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1、The purification of alkaline and thermostable lipase from a newly isolated strain Acinetobacter calcoaceticus 1-7 HaiKuan Wang, HuiJing Ma, ShaoJiong Zhong, YuJie Wei, Wei Qi, Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science an
2、d Technology, Tianjin, PC: 300457; P.R.China E-mail: Abstract A newly isolated moderate thermophilic strain 1-7 producing alkaline and thermostable lipase previously was identified as Acinetobacter calcoaceticus, which was isolated from the soil collected from bay of Bohai in China. The lipase show
3、ed maximum activity at pH 9.0 and high thermostability. The lipase from Acinetobacter calcoaceticus 1-7 was purified by ammonium sulfate precipitation, ion exchange chromatography and Sephadex G-75 chromatography. The molecular mass (Mr) of the lipase was estimated to be 30 kDa using SDS-PAGE. Keywo
4、rdsAcinetobacter calcoaceticus; Thermostable alkaline lipase; Purification. I. INTRODUCTION Lipases (triacylglycerol acylhydrolases; EC3.1.1.3) are one of the most important classes of hydrolytic enzymes that catalyze both the hydrolysis and the synthesis of esters. They have a number of unique char
5、acteristics, including substrate, stereospecificity, regioselectivity and the ability to catalyze a heterogenous reaction at the interface of water soluble and water insoluble systems. They are widely distributed in animals, plants and microorganisms 1. Microbial lipases have currently received cons
6、iderable attention with regard to biotechnological applications for detergents, oil and fat, dairy and pharmaceutical industry 2. Although a number of lipase-producing bacterial sources are available, only a few are commercially exploited as strains, of which the important ones are: Achromobacter, A
7、lcaligenes, Arthrobacter, Bacillus, Burkholderia, Chromobacterium and Pseudomonas 3. As the applications increase, the availability of lipase possessing satisfactory operating characteristic is a limiting factor. Lipase used in detergents needs to be stable under a broad range of temperature and sho
8、uld be active in the presence of surfactants. Hence, thermostable lipases are expected to play an important part in the detergent industry which is used mainly in industrial laundry and in household dishwashers, since they are more thermostable, much higher active at elevated temperature and resista
9、nt to chemical denaturation 4. The industrial demand for lipases with high thermostability continues to stimulate the search for microorganism producers of thermostable enzymes. Thermostable enzymes are usually derived from thermophilic strains, which may be expected to produce intrinsically more he
10、at-stable enzymes than their mesophilic counterparts 5. A few numbers of thermophilic lipase-producing bacteria have been described in the last 6-10. In this study, we conducted the purification on a new thermostable alkaline lipase producing organism, identified as Acinetobacter calcoaceticus. II.
11、MATERIALS AND METHODS A. Materials The thermostable lipase producing strain Acinetobacter calcoaceticus 1-7 was previously isolated from soil collected from bay of Bohai in China. Polyvinyl alcohol (PVA) produced by Beijing Organic Chemical Factory (Beijing, China). Olive oil produced by Moreno Comp
12、any of Spain (SPAIN). p-Nitrophenyl palmitate (pNPP) was purchased from Sigma Chemical Co. Other chemicals were obtained from commercial sources and were of the highest qualities available. B. Lipase fermentation The microorganism from a nutrient agar slant was grown in the seed medium containing (%
13、): peptone 10, yeast extract 5, NaCl 10, pH 7.5 at 37 C for 12 h. One milliliters were inoculated in 250 ml Erlenmeyer flask containing 30 ml of fermentation medium(%): corn flour 10, bean flour 20, K2HPO4 1, NaNO3 5, olive oil 10, with the initial pH of 8.0. Culture conditions were: 37 C, 180 rpm i
14、n a rotary shaker for 30 h. The lipase activity of the culture broth supernatant (centrifuged at 8000 rpm at 4C for 15 min) was measured. C. Lipase activity assay Lipase activity was measured by plate assay method, which was indicated by Victoria Blue. When there is free fatty acid hydrolyzed from o
15、live oil by the lipase, the color of the plate will exchange. The medium of the plates were as follows: 10% emulsion of olive oil (containing 0.2% Victoria blue B) was adjusted to different pH buffers with 2% agar. 4-mm-diameter holes were punched into the agar and filled with 200 l of culture super
16、natant. The plates were incubated for 24 h at different temperature (20 C, 30 C, 40 C, 50 C). Lipase activity was assayed by determined change of the blue zones. This method was useful to test vivid activity but not very accurate. Lipase activity was accurately estimated using a spectrophotometric a
17、ssay method with p-Nitrophenyl This work was supported by Tianjin Natural Science Foundation (No. 09JCZDJC17800 and No. 07JCYBJC07900).) 978-1-4244-4713-8/10/$25.00 2010 IEEEpalmitate (pNPP) as substrate 11. The reaction mixture consisted of two solutions: solution 1 with its composition of 30 mg p-
18、Nitrophenyl palmitate dissolved in 10 ml 2-propanol, solution 2 with its composition of 0.9 ml Triton-100 dissolved in 90 ml 0.2 M phosphate buffer (pH 8.0). The assay mixture contained 900 l of the emulsion of solution 1 and solution 2 (1:9) and 100 l appropriately diluted enzyme solution. The hydr
19、olytic reaction was carried out at 40 C for 15 min, after which the mixture was putted at -20 C for 10 min to end the reaction. The liberated p-Nitrophenol was monitored by the absorbance at 410 nm using a spectrophotometer. One enzyme unit (U) was defined as the lipase activity that released 1 mmol
20、 p-Nitrophenol per min under the standard assay conditions. D. Purification of lipase The purification procedure for lipase from 1-7 strain involves three steps. Step1: Ammonium sulfate precipitation Solid ammonium sulfate was slowly added to the supernatant under stirring to achieve a concentration
21、 of 25% (w/v) saturation at 4C for 10 h. The precipitation was separated by centrifugation at 8000 rpm in a refrigerated centrifuge for 15 min at 4C and dissolved in a minimal amount of 0.2 M phosphate buffer (pH 8.0). Lipase activity both in precipitate and supernatant were determined. The supernat
22、ant was mixed with ammonium sulfate to give saturation of 55% at 4 C. The final precipitate was dialyzed with a bladder membrane for 24 h with 3-4 changes in distilled water. Step2: DEAE-cellulose column chromatography The partially purified enzyme was concentrated with a bladder under PEG and appli
23、ed to an anion DEAE-cellulose column (201.6 cm) previously equilibrated with the 10 mM TrisHCl buffer (pH 8.0). After the washing of three bed volumes with the initial buffer, the elution of bound protein was performed with a negative linear gradient of 01.0 M NaCl in the same buffer at a flow rate
24、of 50 ml h-1. The active fractions were pooled, concentrated by PEG with a bladder Step3: Gel filtration. The concentrate was then chromatographed on a column (80 1.6 cm) of Sephadex G-75, which was pre-equilibrated with 20 mM sodium phosphate buffer pH 7.5 and eluted with the same buffer. The flow
25、rate was adjusted to 50 ml h1. The lipase-rich fractions were pooled, stored at -20C, and used as the purified lipase for polyacrylamide gel electrophoresis and enzyme characterization. E. Determination of molecular weight The molecular mass of the purified lipase was estimated by SDS-PAGE assay, wh
26、ich was performed according to Laemmli 12. Boiling time in sample buffer containing 7.5% 2-mercaptoethanol was 10 min and gels were stained with Coomassie Brilliant Blue G-250. Electrophoresis was performed at 30 mA constant current. For estimation of relative molecular weight 12% SDS-PAGE was run a
27、long with the following standardsrabbit phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa) , rabbit actin (43 kDa), Bovine carbonic anhydrase (31 kDa), trypsin inhibitor (20.1 kDa) and hen egg white lysozyme (14.4 kDa). III. RESULTS AND DISCUSSION Compared with other lipase from Acinetobact
28、er calcoaceticus which have been reported, the lipase from strain Acinetobacter calcoaceticus 1-7 has some distinct virtues, such as good thermostability and with the highest activity at pH 9.0 (Fig 1). Acinetobacter calcoaceticus LP009 isolated from raw milk produced a lipase that had the ability t
29、o improve fat hydrolysis in soybean meal and in premixed animals feed. The lipase exhibited maximum activity at pH 7.0 and 50C 13. The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified, the purified enzyme has optimal activity between pH 7.8 and 8.8 14. Figure 1. The catalytic
30、 activity of lipase from 1-7 strain at different pH by plate method. The four plates were composed with pH 7.0, 8.0, 9.0 and 10.0. The partial purified lipase after centrifugation and ammonium sulfate precipitation was bounded on a DEAE- cellulose column and eluted with a linear gradient of sodium c
31、hloride. In the middle of the gradient, about 300 mM sodium chloride, the fractions with lipase activity were recovered. The active fractions were collected and chromatographed through the Sephadex G-75 with the sodium phosphate buffer at a rate of 50 ml h-1, in the preceding gradient the active fra
32、ction was appeared. After the three-step procedure, the lipase was purified 23.43-fold with a high specific activity of 64.21 u mg-1 and the recovery of 15.8%. The purification results are summarized in Table1. TABLE I. SUMMARY OF PURIFICATION PROCEDURE OF LIPASE FROM ACINETOBACTER CALCOACETICUS Pur
33、ification Step Total activity (U) Total protein (mg) Specific activity (Umg-1) Yield (%) Purificatin (fold) Crude(cell free supernatant) 796 291 2.74 100 1.00 Precipitation (NH4)2SO4 (2555%) 712 79 9.01 89 3.29 Ion exchange 315 11.5 27.51 40 10.04 Gel filtration 125.7 1.96 64.21 15.8 23.43 1-778 9 1
34、0 Figure 2. SDSPAGE (12%) of lipase from Acinetobacter calcoaceticus at different purification steps. Lane 1: Molecular mass standards indicated in kDa; Lane 2: Crude lipase from the supernatant. Lane 3: Partially purified lipase after ammonium sulfate precipitation. Lane 4: Partially purified lipas
35、e after Ion exchange. Lane 5: 30 kDa purified lipase after three steps. The purified lipase showed a single band with a molecular weight of 30 kDa as assessed by SDSPAGE (Fig.2). The lower molecular weight of the lipase is advantageous, as smaller enzymes are more stable due to smaller changes (unfo
36、lding) in tertiary structure 15. The lipase from strain 1-7 was compared with that reported for other lipases previously characterized from other Acinetobacter calcoaceticus. The extracellular lipase from Acinetobacter calcoaceticus BD413 has an apparent molecular mass of 32 kDa 14. The extracellula
37、r lipase of Acinetobacter calcoaceticus 69 V existed as a high molecular complex of about 300 kDa, with a subunit molecular weight of 30.5 kDa being obtained by SDS-PAGE 16. In contrast, many thermostable lipases have been found to possess a higher molecular mass of 4362 kDa 17-19. These results ind
38、icated that the purified lipase was a novel enzyme from Acinetobacter calcoaceticus. IV. CONCLUTIONS In this study, a thermostable lipase producing strain Acinetobacter calcoaceticus 1-7 was isolated from soil. The lipase from Acinetobacter calcoaceticus was purified by three steps: ammonium sulfate
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