Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Isolation and study of two strains ofLeptospirillum-like bacteria from a natural mixed population cultured on a cobaltiferous pyrite substrate

Two strains ofLeptospirillum-like bacteria, L6 and L8, have been isolated from a mixed inoculum, also containingThiobacillus ferrooxidans andT. thiooxidans, cultured for one year with a colbaltiferous pyrite as energy substrate in a 100 I continuous bioleaching laboratory unit. Several physiological properties of the strains are described. The vibrio-shaped microorganisms grew at pH values lower than 1.3. Their growth rate was maximum between 2.5 and 8.0 g l¹ ferrous iron. The optimal growth temperature was 37.5° C. Ferric iron had a stimulative effect on bacterial development up to 8 g l^–1, and growth was as rapid at 14 g l^–1 ferric iron as at 8 g l^–1. The negative influence of cobalt on the final cell concentration was observed at 0.5 g l^–1, but the growth rate was not affected up to 2 g l^–1. The G + C content of strains L8 is 55.6 mol%.     

Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens

D(–)-Lactic acid was produced from cellulose by simultaneous saccharification and fermentation (SSF) in media containing cellulolytic enzymes and Lactobacillus coryniformis subsp. torquens ATCC 25600 at 39 °C and pH 5.4, yielding 0.89 g D(–)-lactic acid g^–1 cellulose at a mean volumetric productivity of 0.5 g l^–1 h^–1. No L(+)-lactic acid was found in the medium.     

Production of crocin using Crocus sativus callus by two-stage culture system

Saffron callus was grown in a two-stage culture on B5 medium supplemented with casein hydrolysate (300 mg l^–1) at 22 °C in dark with naphthalene acetic acid (2 mg l^–1) and 6-benzyladenine (1 mg l^–1) to give maximum biomass (16 g dry wt l^–1), and with indole 3-acetic acid (2 mg l^–1) and 6-benzyladenine (0.5 mg l^–1) for crocin formation. The maximum crocin production (0.43 g l^–1) was achieved by this two-stage culture method, which was three times that by a one-stage method.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Lactosucrose production by various microorganisms harboring levansucrase activity

Production of the artificial sweetener, lactosucrose, by various microorganisms containing levansucrase activity was investigated. Of the tested bacteria, Bacillus subtilis was the most effective producer using lactose as an acceptor and sucrose as a fructosyl donor. Lactosucrose production by this strain was optimal at pH 6.0 and 55 °C whereupon 181 g lactosucrose l^–1 was produced from 225 g lactose l^–1 and 225 g sucrose l^–1 in 10 h.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml^–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml^–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K_mfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005     

Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus

Mycelial growth and extracellular polysaccharide production of Phellinus linteus were optimal at pH 5 and 25 °C. Maximum biomass production (14.2 g l^–1) was after 15 d of cultivation, whereas, extracellular polysaccharide was maximal (3.5 g l^–1) after 21 d. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, decreased plasma glucose, total cholesterol and triacylglycerol concentrations by 49%, 32%, and 28%, respectively, and aspartate aminotransferase activity by 20%. The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.