| dc.description.abstract | The extracellular expression of enzymes in an excellent secretion host such as
Bacillus subtilis is a useful strategy for making enzymes of industrial significance more
affordable by lowering the cost of its downstream processing. Lipases are a category
of hydrolases that catalyze the hydrolysis of triglycerides to glycerol and free fatty
acids. Also, lipases catalyze the hydrolysis and transesterification of other esters as
well as the synthesis of esters. They can perform very specific biotransformation
reactions and are widely used in food, organic synthesis, detergents, cosmetic, and
pharmaceutical industries. Geobacillus stearothermophilus T1.2RQ lipase is an
industrially-desired thermostable lipolytic enzyme with an excellent hydrolytic and
transesterification activity. This study aimed to express T1.2RQ lipase, in B. subtilis
WB800 and ensure its secretion into the extracellular medium. Three signal peptides
(AmyQ, Epr, LipA) and two promoters (Pgrac01 and Pgrac100) were used in the
secretory-expression of T1.2RQ lipase, based on five constructed expression vectors
constructed by restriction cloning and PCR mutagenesis. Sangers sequencing was used
to verify constructs to ensure they are in the correct reading frame.
Recombinants vectors, pHT43 (plasmid control), pHT43-T1.2RQ, pHT43-
T1.2RQ_AmyQ, pHT43-T1.2RQ_Epr, and pHT43-T1.2RQ_LipA, were transformed
by electroporation into Bacillus subtilis WB800. The electroporation was successful
with the optimum condition of 2500V, 200 ohms, 25µF, and 5milliseconds.
Recombinant B. subtilis pHT43 (negative control) and pHT43-T1.2RQlip + signal
peptides were streaked on the lipidic agar, LA+TBN. All four recombinants, including
the negative control (B. subtilis WB800-pHT43), produced clear zones on the TBN
agar plate after overnight incubation (fig 2), indicating the presence of a lipolytic
enzyme in all of the recombinant B. subtilis cells. pHT43-T1.2RQlip + signal peptides
(AmyQ, Epr, and LipA) have clear zones with significantly higher diameters than B.
subtilis WB800-pHT43 (negative control). This phenomenon suggests that the
recombinant thermostable lipase T1.2RQ introduced into these cells was successfully
expressed, thereby increasing the lipolytic capability of its host cells
T1.2RQ lipase was expressed in 50ml terrific broth medium (TB) and the
expression induced with 1mM IPTG. Lipase activity assay using p-nitrophenyl esters
showed that all three signal peptides directed the secretion of T1.2RQ lipase into the
extracellular medium. Signal peptide, LipA, resulted in the highest extracellular
activity of 5.6 U/mL, which corresponds to a 6-fold increase over the parent B. subtilis
WB800 strain. Meanwhile, signal peptides, AmyQ and Epr produced 4.2 U/mL and 3.4
U/mL activity respectively. SDS-Zymogram analysis confirmed that T1.2RQ lipase
was correctly processed and secreted in its original size of 44 kDa. An attempt to further
increase lipase expression level by promoter optimization showed that, contrary to
expectation, promoter Pgrac100 exhibited similar expression levels as the original
Pgrac01. Nevertheless, the demonstration of the successful secretion of T1.2RQ lipase
in this work confirms the latter as a promising approach for the industrial production
of the enzyme | id |
