Alkaline Lipase Research Articles

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH(2)-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca(2+) and bile salts. The specific activities of FSL were about 1,610 and 2,414U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH8.0 and 37°C. The (phospho)lipase enzyme was stable in the pH range of 5-10 and at temperatures below 45°C.

QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms

The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R2adj) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R2cv) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

An organic solvent and thermally stable lipase from Burkholderia ambifaria YCJ01: Purification, characteristics and application for chiral resolution of mandelic acid

Abstract A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S -isomer of mandelic acid with a theoretical conversion yield of 50%, ee p of 99.9% and ee s of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.

Biochemical Characterization of Biomphalaria arabica , the Molluscan Intermediate Host for Schistosomes in Saudi Arabia

Schistosomiasis is the most important human helminth infection in terms of morbidity and mortality. The present study provides a comparative analysis on Biomphalaria arabica , the intermediate hosts of Schistosoma mansoni from two different areas having different prevalence level of Schistosomiasis in Saudi Arabia. B. arabica snails were collected from Riyadh and Hofuf district, and the levels of various enzymes like aspartate and alanine aminotransferase (AST, ALT), Lactate dehydrogenate (LDH), acid and alkaline phosphatase (ACP,ALP), α amylase and lipases were measured in the tissue homogenate of snails, to confirm the relationship between the biochemical and endemic of the disease. Also amino acid profile of B. arabica was determined using amino acid analyzer. The result showed higher levels of AST and LDH in the sample of B. arabica from Hofuf as compared to Riyadh while ALT, ACP, ALK, α amylase and lipase enzymes were found lower in samples from Hofuf as compare to Riyadh. The amino acid profile shows that Riyadh samples have remarkably higher levels of most of the measured amino acids. The present study confirms that biochemical profile of B. arabica is critically important for the success of schistosome life cycle and that snail host definitely plays a part in prevalence of the disease.

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1-7 isolated from Bohai Bay in China for detergent formulation

Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1-7 isolated from Bohai Bay in China for detergent formulation

Enhanced Production of Extracellular Alkaline Lipase by an Improved Strain of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; MTCC 10,055

Problem statement: Lipases are industrially important enzymes having applications in numerous industries. For easy commercialization it is necessary to produce lipases at industrial level which could be achieved by strain improvement and medium formulation. Approach: In the present study strain improvement of Pseudomonas aeruginosa MTCC 10,055 was done by chemical mutagenesis using mutagen 4-nitroquinoline1-oxide for alkaline lipase production. Different fermentation parameters affecting lipase production were optimized using one-variable-at-a-time approach. Results: The selected mutant (M-05) exhibited 3.6-fold higher productivity over wild type. Maximum alkaline lipase was produced when culture was incubated at 35°C with initial medium pH 9.0 in 28 h with inoculum density 0.5% (v/v) (Abs610-1.0). Supplementation of production medium with combination of castor oil and starch as carbon source and Triton-X-100 as surfactant significantly influenced the alkaline lipase production. The composition of fully optimized medium was determined to be (g L-1): (NH4)2SO4, 1.0; KH2PO4, 0.6; MgSO4, 0.4; yeast extract, 0.2; castor oil, 2.0; starch 20.0; gum arabic, 5.0; Triton-X-100, 1.0. An overall 14-fold enhanced production was achieved after complete medium optimization. Conclusion/Recommendations: The improved strain was capable to produce high titer of alkaline lipase at flask level, which can be examined at fermentor level to obtain sufficient enzyme yield to meet the world wide industrial demand.

An organic solvent-tolerant alkaline lipase from Streptomyces sp. CS268 and its application in biodiesel production

In an effort to identify a microbial lipase that can catalyze transesterification reactions used in biodiesel production, an organic solvent-tolerant lipase was purified from Streptomyces sp. CS268. The molecular weight of the purified lipase was estimated to be 37.5 kDa by SDS-PAGE. The lipase showed highest activity at a temperature of 30°C and pH 8.0 while it was stable in the pH range 4.0 ∼ 9.0 and at temperatures ≤ 50°C. It showed the highest hydrolytic activity towards medium-length acyl chain p-nitrophenyl decanoate with Km and Vmax values of 0.59 mM and 319.5 mmol/mg/min, respectively. Also, the lipase showed non-position specificity for triolein hydrolysis. The purified lipase catalyzed transesterification reaction of soybean oil with methanol, suggesting that it can be a potential enzymatic catalyst for biodiesel production.

Optimization of extracellular thermophilic highly alkaline lipase from thermophilic Bacillus sp isolated from Hotspring of Arunachal Pradesh, India

Studies on lipase production were carried out with a bacterial strain (Bacillus sp LBN 2) isolated from soil sample of hotspring of Arunachal Pradesh, India. The cells were cultivated in a mineral medium with maximum production at 1% groundnut oil. The optimum temperature and initial medium pH for lipase production by the organism were 500C and 9.0 respectively. The molecular mass was found to be 33KDa by SDS PAGE. The optimal pH and temperature for activity were 10 and 600C respectively. The enzyme was found to be stable in the pH range of 8–11 with 90% retention of activity at pH 11. The enzyme retained 90% activity at 600C and 70% of activity at 700C for 1h. The lipase was found to be stable in acetone followed by ethanol. The present findings suggested the enzyme to be thermophilic alkaline lipase.

Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1-7 Isolated from bohai bay in china for detergent formulation.

A novel alkaline lipase-producing strain 1–7 identified as Acinetobacter calcoaceticus was isolated from soil samples collected from Bohai Bay, China, using an olive oil alkaline plate, which contained olive oil as the sole carbon source. The lipase from strain 1–7 showed the maximum activity at pH 9.0 under 40 °C. One interesting feature of this enzyme is that it exhibits lipase activity over a broad range of temperatures and good stability. It is also stable at a broad range of pHs from 4.0 to 10.0 for 24 h. Its catalytic activity was highly enhanced in the presence of Ca2+, Mg2+ and K+, but partially inhibited by Cu2+, Al3+, Fe3+, Ba2+and Zn2+. The fact that it displays marked stability and activity in the presence of TritonX-100, Tween-20, Tween-80, SDS, Hydrogen peroxide, Sodium perborate, Sodium hypochlorite, Sodium citrate, Sodium taurocholate, Glycerine and NaCl suggests that this lipase is suitable as an additive in detergent formulations.

Prediction and Process Optimization of Bioscouring of Organic Cotton Fabrics through Specific Mixed Enzymatic System using Artificial Neural Network (ANN)

The objective is to develop an improved enzymatic cotton scouring process on the basis of a fast enzyme reaction to efficiently remove pectin and wax compounds from an organic cotton fabric by using a specific mixed enzymatic system. An attempt has been made to study the pectinolytic activity on the organic cotton fabric using four selective enzymes—alkaline pectinase, protease, lipase, and cellulase—and three process parameters—enzyme concentration, temperature, and reaction time. These process parameters are selected based on an artificial neural network (ANN) using the MATLAB 7.0 software, and the output of the experiment was described in terms of fabric physical properties—weight loss, water absorbency, wetting area, whiteness index, yellowness index, and brightness index. The bioscoured organic cotton fabric was tested for wax content and pectin degradation rate and the results were optimized with minimum error. The test results were analyzed to predict the optimum process parameters to achieve the required bioscouring fabric properties and the required level of pectin degradation rate, and were compared with the results of the actual trials. The performance of the specific mixed enzymatic system during bioscouring was assessed using ruthenium red dye test and FTIR (Fourier transform infrared spectroscopy) to confirm the degradation of pectin in the bioscoured organic cotton fabrics.

Abstract C26: A phase 1 multiple ascending dose study of cabozantinib (XL184) monotherapy in Japanese patients with advanced solid tumors.

Abstract Background: Cabozantinib is a potent orally available small molecule inhibitor of multiple receptor tyrosine kinases with growth promoting and angiogenic properties. The primary targets of cabozantinib are MET and VEGFR2. In preclinical models, cabozantinib demonstrated significant tumor growth inhibition in multiple tumor models including human medullary thyroid cancer (MTC), human breast cancer, human lung carcinoma and rat glioblastoma. Anti-tumor activity in patients has been observed across a broad range of tumor types including MTC, prostate, ovarian and breast cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma, melanoma and glioblastoma. The primary objectives of this open-label phase 1 study are determination of the maximum tolerated dose (MTD) and recommended phase 2 dose in Japanese patients. Methods: Patients with advanced solid malignancies are enrolled in successive cohorts to receive escalating doses of cabozantinib in a 3+3 trial design. Patients are treated with continuous 4-week cycles of cabozantinib administered orally (PO) once daily (QD). Dose limiting toxicities (DLTs) are determined using Cycle 1 data. Response is assessed on Day 29 and every 8 weeks thereafter by modified RECIST v1.0 criteria and patients with stable disease (SD) or objective response receive continued therapy. Pharmacokinetic (PK) parameters of cabozantinib and relevant plasma markers including VEGF-A, sVEGFR2 and PlGF are being evaluated. Results: As of 1 September 2011, 6 patients with advanced malignancies have been enrolled: NSCLC (n=3), colorectal cancer, gastrointestinal stromal tumor (GIST), leiomyosarcoma (n=1 each). Patients were pretreated with a median of 3 prior regimens (range, 2 − 7). Patients have been treated at 2 dose levels of 40mg and 60mg free base equivalents (salt strengths of 50mg and 75mg, respectively) PO QD. There have been no DLTs or serious adverse events reported to date. The MTD has not yet been defined. Observed adverse events were generally mild to moderate and include hypertension, palmer-plantar erythrodysesthesia, diarrhea, mucositis, rash, edema and headache. Laboratory abnormalities include elevated AST/ALT, neutropenia, thrombocytopenia and increases in alkaline phosphatase, LDH, lipase and TSH. Results of preliminary PK analysis at the 40mg QD dose level showed that cabozantinib appeared to reach steady-state plasma concentrations by Day 15 and there was evidence of 6- to 7-fold accumulation on Day 19. One patient with NSCLC has had a confirmed partial response with 38% reduction in the sum of target lesions. Three pretreated patients including 2 with NSCLC and 1 with GIST have had SD with reductions in the sum of target lesions of 20, 13 and 16% respectively, and continue treatment with cabozantinib. Conclusions: Cabozantinib appears well tolerated at doses tested to date. Early signs of antitumor activity including prolonged stable disease and response have been observed in the initial dosing cohorts. Determination of the MTD is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C26.

Cloning and sequence analysis of complete gene encoding an alkaline lipase from Penicillium cyclopium

The complete gene (PG37 lipI) encoding an alkaline lipase (PG37 LipI) was cloned from the genomic DNA of Penicillium cyclopium PG37. The cloned PG37 lipI is 2020 bp in length, consisting of 632 bp of the 5' flanking promoter region and 1388 bp of the downstream fragment that contains 6 exons and 5 short introns. The promoter region harbors putative TATA box, CAAT box and several transcription factor binding sites. The open reading frame (ORF) encodes a PG37 LipI of 285 amino acid residues, which was predicted to contain a 20-aa signal peptide, a 7-aa propeptide and a 258-aa mature peptide with a conserved motif Gly-X-Ser-X-Gly. However, PG37 LipI shows only 32%, 30%, 28% and 26% identity with lipases of Aspergillus parasiticus, Penicillium camembertii, Thermomyces lanuginosus and Rhizomucor miehei, respectively. It was predicted that the main secondary structures of PG37 LipI are alpha-helix and random coil. Three amino acid residues, Ser132-Asp188-His241, compose the enzymatic active center in the tertiary structure.

Isolation, Screening and Production of Extracellular Alkaline Lipase from a Newly Isolated Bacillus sp. PD-12

Isolation, Screening and Production of Extracellular Alkaline Lipase from a Newly Isolated Bacillus sp. PD-12

High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris

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.

Astaxanthin Preparation by Lipase‐Catalyzed Hydrolysis of Its Esters from Haematococcus pluvialis Algal Extracts

Five of 8 fungal lipases screened were found to effectively hydrolyze astaxanthin esters from Haematococcus pluvialis algal cell extracts. Among these, an alkaline lipase from Penicillium cyclopium, expressed in Pichia pastoris, had the highest enzymolysis efficiency. Tween80 was shown to be an effective emulsifier in this lipase hydrolysis system for the 1st time. A series of experiments were performed to find optimal conditions for hydrolysis (pH, temperature, reaction time, lipase dosage). In the optimal reaction system, Tween80 and H. pluvialis extracts (mass ratio 1:1) were emulsified and added to the above lipase at a dosage of 4.6 U/μg (relative to total carotenoids), in phosphate buffer (0.1 M, pH 7.0), and incubated at 28 °C for 7 h, with agitation at 180 rpm. The free astaxanthin recovery ratio under these conditions was 63.2%.

A novel alkaline lipase from Ralstonia with potential application in biodiesel production

With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS–PAGE. The activity was highest at 50–55 °C and pH 8.0–9.5 and was stable at pH 7.0–12.0 and up to 45 °C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K m and V max for p-nitrophenyl palmitate were 2.73 ± 0.6 mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.

Isolation of a Novel Alkaline Lipase Producing Fungus Aspergillus fumigatus MTCC 9657 from Aged and Crude Rice Bran Oil and Quantification by HPTLC

Isolation of a Novel Alkaline Lipase Producing Fungus Aspergillus fumigatus MTCC 9657 from Aged and Crude Rice Bran Oil and Quantification by HPTLC

Alkaline lipase activity from the marine protists, thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.