Method for detecting activity of beta-glucosidase in litters

A technology of glucosidase and litter, applied in the field of biochemistry, can solve the problems of more enzyme solution, less β-glucosidase, easy to be interfered, etc., and achieve the effect of optimizing the reaction system, reducing interfering substances and shortening the operation time.

Pending Publication Date: 2020-09-25
NANJING FORESTRY UNIV
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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

Method used

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  • Method for detecting activity of beta-glucosidase in litters
  • Method for detecting activity of beta-glucosidase in litters
  • Method for detecting activity of beta-glucosidase in litters

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: establish the method for detecting litter β-glucosidase activity

[0041] 1. Reagent preparation

[0042] 1M sodium hydroxide solution: 9.999g sodium hydroxide, dilute to 250mL with deionized water to prepare, store at room temperature.

[0043] Universal buffer stock solution: 3.025g Tris, 3.9g maleic acid, 3.5g citric acid (or 3.825g citric acid monohydrate), 1.56g boric acid, and 122mL sodium hydroxide (1M), dilute with deionized water to 250 mL was prepared.

[0044] Culture buffer: adjust 100mL universal buffer stock solution to the required pH value (pH 4.5) with hydrochloric acid, dilute to 1000mL with deionized water, and sterilize at 4°C for later use.

[0045] Stop buffer (Tris 1M; pH 10-11): 129 grams of Tris was added to 750 mL of deionized water, the deionized water was adjusted to 1000 mL, and stored at room temperature after autoclaving.

[0046] MUB fluorescent standard substance series solution: Weigh 8.8mg MUB and dissolve it into 1mL ...

Embodiment 2

[0059] Example 2: Detection of β-glucosidase activity in the litter of sweetgum pure forest and sweetgum black pine mixed forest

[0060] The litter used in this example was collected from the pure forest of sweetgum sweetgum and mixed forest of sweetgum black pine at the southwestern foot of Zijin Mountain (32°04′N, 118°50′E) in Nanjing City, Jiangsu Province. The collected liquidambar fallen leaves were killed at 105°C for 15 minutes, dried continuously at 80°C for 48 hours, and 20 g of dry weight was weighed and put into a 300-mesh (0.05mm pore size) 15cm*10cm nylon mesh bag. Nylon mesh bags containing dried sweetgum leaves were respectively buried in the pure sweetgum forest and the mixed sweetgum black pine forest, and covered with the original surface soil (0-10cm) about 1m away from the main trunk of the tree. After one year of burial, the semi-degraded litter was taken out, and impurities such as soil and sand were removed. Treatment 1 was the litter of the pure sweetg...

Embodiment 3

[0064] Embodiment 3: Detection of β-glucosidase activity of red eucalyptus plantation litter

[0065] The litter used in this example was collected from the 4-year-old and 9-year-old red eucalyptus plantations in the southern seedling base of Zhanjiang City, Guangdong Province (21°27'N, 110.11'E). The collected fallen leaves of red eucalyptus were killed at 105°C for 15 minutes, dried continuously at 80°C for 48 hours, and 15g of dry weight was weighed and put into a 300 mesh (0.05mm pore size) 15cm*10cm nylon mesh bag. Nylon mesh bags containing dried red eucalyptus leaves were respectively buried in 4-year-old and 9-year-old red eucalyptus plantations, and covered with the original surface soil (0-10cm) about 1m away from the tree trunk. After 4 months of burial, the semi-degraded litter was taken out, and impurities such as soil and sand were removed. Treatment 1 was the 4-year-old red eucalyptus plantation litter, and treatment 2 was the 9-year-old red eucalyptus plantatio...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34
CPCC12Q1/34G01N2333/942
Inventor 刘兵刘怡徐杰耿莉葛炎孙辉
Owner NANJING FORESTRY UNIV
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