Part:BBa_K2783021
Endoglucanase II
Overview
Endoglucanase II from Trichoderma reesei (EGII) cleaves the beta 1-4 bond between glucose monomers in a cellulose polymer, creating two shorter polymers (Figure 1) [1]. This EGII construct includes a dockerin domain, allowing to adhere to an cohesin domain in for example the CipA3 scaffold used by the 2018 Groningen IGEM team. A His6-tag was added at the N-terminus of the sequence to allow easy purification.
Figure 1: Cellulose hydrolysis by EGII. EGII hydrolyzes a beta 1,4 bond in the middle of a long cellulose polymer, producing two shorter polymers.
Experiments
HisEGII was cloned into pSB1C3 in front of a T7 promoter and introduced into E.coli BL21 (DE3 star). Expression was induced for 2.5 hours by adding 1 mM final concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) during exponential growth. Cells were then harvested and disrupted via french press.
After cell disruption, HisEGII was purified using nickel sepharose affinity chromatography. Figure 2 shows a western blot using anti-His antibodies. It is clear that the enzyme expressed well and was soluble, however despite binding well to the nickel beads the purification was not optimal as evident from the low concentration of enzyme in the last two lanes.
Figure 2: Western blot of different HisEGII purification fractions. From left to right: pellet, soluble fraction, soluble fraction mixed with nickel beads, flow through, wash fraction 1 and 2, elution fraction 1 and 2.
To test the activity of EGII we used an enzymatic assay developed at the university of Groningen to measure degradation products of cellulose [2]. The assay uses a mutant Chito-oligosaccharide oxidase that specifically oxidizes the C1 hydroxyl moiety of cellulose degradation products. This leads to the equimolar formation of H2O2, which can be used by horseradish peroxidase to produce a measurable color. Three enzyme samples were used: EGII bought from megazyme, HisEGII, HisEGII diluted 1000 times, HisEGII diluted 10000, cellulase blend bought from sigma and negative control without cellulase. These were incubated with 4 different substrates: ReCell (cellulose from toilet paper reclaimed from sewage), ReCell that was ball milled to mechanically reduce the cellulose fibers to an amorphous structure, microcrystalline cellulose and phosphorylated cellulose. Figure 3 shows the results of this assay. It is clear that HisEGII exhibits activity towards ball milled ReCell, microcrystalline cellulose and phosphorylated cellulose but not raw ReCell. This indicates that preprocessing of the substrate is important for enzyme activity. It confirms however that ReCell is viable as a substrate for this enzyme after it has been reduced to an amorphous structure.
Figure 3: Colorimetric assay for cellulose hydrolysis. A pink color signifies more hydrolysis products. 6 different enzyme samples were tested: EGII bought from megazyme, our own HisEGII, HisEGII diluted 1000 times, HisEGII diluted 10000 times, a cellulase blend bought from sigma (produced by novozymes) and a negative control without any cellulase. Every row was fed with a different substrate: ReCell, ball milled ReCell, microcrystalline cellulose and phosphorylated cellulose. It is clear from this assay that HisEGII indeed has some activity towards ball milled ReCell, microcrystalline cellulose and phosphorylated cellulose but not raw ReCell.
References
[1] Medve, J., Karlsson, J., Lee, D., & Tjerneld, F. (1998). Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: Adsorption, sugar production pattern, and synergism of the enzymes. Biotechnology and Bioengineering. https://doi.org/10.1002/(SICI)1097-0290(19980905)59:5<621::AID-BIT13>3.0.CO;2-C
[2] Ferrari, A. R., Gaber, Y., & Fraaije, M. W. (2014). A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection. Biotechnology for Biofuels. https://doi.org/10.1186/1754-6834-7-37
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 244
Illegal AgeI site found at 1009 - 1000COMPATIBLE WITH RFC[1000]
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