Studies On The Determination Of ?-glucanase Activities In Matrixes

?-glucan is a kind of natural synthetic polysaccharide,which belongs to structural polysaccharide in plant cell wall with D-glucose as its basic unit.?-glucan has a linear molecularnh structure,and present in the aleurone layer and endosperm cells of cereal.?-glucan contains ?-1,3 and ?-1,4 glycosidic bonds,the former causes the irregular sort of cellulose molecule,making these substances have some differences in the physical,chemical properties,and water-soluble.At low concentration,the interaction between B-glucan and water molecules increases the viscosity of the solution.With the increasing of its concentration,the ?-glucan molecules itself formed a netted structure,causing greatly increased of viscosity,and it may form a gel when it reaches a certain level.B-glucanase plays an improtant role in hydrolyzing ?-glucan,which enables it to be degraded to low molecular fragments,lose its hydrophilicity and viscosity.Therefore,?-glucanase obtains a extensively used for food industry,such as the sugar industry,and for fermentation industry,such as beer mashing,beer wort production,and for animal feed industry.However,there is a big problem in application and circulation:inconsistent units of enzyme activity.At present,the method of DNS(3,5-dinitrosalicylic acid)is widely used for determining the activity of ?-glicanase.But the lower sensitivity and higher substrate concentration(usually 1%),resulting in higher costs,and it can not measure the initial velocity.Also,it produces inconsistent oligosaccharide molar absorption coefficient,causing a nonlinear curve of enzyme concentration.The method of MBTH(3-Methyl-2-benzothiazolinonehydra-zone)not only has a high sensitivity,but also requires low substrate concentration,what is more,it can determine the trace enzyme activity.So,it can be widely used in animal feed,food,medicine,textile,plant pathogenic protection,and so on.This paper includes the following researches:1.Establishment of a novel method for the determination of ?-glucanase activityFirstly,we selected the appropriate substrate for different enzyme,and studied the preparation of substrate solutions.And then,we researched the effects of substrate on determining enzymes activity.Lastly,we confirmed the parameters of enzymatic reaction.The results show that:the value of Km is small,so the determination is more accurate and reliable,and can significantly reduce the substrate concentration,thus saving the cost.This method has high precision and sensitivity,and there is no interference from reducing agents on this method,and can be used to determine trace enzyme activity.2.Comparision of these methodsWe compared the currently common methods on the market,such as DNS?S-N,and the new method MBTH,mainly from the recovery rate?detection limit and so on.The result shows that:The shortcomings of S-N are troublesome performance,low sensitivity,and so on.The method of DNS can not determine the initial velocity.The method of MBTH makes up the deficiencies of the two method,and costs reduction.So,it will be widely used for many field.3.application of the novel method to activities assay in the matrixesIn this subject,the substrates mainly were animal feed solution and the extraction of sea cucumber body wall.In this part,there were three steps for obtaining feed solution:dissolved,magnetic stirred(at 4?),centrifuged and so on.About the extraction of sea cucumber body wall:homogeneitied,extracted in buffer,salted-out,dialysis and so on.4.Adaptation of the novel method to the microplate formatFirstly,combined the MBTH and the means of microplate to determine the reducing sugar standard curve;Secondly,established the standard curve of the concentration of B-glucanase;Finally,applied this method to determine enzyme activity in animal feed.The result showed that:the sensitivity was slightly lower than the means of test tube,but the slope of standard curve of ?-glucanase concentration was basically the same with test tube method;the Q1 and D1 was lower,so can be used to determine the trace enzyme activity;the method could deal with a large of samples once,and required less reagent,achieved a high throughput analysis as well as reduced significantly costs.