Cloning and Expression of a Thermostable-α Glucosidase

Yeast is considered as a good host for large scale production of enzymes. This is the first report of α-glucosidase obtained from bacterial source to be expressed in yeast.Seven bacterial isolates were successfully obtained from water sample of Telaga Air Hangat, Langkawi. The optimum growth temperature for these bacterial isolates (L2,L3, L4, GBB1, SR 38, SR 40 and SR 96) was at 55oC. Screening using an α-MUG plate overlay method indicated that 4 out of 7 isolates gave positive α-glucosidase activity (L2, L3, L4 and GBB1). The highest activity was 1.47 U/mL at 55°C from sample L3. This isolate was identified using 16S rRNA as a universal primer and from the BLAST result, the isolate showed 99% similarity to Geobacillus stearothermophilus. The gene encoding α-glucosidase was isolated from this identified bacterium using degenerate primers. A complete gene sequence encoding α-glucosidase (~1.7 kb) was obtained by a DNA walking approach. This gene fragment was successfully cloned and expressed into Escherichia coli Top10 cells using pBAD and pTrcHis2@TOPO TA expression vectors. The intracellular α-glucosidase production by recombinant E. coli was increased 3.4-fold and 2-fold in pBAD and pTrcHis2 compared to the wild type isolate, respectively. The restriction enzymes (RE) based primers were designed to clone the gene into a yeast expression vector pPICZαA and to allow transformation into P. pastoris. Transformation was successfully achieved with the α-glucosidase expression level at 3.3 U/mL before optimization. After optimization, the highest activity obtained was ~10 U/mL. This is about 2-fold higher than the expression by E. coli and 6-fold higher than the wild type isolate. P. pastoris expression system was shown to be effective in increasing the expression yield of the heterologous protei

Engineering T1 lipase for degradation of poly-(R)-3-hydroxybutyrate

Enzymes with broad substrate specificities that can act on a wide range of substrates would be valuable for industrial applications. T1 lipase is known to have broad substrate specificity in its native form, with active site residues that are similar to polyhydroxylalkanoate (PHA) depolymerase (PhaZ). PhaZ6 from Pseudomonas lemoignei (PhaZ6Pl) is one of PhaZs that can degrade semicrystalline poly-(R)-3-hydroxybutyrate [P(3HB)]. The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. Structural analyses on PhaZ6Pl built structure revealed that it does not contain a lid, as opposed to T1 lipase. Therefore, T1 lipase were designed by removing its lid region. This was performed by using Bacillus subtilis lipase A (BSLA) as the reference for T1 lipase modification as the latter does not have a lid region and that its structure fits almost perfectly with T1 lipase based on their superimposed structures. A total of three variants of T1 lipase without lid were successfully designed, namely D1 (without α6–loop–α7), D2 (without α6) and D3 (α6 and loop) in the lid region. All the variants showed PHA depolymerase activity towards P(3HB), with D2 variant exhibiting the highest activity amongst other variants. Further analysis on D2 showed that it was able to maintain its native hydrolytic activity towards olive oil, albeit with decrement in its catalytic efficiency. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only perform triglyceride degradation but also P(3HB) degradation by simply removing the helix 6 which was specifically proven to affect catalytic activity and substrate specificity of the enzyme

Ability of T1 lipase to degrade amorphous P(3HB): structural and functional study

An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well