Team:Stuttgart/Keratinases

Results

Keratinases

BCA Assay

Protein concentration of supernatant and cell disruption of E.coli carrying different keratinase plasmids (KerP, KerUS and KerA in different vectors) was measured with BCA Assay method (see chapter methods). The figure 1 below shows the BSA standard curve (blank corrected, arithmetic average of triplicates). Table 7 and 8 show the results of sample measurements (OD values: blank corrected, arithmetic average of triplicates). The concentrations are calculated with the calibration curve (BSA standard curve). Expectetly protein concentration is much more higher in cell disruption than in supernatants. It is also noticeable that protein concentraion of E.coli wild type is very high compared to engineered cultures. Unfortunately we do not have OD values, but we explain it by a higher amount of wild type cells after cultivation.

Table: supernatant (dilution 1:25):

sample	OD562	concentration [µg/mL]
KerP psD1K3	0,194	3066,667
KerP psD1C3	0,195	3091,667
KerUS pet28	0,191	2991,667
KerUS psD1C3	0,207	3391,667
KerUS BB	0,226	3866,667
KerA pet28	0,194	3083,333
KerA psB1C3	0,213	3550,000
KerA BB	0,201	3241,667
wild type E.coli	0,248	4416,667

Table: cell disruption (dilution 1:25):

sample	OD562	concentration [µg/mL]
KerP psD1K3	0,514	11075,000
KerP psD1C3	0,611	13491,667
KerUS pet28	0,405	8358,333
KerUS psD1C3	0,285	5350,000
KerUS BB	0,385	7841,667
KerA pet28	0,482	10266,667
KerA psB1C3	0,585	12858,333
KerA BB	0,410	8475,000
wild type E.coli	0,615	13600,000

Figure 1: BSA standard calibration curve.

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Figure 1: : Extinction of Azo-keratine conversion over time of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3). Measurements of enzyme activity was performed at three timepoints (0h, 3h, 24h). Intracellular enzyme activity was measured.

Figure 1: : Extinction of Azo-keratine conversion over time of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3). Measurements of enzyme activity was performed at three timepoints (0h, 3h, 24h). Extracellular enzyme activity was measured.

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Figure 1: Intracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).

Figure 1: Extracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).

	Keratinases	Percentage extracellular protein
KerUS pet 28	26%
KerUS psB1C3	39%
KerA pet 28	23%
KerA psB1C3	22%
KerP psB1C3	22%
KerP psB1C3	19%

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Figure 1: Ratio extinction azokeratine/protein concentraion of intracellular keratinases over time. To see is extinction (440nm) of converted azo-keratine in relation to total protein concentration (c) of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).

Figure 1: Ratio extinction azokeratine/OD600 of extracellular keratinases over time. To see is extinction (440nm) of converted azo-keratine in relation to total protein concentration (c) of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).

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Figure 1: Digest of kerP plasmids

FÜLL MICH MIT WORTEN

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Figure 1:

SCHREIB WAS REIN :-)

Figure 1: Agaraose-gel electrophoresis of purified rose-PCR products.

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