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=== Experimental Data and Discussion === | === Experimental Data and Discussion === | ||
[[Image:Absorbancelongph7cold.png|500px|left|]] | [[Image:Absorbancelongph7cold.png|500px|left|Absorbance at 405nm of a PNPA+Protein solution with pH = 7 and sodium chloride in a cold environment.]] | ||
50uL of purified protein from the first elution was added to 3mL of cold 1mg/mL PNPA at pH=7.30. Chloride ion cofactor was provided by the 2M HCl. A change in absorbance at 428.7nm over time was noted. The time range of the run did not produce a linear response. The absorbance increased for the first ten minutes and plateaued. The time 0 to 10 minutes was used to create the third figure with a more refined linear range. | 50uL of purified protein from the first elution was added to 3mL of cold 1mg/mL PNPA at pH=7.30. Chloride ion cofactor was provided by the 2M HCl. A change in absorbance at 428.7nm over time was noted. The time range of the run did not produce a linear response. The absorbance increased for the first ten minutes and plateaued. The time 0 to 10 minutes was used to create the third figure with a more refined linear range. | ||
[[Image:Absorbancecoldph7try.png|500px|left|]] | [[Image:Absorbancecoldph7try.png|500px|left|A trendline showing the potential relationship between absorbance and time from the data gathered from the PNPA+Protein solution with pH = 7, sodium chloride, and a cold environment.]] | ||
A linear equation of y=9.275x10^-7x+1x10^-4 with a coefficient of determination of 0.8492 was calculated using linear regression. | A linear equation of y=9.275x10^-7x+1x10^-4 with a coefficient of determination of 0.8492 was calculated using linear regression. | ||
[[Image:Tablebeerlaw.png| | [[Image:Tablebeerlaw.png|500px|left|thumb|Beer-Lambert Law sample calculation]] | ||
[[Image:Concentrationcoldmolarabsorb12000ph7.png|500px|left|thumb|]] | [[Image:Concentrationcoldmolarabsorb12000ph7.png|500px|left|thumb|A trendline showing the potential relationship between time and product concentration from the data gathered from the PNPA+Protein solution with pH = 7, sodium chloride, and a cold environment.]] | ||
Absorbance values can be transformed to units of concentration via the Beer-Lambert law. We must accept the approximation of the Molar Extinction Coefficient for PNPA hydrolysis at 428.7nm as 12000 M^-1 cm^-1. An example calculation is supplied in the table. Graphing time versus concentration and determining the slope of the line yields the enzyme's velocity in M/min. 1mg/mL of PNPA is saturating conditions which implies the Vmax is also the slope. The reaction volume total times Vmax yields Units of Enzyme Activity. This value can be used as a relative comparison tool for enzyme performance in given conditions. | Absorbance values can be transformed to units of concentration via the Beer-Lambert law. We must accept the approximation of the Molar Extinction Coefficient for PNPA hydrolysis at 428.7nm as 12000 M^-1 cm^-1. An example calculation is supplied in the table. Graphing time versus concentration and determining the slope of the line yields the enzyme's velocity in M/min. 1mg/mL of PNPA is saturating conditions which implies the Vmax is also the slope. The reaction volume total times Vmax yields Units of Enzyme Activity. This value can be used as a relative comparison tool for enzyme performance in given conditions. | ||
[[Image:Iceenzymeefficiencyscinotation.png|600px|left|thumb|Enzyme Activity at neutral pH at cold temperature with Chloride cofactor]] | [[Image:Iceenzymeefficiencyscinotation.png|600px|left|thumb|Enzyme Activity at neutral pH at cold temperature with Chloride cofactor.]] | ||
The cold 4 degrees C reaction was changed to room temperature 20 degrees C. All other conditions remained constant to evaluate the effect of temperature on enzyme. Temperature increase negatively affects enzyme performance. Considering the cold loving nature of Chitinophaga pinensis, the enzyme being more active at a lower temperature is a reasonable conclusion. | The cold 4 degrees C reaction was changed to room temperature 20 degrees C. All other conditions remained constant to evaluate the effect of temperature on enzyme. Temperature increase negatively affects enzyme performance. Considering the cold loving nature of Chitinophaga pinensis, the enzyme being more active at a lower temperature is a reasonable conclusion. | ||
[[Image: | [[Image:Roomtempconcentrationtime.png|600px|left|thumb|Enzyme Activity at neutral pH at room temperature with Chloride cofactor.]] | ||
[[Image: | [[Image:Sigfigroomtempenzymeactivity.png|600px|left|thumb|Enzyme Activity at neutral pH at room temperature with Chloride cofactor.]] | ||
[[Image: | [[Image:Enzyme units percentage increase.png|600px|left|thumb|4 degrees C yields 42.2% increase in Units of Enzyme Activity цmol/minute.]] | ||
=== Conclusion === | |||
=== | |||
42.2% increase in units of enzyme activity was a considerable gain. No replicates were done for this study. A future experiment would include replicates so t-test can be run to determine significance of the increase. Overall the temperature decrease had an effect on the conversion of PNPA to PNP. | 42.2% increase in units of enzyme activity was a considerable gain. No replicates were done for this study. A future experiment would include replicates so t-test can be run to determine significance of the increase. Overall the temperature decrease had an effect on the conversion of PNPA to PNP. | ||
