Method for detecting activity of laccase in litter

A litter and activity technology, applied in the field of enzyme activity detection, can solve the problems of non-degrading microbial colonization of surface litter, large variation in calculation time, too long detection time, etc., and achieves cumbersome operation steps, improved sensitivity, Precise results

Active Publication Date: 2020-06-05
NANJING FORESTRY UNIV
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AI Technical Summary

Problems solved by technology

However, due to the complex environment such as the complexity of soil colloids and adsorption characteristics, the inhibition of laccase activity, the difficulty in grasping the substrate concentration, and the large variation in calculation time, and the determination of soil laccase activity uses a combination of enzyme activity and kinetics. Due to the long detection time (6-8 hours) caused by the measurement method, the current method for the determination of soil laccase activity is not suitable as a standardized operation procedure for the determination of litter laccase activity.
And because the composition of the original litter in the forest is complex: some surface litters may have no or few degrading microorganisms colonized, microorganisms secrete less extracellular laccase, and some litters may be in the final stage of degradation

Method used

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  • Method for detecting activity of laccase in litter
  • Method for detecting activity of laccase in litter
  • Method for detecting activity of laccase in litter

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: establish the method for detecting litter laccase activity

[0036] 1. Reagent preparation:

[0037] 1M sodium hydroxide solution: 9.999g sodium hydroxide, dilute to 250mL with deionized water to prepare, store at room temperature.

[0038] Universal buffer stock solution: Prepare with 3.025g Tris, 3.9g maleic acid, 3.5g citric acid (3.825g citric acid monohydrate), 1.56g boric acid, and 122mL sodium hydroxide (1M) to 250mL with deionized water .

[0039] Culture buffer: adjust 100mL of universal buffer stock solution to pH 4.5 with hydrochloric acid, dilute to 1000mL with deionized water, and store at 4°C after sterilization.

[0040] 1330 μM ABTS working solution: Weigh 73.17 mg ABTS and dissolve it in 100 mL culture buffer, store at 4°C in the dark for 4 weeks before use.

[0041] 2. Operation steps for detection of litter laccase activity:

[0042] 1) After the litter samples to be tested were cut into pieces and passed through a 2mm sieve, weigh 3...

Embodiment 2

[0051] The litter branches used in this example were collected from the pure sweetgum forest and the 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 branches with a diameter of 5 mm were killed at 105°C for 15 minutes, dried continuously at 80°C for 48 hours, and 7g of dry weight was weighed and put into a 300-mesh (0.050mm aperture) 10cm*8cm nylon decomposition bag. Nylon decomposition bags containing dried sweetgum branches 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 and compacted lightly. After 1 year of burial, the semi-degraded litter branches were taken out, and impurities such as sediment were removed. Treatment 1 was the litter branches of sweetgum pure forest, and treatment 2 was the litter branches of liquidamba...

Embodiment 3

[0064] The litter and dead branches used in this example were collected from the 4-year-old and 9-year-old Eucalyptus grandis plantations in the southern seedling base (21°27'N, 110°11'E) of Zhanjiang City, Guangdong Province. The collected dead branches of Eucalyptus grandis litter with a diameter of 5mm were killed at 105°C for 15min, dried continuously at 80°C for 48h, and 10g of dry weight was weighed and put into a 300 mesh (0.05mm aperture) 10cm*8cm nylon decomposition bag. The nylon mesh bags containing the dried dead branches of Eucalyptus grandis were respectively buried in the 4-year-old and 9-year-old Eucalyptus grandis plantations, covered with the original surface soil (0-10cm) about 1m away from the main trunk of the tree and compacted lightly. After 4 months of burial, the semi-degraded litter was taken out, and impurities such as sediment were removed. Treatment 1 was the litter branches of the 4-year-old Eucalyptus grandis plantation, and treatment 2 was the li...

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Abstract

The invention discloses a method for detecting activity of laccase in litter, and belongs to the technical field of enzyme activity detection. The method for detecting the activity of laccase in litter provided by the invention comprises the steps of weighing sieved litter chips into a centrifugal filter pipe, adding a culture buffer solution, incubating at a low temperature, performing centrifuging at a high speed, mixing enzyme filtrates, and adjusting the mixture to a certain volume; heating a diluted enzyme solution in the previous step under a dark condition to prepare a contrast inactivated enzyme solution; adding a to-be-detected diluted enzyme solution and a contrast inactivated enzyme solution into an enzyme activity hole and a negative hole respectively, then adding an ABTS substrate working solution, and carrying out shaking incubation at 22 DEG C under the dark condition; measuring the light absorption value OD under the wavelength of 420 nm; calculating a dilution factor F; and calculating the activity of laccase. The method has the advantages that the centrifugal filter pipe with small aperture is adopted, so that interfering substances in the enzyme solution are greatly reduced; after the reaction system is optimized, the treatment reaction conditions are consistent, the sensitivity is high, and the result is reliable; the result data is obtained in batches, theon-machine time is shortened, and the efficiency is high.

Description

technical field [0001] The invention belongs to the technical field of enzyme activity detection, and more specifically relates to a method for detecting laccase activity of litter. 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. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26G01N21/75G01N21/31
CPCC12Q1/26G01N21/75G01N21/31G01N2333/90232
Inventor 刘兵葛炎李小丽李世杰马阳孙辉
Owner NANJING FORESTRY UNIV
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