liquid amended medium (AM) culture. To improve MS production, the culture medium was varied. Three quantitative variables, carbon, nitrogen, and basal salt sources, were investigated using the AM. An orthogonal experiment [L₉ (3)³] design was applied to optimize the culture medium. Maximum MS production (21.9 × 10⁴ MS ml^?1) was obtained using the optimized culture medium (32.0 g l^?1 of glucose, 2.0 g l^?1 of ammonium citrate, and 0.15 g l^?1 of ferrous sulfate). Subsequently, viability, including thermotolerance, shelf-life, and infection, were investigated to evaluate the potential of MS for use as a mycoinsecticide. The MS, collected from the optimized culture medium, exhibited virulence and greater thermotolerance than conidia, and maintained 86.4 % germinability after one-year room temperature storage. These results suggested that the MS obtained through optimization production can be used as a mycoinsecticide.     

Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H₂ production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H₂ production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H₂ production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)^?1 and Hyd-1 activity was 0.1 U (mg protein)^?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)^?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)^?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H₂ production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.     

Enrichment of biologically active 18-β glycyrrhetinic acid in Glycyrrhiza glabra root by solid state fermentation

This paper describes the in situ bioconversion of glycyrrhizin of Glycyrrhiza glabra root to 18-β glycyrrhetinic acid by solid state fermentation. Fermentation was carried out with two different fungal strains, Penicillium chrysogenum and Rhizopus oryzae. The solid state fermentation was carried out under stationary state and under rotating state. Penicillium chrysogenum is a better producer of 18-β glycyrrhetinic acid than Rhizopus oryzae. The induced P. chrysogenum seed culture produces higher 18-β glycyrrhetinic acid with 2.955 mg g^?1 and maximum β-glucuronidase activity of 3,583.8 U ml^?1 under stationary solid state fermentation. The mycelium growth and bioconversion rate is highest at pH of 5.5 and 4.5, respectively. G. glabra root supplemented with a solution of dextrose 9 g l^?1, MnSO₄?·?H₂O 3 g l^?1 and (NH₄)₂SO₄ 0.540 g l^?1 produces 48.580 mg of 18-β glycyrrhetinic acid per gram of G. glabra root, i.e. 86.74 % bioconversion by P. chrysogenum in 96 h under stationary state solid state fermentation.     

Optimisation of submerged culture conditions for the production of mycelial biomass and exopolysaccharide byPleurotus nebrodensis

The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) fromPleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth and EPS production was as follows: 200 g l^?1 bran, 25 g l^?1 maltose, 3 g l^?1 yeast extract, 1 g l^?1 KH₂PO₄, 1 g l^?1 MgSO₄ 7H₂O. Under the optimal conditions, the mycelial biomass (4.13 g l^?1) and EPS content (2.40 g l^?1) ofPleurotus nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively.     

Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control

Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-d-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml^?1) and cell biomass (7.95 g DCW l^?1). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml^?1, nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml^?1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein ^?1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The K_m and V_max of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min^?1 mg^?1, respectively.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

Effects of pulse feeding of beet molasses on recombinant benzaldehyde lyase production by Escherichia coli BL21(DE3)

The effect of fed-batch operation (FBO) strategy was investigated using pretreated-beet molasses, containing galactose that induces the lac promoter, on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21(DE3)pLySs. After batch cultivation with 30 g l^?1 pretreated-beet molasses consisting of 7.5 g l^?1 glucose and 7.5 g l^?1 fructose, three FBO strategies were applied at dissolved oxygen (=40%) cascade to air-flow rate. In FBO1 when air-flow rate decreased considerably, feed was given to the system in pulses in such a way that pretreated-beet molasses concentration increased by 10 kg m^?3 (containing 2.5 g l^?1 glucose+2.5 g l^?1 fructose); however, decrease in air-flow rate demonstrated only the absence of glucose but not fructose. Thus, in FBO2 when fructose and glucose were completely utilized, pretreated-beet molasses was pulse-fed and its concentration increased by 10 g l^?1. In FBO3 with the decreased amount of pretreated-beet molasses (6 g l^?1), shift response time from glucose to fructose consumption was avoided, and glucose and fructose consumptions were well correlated with air-flow rate, and the highest C _X (8.04 g l^?1) and BAL (2,315 U ml^?1) production were obtained (t?=?24 h) with the highest substrate yield on cell and product formation.     

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l^?1 cell mass and 1.488 g l^?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l^?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R²) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg^?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.     

Changes in growth and size of Keratella cochlearis (Gosse) in relation to some environmental factors in cultures

Keratella cochlearis (Gosse) was cultured non-axenically in Carefoot medium diluted with Erken water at 5 °C, 15 °C and 20 °C with Rhodomonas minuta (Skuja) as a food alga. The rotifer reached ca. 120 ind. ml^?1, having generation times of 2–7 days, a Q₁₀-value of ca. 2, and at the lowest temperature >20% longer posterior spines. When co-cultured with Chlorella sp., at 0–30 mg Ca l^?1 and 1.6 meq NaHCO₃ l^?1 in medium L 11 at 20 °C, the maximum generation time and individual numbers were 3–4 days and up to 100 ind. ml^?1, respectively. Animal numbers increased in relation to nutrient multiples, up to two multiples, of the culture medium L 16. Growth and length were reduced, although the width increased above two multiples of this culture medium. The trace metal tolerance was broad and increased additions of a metal mixture (L 11) slightly increased the length of the rotifers. No major changes in the length were observed when HCO₃ or Ca were varied in the culture medium (L 11), although a decrease in the length was noted in old cultures.     

Immobilization of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B onto Purolite<Superscript>®</Superscript> MN102 and its application in solvent-free and organic media esterification

The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite^® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters—isoamyl acetate and l-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g^?1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g^?1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml^?1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of l-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.