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Results

Bioinformatic Design Results

Genes of the enzymes have been cloned to the pET29b expression vector between NdeI and XhoI restriction sites.

Creation of Recombinant DNA

The gene was cut from the enzyme restriction sites which are different from each other using appropriate enzymes. 1μL restriction enzyme was used. The expression vector, which has been used as backbone, also digested from the same restriction sites. Then the gene sequence is ligated with the backbone. The enzyme was added last on ice and an enzyme buffer that is suitable for both, was used.

First, we measured the mass of the gel. Then added Buffer GB at a 1:1 ratio, assuming a volume of 100μl per 0.1 gram. Next, we centrifuged the tube to ensure that the gel has descended into the Buffer. We incubated the gel at 50 °C until it melted and, for a better result, we inverted the tube from time to time. We passed the liquid to the Column in the collection tube that comes in the kit (We used GF-1 DNA Recovery kit). We centrifuged it at 10000 x g for 1 min. Then, we discarded the liquid in the collection tube. We added 650μl Wash Buffer to the colon, centrifuged at 10.000 rpm for 1 min. We discarded the liquid in the collection tube one more time.

We centrifuged the empty column one more time to remove any ethanol residue. Next, we did ligation. The mix contained: T4 DNA Ligase Buffer, Vector DNA, Insert DNA,T4 DNA Ligase and water. We mixed the reaction by shaking it up and down. For the sticky ends, we made an incubation process for 10 minutes at room temperature. It should have been deactivated by preserving it at 65 °C. Then we cooled it on ice. We ran the end product on agarose gel on convenient voltage and duration

Protein Expression

Induction was done with IPTG. This was done at a density of 30 μmoles and 50 μmol. The last concentration was 0,5 μmol. Right after IPTG, protein isolation was done. At the end of the isolation, we did a Bradford Analysis which determined the concentration of the enzyme
CelAB, EGII and EGII (C99V) protein concentrations were observed as 0.127, 0.1 and 0.102, respectively.

	1	2	3	4	5	6
A	Standart	Standart	Standart	Standart	Standart	Standart
B	CelAB	CelAB	CelAB	EGII	EGII	EGII
C	EGII C99V	EGII C99V	EGII C99V	-	-	-

Enzyme	Absorbance	Concentration (mg/mL)
CelAB	0.3726625	0.127356103179867
EGII	0.2968375	0.3006875
EGII (C99V)	0.100889210792698	0.102233062236029

Enzyme Activity Determination

To test the enzymes we conducted a CMCase activity analysis test. Briefly, we prepared standards that we know their glucose concentrations and measured their absorbance at 540 nm after DNS treatment.
Depending on the concentration-absorbance, we made graphs. We then measured the absorbance of the samples we treated with the enzymes. We calculated the amount of product we obtained with the enzymes by substituting the absorbance values we obtained in the equation we obtained from the standard graph. Then, by drawing the product formation graph against the dilution amount, we calculated from the graph equation how much product we could produce when we use the enzyme without dilution. We wrote the amount of product we obtained in milligrams in μmol. We have calculated the Enzyme Activity by taking into account the reaction time.

Different from the CMCase Enzyme Activity protocol we diluted 2 enzymes as 100-times and 1000-times.

For CelAB, we got 189,3 mg product yield. This quantity is equal to 1051 µmol product formation. The unit value is 17,5 U/min.
For EGII, we got 268,5 mg product yield. This quantity is equal to 1491 µmol product formation. The unit value is 24,85 U/min.
For EGII(C99V), as shown (Figure 4) we got 208,24 mg product yield. This quantity is equal to 1156 µmol product formation. The unit value is 19,26 U/min.

All of the enzymes got pretty good enzymes activities. Wild type EGII got the highest U/min activity and CelAB got the lowest activity with 17,5 U/min.

Even though we proved enzyme’s activities, the activity results can be enhanced with optimization of protein induction and substrate-enzyme treatment conditions.

Demonstration of CelAB, EGII and EGII C99V Enzyme Effects on Filter Paper

CelAB, EGII and mutant EGII C99V genes originating from the specified organisms were inserted into the pET29b(+) plasmid vector, and enzyme production was performed by inducing lactose in Escherichia coli BL21 host organism. CelAB is a cellulase type enzyme that breaks down the cellulose molecule, which is a polysaccharide, into monosaccharides such as β-glucose. EGII is a glucanase type enzyme and is also responsible for the depolymerization of cellulose, glucose is released as the end product. Glucose gives its highest absorbance in the wavelength range of 260-270 nm. Filter papers in the laboratory are used to see the effectiveness of enzymes that break down cellulose, since they are in cellulose structure. In the experiment, filter papers of equal size and approximately equal weight were placed in enzyme lysates with equal concentrations, the control group included the enzyme-free lysate solution (Table.1). The tubes were incubated for 72 hours at 50°C with shaking. The appearance of the tubes after 20 hours and 72 hours (after removing the filter papers) is shown in Figure.1 and Figure.2, respectively.

Table.1: Enzymes used in the experiment, their concentrations and filter papers

Enzymes Samples	Enzyme concentrations	Filter paper size	Filter paper weight
Control	-	1x6cm	37.9mg
CelAB	3 mg/ml	1×6 cm	35 mg
EGII	3 mg/ml	1×6 cm	39.6 mg
EGII (C99V)	3 mg/ml	1×6 cm	37.3 mg

Figure.1: Image of test tubes after 20 hours

Figure.2: Image of test tubes after 72 hours

In the qualitative determination, the depolymerization effect of the enzymes on the filter paper is observed by looking at the images of the tubes and the pieces of paper accumulated at the bottom.

In the quantitative determination, the pre- and post-experiment masses of the papers used as the substrate, the shredded paper masses accumulated in the tubes, and the glucose end product absorbance in the liquid in the tubes were measured using a spectrophotometer.

Spectrophotometer measurements were made triplicate at 260 and 270 nm wavelengths, the raw absorbance data are given in Table.2. The absorbance value of the released glucose depending on the samples is shown in Graph.1.

Samples	λ 1	Abs. 1	λ 2	Abs. 2
Blank 1	0,000	270	0,000	260
Control	0,036	270	0,055	260
Control 1	0,057	270	0,077	260
Control 2	0,050	270	0,067	260
CelAB	18,48	270	22,50	260
CelAB 1	20,31	270	22,50	260
CelAB 2	18,61	270	22,50	260
EGII	14,57	270	16,97	260
EGII 1	14,26	270	16,60	260
EGII 2	14,36	270	16,77	260
EGII C99V	16,47	270	19,10	260
EGII C99V 1	17,18	270	19,91	260
EGII C99V 2	16,99	270	19,66	260

As seen in the graph, the highest glucose absorbance and thus the highest glucose end product formation was observed in the treatment with CelAB enzyme. It can be said that CelAB has the highest cellulose degradation capacity among the three enzymes. EGII was found to have lower efficacy than CelAB. It was observed that the ability of the EGII C99V enzyme, which was obtained as a result of the mutation in the EGII enzyme, in cleaving glucose monomer from cellulose structures improved compared to wild type EGII and approached CelAB, as expected.

The before and after mass of filter papers used in the experiment were weighed. Weighing results are given in Table.3. Despite drying in an incubator at 50°C and room temperature for one week and in microwave for 20 seconds, the post-experiment mass of the papers was higher than before the experiment. It was observed that the solution and enzyme used in the experiment could not be completely removed from the papers by these drying methods.

Table.3: Masses of filter papers before and after the experiment

Samples	Pre-experimental mass of paper (mg)	Post-experimental mass of paper (mg)
Control	37,9	42,3
CelAB	35	44,4	0,055	260
EGII	39,6	45,2
EGII C99V	37,3	45,3

Despite the fact that the mass of the papers increased, the sedimentation masses formed by the shredded papers in the tube were compared, since fragmentation was clearly observed in the tubes. After the papers were taken from the test tubes, the tubes were centrifuged and the liquid part was discarded, so the mass of the precipitates was determined by comparing it with the mass of the empty tube. The results are given in Table.4 and Graph.2.

Table.4: Masses of tubes and sediments accumulating at the bottom before and after the experiment

Samples	Empty tube mass (g)	Tube mass after test (g)	Mass of precipitate (g)
Control	6,9306	6,9328	0,0022
CelAB	6,7575	6,8782	0,1207
EGII	6,9053	6,9686	0,0633
EGII C99V	37,3	45,3	0,0403

Graph.2: Masses of shredded paper precipitates according to samples

The precipitated paper masses plot gives proportionally consistent results with the spectrophotometer measurements for CelAB and EGII samples. Again, the highest degradation, that is, the highest enzyme activity, was observed in CelAB. Unlike the spectrophotometer results, the amount of paper degraded by the EGII C99V enzyme was the lowest. The reason for this is the possibility that, due to the mutation in the enzyme structure, it exhibited the behavior of cleaving the final product, glucose monomers, rather than completely physically breaking down the substrate compared to other enzymes. Another reason may be an experimental error that occurred while getting rid of the liquid portion after settling.