Method for detecting activity of beta-glucosidase in litters

Abstract

The invention discloses a method for detecting the activity of beta-glucosidase in litters, and belongs to the technical field of biological chemistry. The method for detecting the activity of the beta-glucosidase in the litters, which is provided by the invention, comprises the main steps of: weighing the litters which are cut up and sieved, placing the litters into a centrifugal filtration pipe,adding culture buffer solution to carry out incubation, after high-speed centrifugation, mixing supernate, regulating a volume of the obtained product to a certain volume and using the obtained product as enzyme activity to-be-detected solution; respectively adding the enzyme activity to-be-detected solution and inactivated enzyme activity to-be-detected solution into a sample activity hole and anegative control hole, then adding fluorogenic substrate working solution 4-MUB-beta-D-glucoside solution, and carrying out oscillation incubation under the dark condition; then adding Tris stop solution into an enzyme-labeled hole; carrying out fluorescence detection by adopting a multifunctional enzyme labeling instrument; and calculating the activity of the beta-glucosidase. The method has theadvantages that after a reaction system is optimized, sensitivity is high, a volume of the to-be-detected enzyme solution consumed by detection is small, operation time is shortened, and mass measured data can be acquired in short time; and the method is simple to operate and high in efficiency.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and more specifically relates to a method for detecting the activity of litter β-glucosidase. Background technique [0002] Litter decomposition is the basis of forest soil material transformation, the main source of plant and microbial nutrients, and plays an important role in maintaining carbon and nutrient cycles in forest ecosystems. Microorganisms are the main contributors to the decomposition of forest litter. During the process of litter degradation, microorganisms colonized on litter will secrete various extracellular enzymes, which will change the composition and structure of litter, thereby promoting the degradation of litter. As the most important biologically active substance involved in litter decomposition, enzyme activity involves various biochemical processes, is directly related to litter decomposition, and largely reflects the nutrient cycle status of soil C, N, P, etc. Va...

AI Technical Summary

Problems solved by technology

[0004] The determination of β-glucosidase activity in litter in my country is an extension of the soil enzymatic method, but the research on the determination of β-glucosidase activity in forest litter has extensive and single methods, derailment from international advanced methods, and experimental equipment. There are many problems such as slow replacement and replacement of β-glucoside activity with total cellulase activity

Wherein with salicin as the Barush and Swiain method of substrate, the enzymatic hydrolysis product is made chromogen with 4-aminoantipyrine, then uses spectrophotometric colorimetric determination, but reaction is easily disturbed, and detection sensitivity is low; The colorimetric method using phenyl-β-glucoside as the substrate for enzymatic hydrolysis is widely used. Although this method is simple to operate and has good reproducibility, it is susceptible to interference, consumes too much enzyme solution, and takes too long overall

Moreover, due to the complex composition of the original litter in the forest: there may be no or few degrading microorganisms in the surface litter, the microorganisms secrete less extracellular β-glucosidase, and some litters may be in the final stage of degradation

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