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PULLG6 assay for the measurement of pullulanase employs a water soluble defined substrate, namely 4,6-O-benzylidene-4-nitrophenyl-6³-α-D-maltotriosyl-maltotriose (BPNPG3G3), coupled with the ancillary enzymes α-glucosidase and β-glucosidase. Upon hydrolysis of the substrate at the 1,6-α-linkage by pullulanase or limit-dextrinase, the released 4-nitrophenyl-β-maltotrioside is immediately hydrolysed to glucose and 4-nitrophenol by the concerted action of the α-glucosidase and β-glucosidase enzymes in the reagent mixture. The reaction is terminated and phenolate ions are developed by addition of dilute alkali. The absorbance is read at 400 nm and the value obtained correlates directly with pullulanase activity.

Colourimetric method for the determination of pullanase
or limit-dextrinase

Principle:
(pullulanase/limit-dextrinase)
(1) Benzylidene-G₃-(α-1,6)-G₃-β-PNP + H₂O → Benzylidene-G₃
+ G₃-β-PNP

(thermostable α-glucosidase and β-glucosidase)
(2) G₃-β-PNP + H₂O → D-glucose + PNP

(alkaline solution)
(3) PNP → phenolate ion (yellow colour)

Note: PNP = 4-nitrophenol

Kit size:                              100 assays
Method:                              Spectrophotometric at 400 nm
Total assay time:                10 min for pullanase preparations
                                           30 min for malt extracts containing
                                           limit-dextrinase
Detection limit:                   0.18 U/mL for pullulanase preparations
                                           (50-fold dilution)
                                           0.01 U/g for limit dextrinase in milled malt
Application examples:         Assay of microbial pullulanase preparations
                                           Measurement of limit-dextrinase in 
                                           malt extracts
Method recognition:            Novel method

Advantages

• High sensitivity
  
• Suitable for manual and auto-analyser formats
  
• No transglycosylation interference
  
• Very cost effective
  
• All reagents stable for > 1 year after preparation
  
• Very specific

• Simple format

• Standard included

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