Team:SZ-SHD/Engineering
Engineering
Chitin is a stable natural polymer secreted by insects and many other organisms for protection and support. Such as the lining of insect pregut, which is plated with chitin to protect the soft tissue from harmful substances. It has been characterized that the presence of chitinase could degrade this chitin layer and reduce the growth rate of insects (i.e.: locusts) and eventually kill the individual. The lethality can be further enhanced with the aid of Bt toxins that disrupts the epithelial cells on intestines. Therefore, we introduce chitinase to our project, a type of enzyme that catalyst the hydrolysis of chitins to weakens and degrades this protective layer.
The first chitinase used is ifchit, an endochitinase with molecular mass ~46kDa that originates from insect pathogenic fungi, Isaria fumosorosea. Which we have codon-optimized the gene, and transformed into E. coli BL21 for expression.
In order to characterize the production of ifchit, we assembled the plasmid pET28a-ifchit1 (pSS4) which allows the expression to be regulated under pTac system. Hence, we initiate protein synthesis by adding isopropyl β-D-1-thiogalactopyranoside(IPTG) when OD 600 ~0.6
We first attempted the expression, under 37℃ for 10 hours with 1mM IPTG. Then SDS-PAGE has been performed to compare the protein composition before and after induction (detailed in our protocols and methods).
We use Ni-NTA purification to extract the desired chitinase by adsorbing its 6*His-tag. But the electrophoresis suggested that there are multiple peptides present in the elusion buffer, whereas the length of them varied little in repeated groups. Nevertheless, we evaluated the chitinase activity of bacterial lysate which a negligible value was returned:
These results suggested faulty expressions have occurred, whereas the causes are still mysterious to us. It could be assumed that the characterization of this ifchit would be successful if the temperature lowered below 16℃, but the yield won’t be high either. Therefore, we decided to express another chitinolytic enzyme with a prokaryotic origin: chitinase from Xenorhabdus nematophila (strain 19061), abbreviate as xncht.(~76kDa)
The expression condition used for xncht is 37℃ 4 hours with 1mM IPTG, which we have identified the band between 70 and 90 kDa result of SDS-PAGE:
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As characterized in the figure below, the a significant chitinase activity could be visualized for both homogenate of and T4 lysate of induced bacterial culture (IPTG+).
The result of chitinase activity analysis of different samples. Control: bacteria lysate of uninduced culture; homogenate: lysate of recombined strain (only pET28-xncht) by ultrasonic disruption; T4 lysate: culture supernatant of induced (IPTG+, arabinose+) co-transformed strain with both pET28a-xncht and pSB1C3-pBAD-T4 (mediate cell death when L-arabinose present). The result shows a significant degradation of colloidal chitin when incubate with bacteria lysate which suggests a decent amount of chitinase activity: 0.43μMh-1 for homogenate, 0.29 μMh-1 for T4 lysate.
Based on these results above, we completed our designed model: the bulk release of active chitinase as an insecticide.
Reference:
[1]: Huang, Z., Hao, Y., Gao, T., Huang, Y., Ren, S., & Keyhani, N. O. (2016). The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea. Applied microbiology and biotechnology, 100(12), 5491-5503.
