Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

产低温脂肪酶菌株CZW001发酵培养基优化研究

【目的】研究产低温脂肪酶菌株CZW001发酵培养基。【方法】在单因素试验的基础上, 采用Plackett-Burman (P-B)设计, Box-Behnken (B-B)设计和响应面试验设计(RSM), 在20 °C、pH 8.0、160?r/min发酵2 d条件下, 对发酵培养基进行优化。【结果】该菌株最适产酶培养基为(g/L): 葡萄糖7.68, 橄榄油21.93, 硫酸铵2.0, 磷酸二氢钾1.0, 硫酸镁0.27, 氯化钙0.3, 氯化钠20.0, 吐温-80 1.0。其最高酶活为62.8 U/mL, 比优化前提高了3.14倍。【结论】通过对产低温脂肪酶菌株CZW001发酵培养基优化研究, 明显提高低温脂肪酶活力。     

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.     

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O_m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W_m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O_m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

Optimized production of a thermostable lipase by recombinantPseudomonas putida 1641

A thermostable lipase gene fromPseudomonas fluorescence was cloned into a multicopy plasmid and electroporated intoP. putida. The recombinantP. putida produced soluble lipase into the broth. Using these recombinant cells, the optimum conditions for lipase production were investigated. Olive oil was the most effective carbon source, producing upto 60 unit/ml. Supplementation of the medium with a surfactant, such as Tween-80, increased the lipase productivity significantly. The optimum temperature and pH for lipase production were 26°C and 7.0, respectively.     

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH₄)₂SO₄, CaCl₂, MgSO₄, olive oil, peptone, K₂HPO₄, NaNO₃, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K₂HPO₄, NaNO₃, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K₂HPO₄, NaNO₃, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.     

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2–0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.     

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.     

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 °C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Effect of tweens on the lipase activity of Oospora lactis

Oospora lactis was found to use Tweens 20, 40, 60 and 80 as a source of carbon in a nutrient medium for lipase biosynthesis. The highest production of lipase was registered in the medium with Tween-80. The lipase activity of the culture increased if even small quantities of Tweens 40, 60 and, particularly, 80 (0.05--1.0%) were added to the medium containing cottonseed oil. All the tested Tweens stimulating the biosynthesis of lipase were also, at a particular concentration, the inhibitors of this enzyme.     

Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01

An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.     

Upstream and Downstream Processing of Lovastatin by Aspergillus terreus

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 μg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.     

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l^?1, 40 g Tween 80 l^?1 and 30 g peptone l^?1, TliA was produced at a level of 2,200 U ml^?1 in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml^?1) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Enhanced production of Penicillium expansum PED-03 lipase through control of culture conditions and application of the crude enzyme in kinetic resolution of racemic Allethrolone

Alkaline lipase production was performed in submerged fermentation by Penicillium expansum PED-03. It was found that the suitable carbon source and nitrogen source for lipase production were 0.5% starch and 4.0% soybean meal, respectively. The maximal lipase activity (850 U/mL) of production was achieved at initial pH 5.5-6.0, 26 degrees C, 72 h. Tween-80 was an effective enhancer for lipase production. Agitation speed of the fermentor played an important role, and the suitable agitation speed for lipase production was 500 r/min. The lipase was stable within the range of pH 7.0-10.0 and 20-40 degrees C, and the optimum conditions for the enzymatic reaction were 35 degrees C and pH 9.5. The enzymatic resolution of racemic allethrolone (4-hydroxy-3-methyl-2-(2-propenyl)-2- cyclopenten-1-one) was carried out by the lipase from P. expansum PED-03, and the conversion reached 48% with excellent enantioselectivity (E > 100), which showed a good application potential in the production of optically pure allethrolone.     

Fluorene biodegradation and identification of transformation products by white-rot fungus Armillaria sp. F022

A diverse surfactant, including the nonionic Tween 80 and Brij 30, the anionic sodium dodecyl sulphate, the cationic surfactant Tetradecyltrimethylammonium bromide, and biosurfactant Rhamnolipid were investigated under fluorine-enriched medium by Armilaria sp. F022. The cultures were performed at 25 °C in malt extract medium containing 1 % of surfactant and 5 mg/L of fluorene. The results showed among the tested surfactants, Tween-80 harvested the highest cell density and obtained the maximum specific growth rate. This due Tween-80 provide a suitable carbon source for fungi. Fluorane was also successfully eliminated (>95 %) from the cultures within 30 days in all flasks. During the experiment, laccase production was the highest among other enzymes and Armillaria sp. F022-enriched culture containing Non-ionic Tween 80 showed a significant result for laccase activity (1,945 U/L). The increased enzyme activity was resulted by the increased biodegradation activity as results of the addition of suitable surfactants. The biotransformation of fluorene was accelerated by Tween 80 at the concentration level of 10 mg/L. Fluorene was initially oxidized at C-2,3 positions resulting 9-fluorenone. Through oxidative decarboxylation, 9-fluorenone subjected to meta-cleavage to form salicylic acid. One metabolite detected in the end of experiment, was identified as catechol. Armillaria sp. F022 evidently posses efficient, high effective degrader and potential for further application on the enhanced bioremediation technologies for treating fluorene-contaminated soil.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Lipase from Pseudomonas aeruginosa LP602: biochemical properties and application for wastewater treatment

Lipase from Pseudomonas aeruginosa LP602, a bacterial strain isolated from a domestic wastewater sample, was preliminarily characterized. The enzyme exhibited maximum lipolytic activity at pH 8.0 where it was also stably maintained. At 55°C, the lipase had the highest activity but not stability. The enzyme was insensitive to EDTA and to many ions tested except Zn²⁺. It was sensitive to SDS but not to Tween-20, Tween-80 or Triton X-100. The enzyme was active towards a number of commercial food grade fats and oils. A suitable medium formula for lipase production was MMP containing 6.25% whey as a carbon source, 1% soybean oil as inducer and 0.5% yeast extract supplement. The culture was fed with glucose to a final concentration of 0.1% at the 15th hour of incubation. Lipase production under this condition was 3.5 U ml⁻¹. Both P. aeruginosa LP602 cells and the lipase were shown to be usable for lipid-rich wastewater treatment. Received 21 April 1998/ Accepted in revised form 6 August 1998