Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y
One thousand and two hundred base pairs (bp) of open reading frame (ORF) encoding for an organic solvent tolerant lipase gene was cloned and expressed intra- and extracellularly. The intracellular expression was done using pBAD TOPO TA expression vector with 0.02% (vlv) of L-arabinose as optimum...
| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2005
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/5905/1/FBSB_2005_6%20IR.pdf |
| _version_ | 1825943950501871616 |
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| author | Sulong, Moohamad Ropaning |
| author_facet | Sulong, Moohamad Ropaning |
| author_sort | Sulong, Moohamad Ropaning |
| collection | UPM |
| description | One thousand and two hundred base pairs (bp) of open reading frame (ORF)
encoding for an organic solvent tolerant lipase gene was cloned and
expressed intra- and extracellularly. The intracellular expression was done
using pBAD TOPO TA expression vector with 0.02% (vlv) of L-arabinose as
optimum inducer after 4 h of incubation. at 37°C with an optimum lipase
activity of 0.5 Ulml. The extracellular expression was obtained by cotransforming
pJL3 expression vector encoding bacteriocin release protein
(BRP) into E. coli TOP10 harbouring the recombinant pBAD TOPO TA. The
secretory expression of recombinant organic solvent tolerant 205y lipase
increased the lipase activity tremendously to 2.5 Ulml.
The 205y lipase was purified to 8-fold and 32% recovery using two steps
purification, ultrafiltration and hydrophobic interaction chromatography (HIC).
The molecular mass of the purified 205y lipase revealed homogeneity on
SDS-PAGE at approximately 30 kDa.
The optimum pH for the purified 205y lipase was found at 7.0-8.0 and its
stability showed a broad range of pH value between pH 5.0 to pH 13.0 at 37
"C. The purified 205y lipase exhibited an optimum temperature of 55°C. The
lipase activity of the purified 205y lipase was enhanced in the presence of
alkaline metal such as (Na) and alkaline earth metal such as ( ~ g ~c'a,2 ' and
~ a ~ ' )H.o wever, the 205y lipase activity was inhibited in the presence of
transition metal ions, zn2', cu2' and ~ e ~ 'T.h e chelating agent,
ethylenediaminetetraacetic acid (EDTA), did not affect the purified 205y
lipase activity while serine hydrolase inhibitor, phenylmethane sulfonoyl
fluoride (PMSF), inhibited the lipase activity.
The activity of the purified 205y lipase demonstrated good stability in the
presence of methanol, p-xylene and n-decane with Dimethylsulfoxide
(DMSO) being the most stabilizing. The purified 205y lipase showed a
preference toward hydrolysing medium carbon chain length of triglycerides,
tricaprin (C10). The purified 205y lipase also exhibited 1,3- regiospecific
nature of the enzyme. |
| first_indexed | 2024-03-06T07:08:18Z |
| format | Thesis |
| id | upm.eprints-5905 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T07:08:18Z |
| publishDate | 2005 |
| record_format | dspace |
| spelling | upm.eprints-59052022-02-03T02:20:04Z http://psasir.upm.edu.my/id/eprint/5905/ Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y Sulong, Moohamad Ropaning One thousand and two hundred base pairs (bp) of open reading frame (ORF) encoding for an organic solvent tolerant lipase gene was cloned and expressed intra- and extracellularly. The intracellular expression was done using pBAD TOPO TA expression vector with 0.02% (vlv) of L-arabinose as optimum inducer after 4 h of incubation. at 37°C with an optimum lipase activity of 0.5 Ulml. The extracellular expression was obtained by cotransforming pJL3 expression vector encoding bacteriocin release protein (BRP) into E. coli TOP10 harbouring the recombinant pBAD TOPO TA. The secretory expression of recombinant organic solvent tolerant 205y lipase increased the lipase activity tremendously to 2.5 Ulml. The 205y lipase was purified to 8-fold and 32% recovery using two steps purification, ultrafiltration and hydrophobic interaction chromatography (HIC). The molecular mass of the purified 205y lipase revealed homogeneity on SDS-PAGE at approximately 30 kDa. The optimum pH for the purified 205y lipase was found at 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to pH 13.0 at 37 "C. The purified 205y lipase exhibited an optimum temperature of 55°C. The lipase activity of the purified 205y lipase was enhanced in the presence of alkaline metal such as (Na) and alkaline earth metal such as ( ~ g ~c'a,2 ' and ~ a ~ ' )H.o wever, the 205y lipase activity was inhibited in the presence of transition metal ions, zn2', cu2' and ~ e ~ 'T.h e chelating agent, ethylenediaminetetraacetic acid (EDTA), did not affect the purified 205y lipase activity while serine hydrolase inhibitor, phenylmethane sulfonoyl fluoride (PMSF), inhibited the lipase activity. The activity of the purified 205y lipase demonstrated good stability in the presence of methanol, p-xylene and n-decane with Dimethylsulfoxide (DMSO) being the most stabilizing. The purified 205y lipase showed a preference toward hydrolysing medium carbon chain length of triglycerides, tricaprin (C10). The purified 205y lipase also exhibited 1,3- regiospecific nature of the enzyme. 2005-11 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/5905/1/FBSB_2005_6%20IR.pdf Sulong, Moohamad Ropaning (2005) Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y. Masters thesis, Universiti Putra Malaysia. Lipase Bacillus sphaericus |
| spellingShingle | Lipase Bacillus sphaericus Sulong, Moohamad Ropaning Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title | Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title_full | Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title_fullStr | Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title_full_unstemmed | Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title_short | Expression, Purification And Characterization Of Organic Solvent Tolerant Lipase From Bacillus Sphaericus 205y |
| title_sort | expression purification and characterization of organic solvent tolerant lipase from bacillus sphaericus 205y |
| topic | Lipase Bacillus sphaericus |
| url | http://psasir.upm.edu.my/id/eprint/5905/1/FBSB_2005_6%20IR.pdf |
| work_keys_str_mv | AT sulongmoohamadropaning expressionpurificationandcharacterizationoforganicsolventtolerantlipasefrombacillussphaericus205y |