Characteristics and determination scheme of Worthington pectinase

AI Meijie Worthington  Purified pectinase is a multi-component preparation , It can efficiently depolymerize vegetable pectin with different degrees of esterification . This product contains a lot of hemicellulase 、 Cellulase 、 Pectin esterase and xylanase activity , It works with pectin lyase and polygalacturonase to digest plant cell wall tissue . When and  Worthington  When used with purified cellulase , Purified pectinase has been found in maize 、 soybean 、 Red beet 、 Sunflower 、 Several plant systems, such as tomatoes and citrus, have successfully produced high-yield living protoplasts . Generally speaking , The concentration range is  0.1%  to  0.5%  Pectinase of （ With  0.5%  to  1.5%  Cellulase ） stay  24°C  to  37°C  Next use  1  to  16  Hours will produce good results .

The main ：  

Pectin lyase  (4.2.2.10)

Polygalacturonase  (3.2.1.15)

Galactan  1,4-α- Galacturonidase  (3.2.1.67)

Secondary ：

Cellulase （ Endocellulase （3.2.1.4）、 Exocellulase （ 3.2.1.91） 、 Cellobiase （3.2.1.21））

Hemicellulase  (3.1.1.73)

Pectin esterase  (3.1.1.11)

Xylanase  (3.2.1.8)

AI Meijie Worthington  Characteristics of Pectinase from Aspergillus niger ：

In addition to its high enzyme activity , Purified pectinase is also specially used in plant protoplast culture . Use quantitative measurements D- Galacturonic acid is released from polygalacturic acid to determine enzyme . The method is based on the determination of reducing sugars by using neocuprine hydrochloride chromogenic reagent . When and  Worthington  When used with purified cellulase , Purified pectinase has been found to be useful in several plant systems （ For example, corn 、 soybean 、 Red beet 、 Sunflower 、 Tomatoes and oranges ） Live protoplasts that produce good yields in . Generally speaking , The concentration range is  0.1%  to  0.5%  Pectinase of （ With  0.5%  to  1.5%  Cellulase ） stay  24°C  to  37°C  Next use  1  to  16  Hours will produce good results .

Store :  Store in  2-8°C. Moisture-proof . If you don't use the whole bottle at one time , Weigh into one-time aliquots on arrival , And cover tightly 、 dry 、 Cold storage . The material is very hygroscopic , If exposed to moisture, it will become sticky and difficult to weigh .

stability / Store ： Moisture-proof . If you don't use the whole bottle at one time , Weigh into one-time aliquots on arrival , And in  2-8°C  Cover tightly and store dry . The material is very hygroscopic , If exposed to moisture, it will become sticky and difficult to weigh .

Unit definition ： A unit in  37°C、pH 5.0  Release from polygalacturic acid every minute  1µmole D- Galacturonic acid .

Technical specification ： Pectinase has J Strong water absorption ; Store dry to prevent moisture .

AI Meijie Worthington Pectinase Determination ：

Method ： stay  37°C,pH 5.0  Under the condition of , One unit is released from polygalacturic acid per minute  1 umole D- Galacturonic acid .

Reagents ：

0.1 M  Citric acid / Phosphate buffer （ Determine buffer ）,pH 5.0

Color reagent  A： take  40g  anhydrous  Na 2 CO 3 dissolution stay  600 ml  reagent grade  H 2 O  in . Join in  16 g  Glycine and stir until dissolved . Join in  0.450 gm CuSO 4 5H 2 O, Stir until dissolved . Use reagent grade  H 2 O  Add to  1  l .

Color reagent  B： take  1.2 g neocuprine - HCl  Dissolve in  1  l  H 2 O  Grade reagent . stay  4°C  Store it in a brown bottle .

D- Galacturonic acid standard ,1mg/ml

0.5%  Polygalacturonate substrate ：

Heat on a hot plate  500 ml  Test buffer . While heating and stirring , Slowly join  2.5 gm  Polygalacturonic acid . Heat and stir until dissolved . It will be a little sticky and opaque . Don't let the solution boil . If necessary, , Cool and restore volume . Store in  4°C.

enzyme ：

Fresh makeup every day . Put the solution on ice until it is used . Dilute continuously in the test buffer to  0.01 mg/ml.

Program ：

Prepare three test tubes , Each test tube contains  1 ml  Buffer and  6 ml  substrate （ Reagent blank ）. Prepare three test tubes , Each test tube contains  6 ml  Substrate and  1 ml 0.1 mg/ml  Enzyme samples （ test ）. Prepare three test tubes , Each test tube contains  6 ml  Buffer and  1 ml  The concentration is  0.1 mg/ml  Enzyme samples （ Sample blank ）.

Put the test tube in  37°C  Incubate in a water bath  60  minute ± 1  minute . Use  10 μg  to  125 μg  Of  D- Galacturonic acid standard is divided into standard curve tubes , triplicate .

Tube incubation  60  Minutes later , Immediately put the scaffold into ice water to stop the reaction . From each tube  100 μl  Sub assemble into another group of tubes （ Also on the ice ）. Add  2 ml Color Reagent A  and  2 ml Color Reagent B. Invert and mix well . Put the rack into a boiling water bath  13  minute ±1  minute . Cool the test tube , Then join  2ml H 2 O. Invert and mix well . Read with disposable cuvette  A 450 Absorbance with water blank . Do not use a flow cell ; Orange is attached to the glass . Calculate the average absorbance of each group in triplicate .

Calculation ：

Draw a standard curve ：

 

 

Calculate the rate of change . The curve should be linear , until  100 ug D- Galacturonic acid , If not , Repeat the curve with fresh reagent .