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1.method:
Will 2 ml 0.02 mol/l PH 5.0 acetic acid salt buffer makes up to 3% dextran 70 KDa 35 ¡ã C heat preservation solution in 10 min, add the liquid enzymes diluted properly 0.5 ml insulation 1 hr later, using 3, 5 two nitro salicylic acid (DNS) method for the determination of the generation of reducing sugar.  In the above conditions ,a Enzymes living unit (u) means every minute produce 1 umol reducing sugar (glucose equivalent) need enzyme quantity.
2.Detecting steps:
(1), sample with the control group
1, fermented liquid treatment: fermented liquid in condition 4000 RPM conditions centrifugal 10 min, draw on 1 ml clear liquid, with purified water dilute 20-400 times, remember as 1 # solution.
2, take 2 ml PH value of 5.0 acetic acid salt buffer made 3% dextran 70 KDa solution in 10 ml centrifugal liquid pipe, 35 ¡æ water bath 10 min, remember as 2 # solution.
3,draw  375 ul 3, 5 two nitro salicylic acid (DNS)  solution in the 20 ml than color dish with 1000 ul pipettor, buckle plug to spare, remember as 3# solution.
4,draw 0.5 ml liquor from 1# solution add to 2# solution, fast vibration well,  remember as  4 # solution.
5, draw 0.5 ml liquor from 4# solution add to 3# solution, remember as #5 solution.
6, 4# solution remaining at 35 ¡æ water bath 60 min, written as #6 solution.
7, 5# solution shake well and put into 100 ¡æ water bath, timing, 5 min enzyme liquid inactivated after processing,cooling in cold water immediately, then add 5375 ul purification water blending, and remember as 7# solution, as zero proofreading group. Set aside.
(2), fermentation sample processing
1,draw  375 ul 3, 5 two nitro salicylic acid (DNS)  solution in the 20 ml than color dish with 1000 ul pipettor, buckle plug spare (written for 8# solution).
2,draw  0.5 ml liquid from 6# solution into 8# solution, shake well and put  into 100¡æ water bath, timing 5 min enzyme liquid inactivated after processing, immediately into cold water sufficient cooling, add 5375 ul purification water blending (written for 9# solution).
(3), sample testing
Spectrophotometer open after 20 min in advance, will be rotated to 540 nm wavelength, 7# solution as control, 9# solution as samples, detection, record sample absorb light value, each sample determine three time absorb light value, take average.
(4), enzyme activity calculation
Formula: the enzyme live (u/ml) = (absorb light value / 0.6023) * diluted times / 60/180 * 1000 * 10
(5) formula explanation: diluted times is refers to the fermentation industry diluted times, diluted times depends on how much enzyme unit and can't let live enzyme solution liquid absorb light a value greater than 1; 0.6023 is a calculation constant; 60 is enzyme liquid reaction time; 180 is glucose molecular weight; 1000 is Moore and micro switch between Moore; 10 is 0.1 ml enzyme solution and 1 ml enzyme convert between liquid.
5Â¥2012-07-17 10:44:41
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