Catalytic properties of mycelium-bound lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> MYA 135

A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0–6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4–8°C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37°C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.     

Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and 170 rpm. Under the optimal conditions, over 60 U ml⁻¹ of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.     

Acid phosphatase production by recombinant <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g⁻¹ DYB) and laboratory fermenter (18,465 U g⁻¹ DYB), respectively.     

Influence of culture conditions on thermostable lipase production by a thermophilic alkalitolerant strain ofBacillus sp

The effect of different culture conditions on thermostable lipase production byBacillus sp. was studied in shake flasks. A maximum enzyme activity of 67–75 nkat/mL was observed in a medium consisting of 0.5% soybean flour and 0.1% stearyl glycerol esters or natural fats. A lipase activity of about 117 nkat/mL was established when the cultivation was carried out in laboratory fermentor at 20% minimal dissolved oxygen level, the enzyme production being increased 1.5 fold compared to that in a flask culture.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> SR1 as a Source of Chitinase Having Potentiality of Using as a Biocontrol Agent

Serratia marcescens, strain SR₁ was isolated from the local soil of a cultivated farm and it was screened as potent strain for chitinase production. Maximum chitinase production (77.3 u Mh⁻¹ 100⁻¹) was observed after 96 h of incubation period with pH 5.5 at 30°C under shake conditions (120 rpm). Compare to still flasks, shake culture with prawn fish colloidal chitin of 0.5% (w/v) concentration, showed a better enzyme yield. Crude enzyme showed antifungal activity against plant pathogens.     

Enhanced production of recombinant galactose oxidase from <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had kinetic properties similar to those of the fungal wild-type enzyme.     

Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures

The nutritional medium requirement for biomass and triterpenoid production by Antrodia cinnamomea AC0623 strain was optimized. Box–Behnken was applied to optimize biomass and triterpenoid production. According to response surface methodology (RSM), the optimum concentrations of N-source were determined. The results indicate that when a submerged culture in shake flasks was operated at 28°C, initial pH 5.5, and rotation speed 105 rpm, the biomass and triterpenoid content in dry basis could be increased to 3.20% (w/w) and 31.8 mg/g, respectively. The experiments were further scaled up to 100- and 700-l fermentors. Higher content of triterpenoids (63.0 mg/g) was obtained in 700-l fermentations by means of the control of cultural conditions and the modification of medium composition based on the RSM.     

Production of levanase byRhodotorula sp.

Rhodotorula sp. produced a high yield of levanase (12.5 nkat/mL) in shake flasks in basal medium containing 1% maltose as the sole carbon source. Among the different carbon sources used, maltose was found to be the best for levanase production. The optimum temperature and pH for levanase production were 30°C and 6, respectively. In a batch reactor the enzyme productivity was higher (500 nkat L⁻¹ h⁻¹) than in shaken flasks (347 nkat L⁻¹ h⁻¹).     

Optimization of <Emphasis Type="Italic">Serratia marcescens</Emphasis> lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester

Lipase production and cell growth of Serratia marcescens ECU1010 were optimized in shake flasks, with lipase production being enhanced 9.5-fold (4,780 U/l) compared with the initial activity (500 U/l). Optimal carbon and nitrogen sources were Tween-80 and peptone, and the optimal ratio of Tween-80 to peptone was 1:3. The optimized cultivation conditions were 25°C and pH 6.5. Lipase activity, particularly specific activity, could be improved by decreasing the cultivation temperature from 35 to 25°C. Enzyme stability was significantly improved by simple immobilization with synthetic adsorption resin no. 8244. After five reaction cycles, enzyme activity decreased only very slightly, while enantioselectivity of the preparation remained constant, and the ee_s (enantiomeric excess of the remaining substrate) achieved in all cases was higher than 97%. The resin-8244-lipase preparation can be used for efficient enantioselective hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of Diltiazem.     

A thermoalkaliphilic lipase of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. T1

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na⁺, Ca²⁺, Mn²⁺, K⁺ and Mg²⁺ , but inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T _m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001