characteristics of a newly isolated lipase from thermophilic burkholderia pseudomallei

1、<p>　　Characteristics of a Newly Isolated Lipase from Thermophilic Burkholderia Pseudomallei</p><p>　　Abstract. A lipase producing thermophilic Burkholderia pseudomallei bacterial strain was isolated fr

2、om Saudi Arabian environment. Based on this strain, a lipase protein was purified to homogeneity 128.2 fold as a fusion protein with glutathione S-transferase. SDS- PAGE of the purified enzyme revealed it has Mr of 32 kD

3、a. The recombinant lipase was efficiently immobilized in calcium alginate gelatin composites. The optimum temperature for free enzyme was recorded at 65 ℃. However the immobilized </p><p>　　Key words: Clonin

4、g, Expression, Purification, Immobilization, Characterization, Glutathione S transferase. </p><p>　　1. Introduction </p><p>　　The production of fatty acids by the hydrolysis of natural oils and

5、fats is a very important component in the economic utilization of these naturally produced renewable raw materials. These products include oils from corn, sunflower, palm, coconut, olives and rice bran. A significant num

6、ber of high value products require fatty acids in their manufactures. These include coatings, adhesives, specially lubricating oils, shampoos and other personal care products. Oils and fats are part of a group of</p&g

7、t;<p>　　In this work we describe the cloning and overexpression the lipase enzyme from thermophilic Burkholderia pseudomallei by using the molecular biology tools. The overexpressed protein is purified, immobilize

8、d and optimal pH, temperature, thermal staability and the effect of different cations are studied for both free and immobilized enzyme. 　　2. Materials and methods </p><p>　　E. coli Lipase was streaked onto

9、LBA plates and incubated overnight at 37℃. A single colony was used to inoculate 10ml of LB broth supplemented with 100μg/ml ampicillin, and grown overnight at 37℃, 200rpm in a shaking incubator. The overnight cultures w

10、ere used to inoculate 100 ml LBA media. The cultures were incubated at 37℃ and 200rpm, until they reached to the mid-logarithmic growth phase OD650nm of 0.4- 0.6, at which point isopropyl-1-thio-B-galacto-pyranoside (IPT

11、G) was added to a final con</p><p>　　3. Results and analysis </p><p>　　3.1 PCR amplification of the lipase gene </p><p>　　As shown in Fig. 1, the PCR oligonucleotide forward and rev

12、erse primers were utilized to amplify the entire lipase gene of bacterial strain B. pseudmallei 21bp upstream of the lipase gene to 14bp downstream of the lipase gene with EcoR I and BamH I restriction sites. The primers

13、 incorporated with BamH I and EcoR I restriction sites to facilitate the subsequent cloning of the lipase gene. To minimize the mutagenic effect of the PCR procedure we utilized cloned Pfu DNA polymerase as a proof readi

14、n</p><p>　　3.2 Purification of GST-Lipase B. pseudmallei fusion protein </p><p>　　As shown in Fig. 2, the purified GST-lipase fusion protein of B. pseudomallei appear to be homogeneous, yielding

15、 a strong homogenate protein band in polyacrylamide gel electrophoresis, which stained for protein. The apparent molecular weight of GST-lipase B. pseudomallei fusion protein is 58.0 kDa (Figure 5 lane 4). The molecular

16、mass of GST is 26 KDa and the molecular mass of B. pseudomallei lipase estimated to be 32 kDa. 　　3.3 Effect of temperature </p><p>　　The results are illustrated in Figure 7. It is apparent that the optimal

17、temperature for the free enzyme activity is recorded at 65 ℃. Any further increasing in the temperature beyond the optimal leads to a significant decrease in the enzyme activity. The optimum temperature for the immobiliz

18、ed lipase is shifted to higher temperature and recorded at 70℃ The optimum temperature for the immobilized enzyme is higher than that recorded with the free enzyme. </p><p>　　4. Discussion and conclusion <

19、;/p><p>　　The molecular mass of lipase purified from B. pseudomallei is similar to the molecular mass of lipases obtained from other bacterial strains as Acinetobacter sp. RAG.1 (33 kDa, Snellman et al., 2002),

20、 Pseudomonas sp. KWI.56 (33 kDa, Brune and Gotz 1992) and P. fluorescens AK 102 (33 kDa, Kojima et al., 1994). The molecular mass of lipases of B. pseudomallei is lower than that of the lipase protein purified from other

21、 microorganisms e.g. Bacillus sp.THLO27 (69 kDa, Dharmsthiti and Luchai 1999) an</p><p>　　[1] Abdou, A. M. (2003). Purification and partial characterization of psychrotrophic Serratia marcescens lipase. J Da

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24、crobial lipases. Enzyme and Microb Technol, 39:235.251. </p><p>　　[5] Kanjanavas, P., Khuchareontaworn, S., Khawsak, P., Pakpitcharoen, A., Pothivejkul, K., Santiwatanakul, S., Matsui,K., Kajiwara,T. and Cha

25、nsiri, K. (2010). Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2. Int J Mol Sci. 11(10): 3783–3792. </p><p>　　[6] Nawani, N., Singh, R

26、. and Kaur, J. (2006). Immobilization and stability of a lipase from thermophilic Bacillus sp.: The effect of a process parameters on immobilization of enzyme. Electro.J. Biotechnol., 9(5): 563.565. </p><p>

27、　　[7] Snellman, EA, Sullivan ER, Colwell RR (2002) Purification and properties of the extracellular lipase, Lip A, of Acinetobacter sp. RAG.1. Eur J Biochem 269:5771–5779. </p><p>　　[8] Rathi P, Saxena R. K,

28、 Gupta R. (2001). A novel alkaline lipase from Burkholderia cepacia for detergent formulation. Process Biochem 37:187–192. </p><p>　　[9] Surinenaite, B., Bendikiene, V., Juodka, B., Bachmatova, I., Marcinkev