Method for in-situ determination of wood fiber biomass enzyme accessibility

Abstract

The invention belongs to the field of environmental biological energy and relates to a method for in-situ determination of cellulase accessibility of wood fiber biomass. The method comprises the following steps of: 1) marking general enzyme binding sites of a wood fiber biomass sample; 2) in-situ observing the distribution and the quantity of the general enzyme binding sites, and calculating to obtain the quantity of fiber globule fluorescent protein probes which are attached onto the surface of the sample; 3) releasing the general enzyme binding sites; 4) closing the sample obtained in the step 3 by using lignin attachment proteins, and marking the cellulase binding sites in the sample to obtain the distribution and the quantity of the cellulase binding sites and the quantity of the fiber globule fluorescent protein probes which are attached onto the surface of the sample; and 5) comparing results of the step 4 and the step 2, and evaluating the cellulase accessibility of the wood fiber biomass. The method provided by the invention has the advantages that the enzyme binding sites can be observed in situ and counted, visualization is realized and the enzyme accessibility of the wood fiber biomass can be intuitively evaluated.

Description

technical field

[0001] The invention belongs to the field of environmental bioenergy, and relates to a method for in situ measuring the cellulase accessibility of lignocellulosic biomass. Background technique

[0002] With the exacerbation of energy shortage, it is necessary to find alternative new energy sources. As a large agricultural country in my country, the annual solar energy fixed by lignocellulosic biomass is about 10 times the total energy consumption of human beings; therefore, lignocellulosic biomass will occupy an important position in the future energy structure. However, the lignin content of lignocellulosic biomass, the degree of polymerization, crystallinity, particle size, porosity and other physical and chemical composition and structure of lignocellulosic biomass largely limit the biotransformation and utilization of lignocellulosic biomass. Cellulase accessibility of a lignocellulosic biomass substrate refers to the amount of cellulase binding sites pe...

Examples

Embodiment 1

[0062] (1) Take a 6mm×4mm×1mm small fragment of dewaxed straw in a low-protein adsorption tube (dewaxed straw components: cellulose 38.44, hemicellulose 33.60, lignin 5.73, ash and others 22.23), add pH The value was 6.0, 500 μL of phosphate buffer solution with a concentration of 100 mM, and the initial concentration Et of cellulosomal fluorescent protein probe GFP-CBM3 was 4.0 μM / L, and labeled in the dark for 1.5 h at a constant temperature of 55 °C.

[0063] (2) In situ observation and analysis of dewaxed rice straw samples using laser confocal microscopy (LCSM, Leica TCS SP5) and fluorescent microplate reader, to obtain the total enzyme binding site distribution and cellulosomal fluorescent protein probe adsorption amount of the sample ; The distribution of the total enzyme-binding sites is shown in Figure 1(b), with an average fluorescence value of 34.6; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E fa 2.7...

Embodiment 2

[0068] (1) Take a 3mm×8mm×200μm small fragment of pretreated straw in a low-protein adsorption tube (pretreated straw components: cellulose 50.04, hemicellulose 5.74, lignin 14.45, ash and others 24.77), add pH The value was 5.0, 1 mL of sodium citrate buffer with a concentration of 50 mM, and the initial concentration of cellulosomal fluorescent protein probe GFP-CBM3 was 2.0 μM / L, and labeled in the dark for 2.0 h at a constant temperature of 15°C.

[0069] (2) The measurement method is as described in step (2) in Example 1. The distribution of the total enzyme binding sites is shown in Figure 1(d), with an average fluorescence value of 31.8; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E fa 0.90μM / L, E a is 1.10 μM / L (calculated using the Langmuir equation), and the adsorption amount of the cellulosomal fluorescent protein probe per unit area of ​​the sample is E ua 4.58×10 4 μM / m 2 ;

[0070] (3) Add 0.5...

Embodiment 3

[0074] (1) Take a 5mm×5mm×100μm small fragment of pretreated straw in a low-protein adsorption tube (pretreated straw components: cellulose 69.30, hemicellulose 21.24, lignin 1.52, ash and others 7.80), add pH The value was 7.0, 200 μL of phosphate buffer solution with a concentration of 200 mM, and the initial concentration Et of cellulosomal fluorescent protein probe YFP-CBM was 8.0 μM / L, and labeled in the dark for 0.5 h at a constant temperature of 60 °C.

[0075] (2) In situ observation and analysis of dewaxed rice straw samples using laser confocal microscopy (LCSM, Leica TCS SP5) and fluorescent microplate reader, to obtain the total enzyme binding site distribution and cellulosomal fluorescent protein probe adsorption amount of the sample ; The distribution of the total enzyme-binding sites is shown in Fig. 1(f), with an average fluorescence value of 142.4; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E f...

technical field

[0001] The invention belongs to the field of environmental bioenergy, and relates to a method for in situ measuring the cellulase accessibility of lignocellulosic biomass. Background technique

[0002] With the exacerbation of energy shortage, it is necessary to find alternative new energy sources. As a large agricultural country in my country, the annual solar energy fixed by lignocellulosic biomass is about 10 times the total energy consumption of human beings; therefore, lignocellulosic biomass will occupy an important position in the future energy structure. However, the lignin content of lignocellulosic biomass, the degree of polymerization, crystallinity, particle size, porosity and other physical and chemical composition and structure of lignocellulosic biomass largely limit the biotransformation and utilization of lignocellulosic biomass. Cellulase accessibility of a lignocellulosic biomass substrate refers to the amount of cellulase binding sites pe...

Examples

Embodiment 1

[0062] (1) Take a 6mm×4mm×1mm small fragment of dewaxed straw in a low-protein adsorption tube (dewaxed straw components: cellulose 38.44, hemicellulose 33.60, lignin 5.73, ash and others 22.23), add pH The value was 6.0, 500 μL of phosphate buffer solution with a concentration of 100 mM, and the initial concentration Et of cellulosomal fluorescent protein probe GFP-CBM3 was 4.0 μM / L, and labeled in the dark for 1.5 h at a constant temperature of 55 °C.

[0063] (2) In situ observation and analysis of dewaxed rice straw samples using laser confocal microscopy (LCSM, Leica TCS SP5) and fluorescent microplate reader, to obtain the total enzyme binding site distribution and cellulosomal fluorescent protein probe adsorption amount of the sample ; The distribution of the total enzyme-binding sites is shown in Figure 1(b), with an average fluorescence value of 34.6; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E fa 2.7...

Embodiment 2

[0068] (1) Take a 3mm×8mm×200μm small fragment of pretreated straw in a low-protein adsorption tube (pretreated straw components: cellulose 50.04, hemicellulose 5.74, lignin 14.45, ash and others 24.77), add pH The value was 5.0, 1 mL of sodium citrate buffer with a concentration of 50 mM, and the initial concentration of cellulosomal fluorescent protein probe GFP-CBM3 was 2.0 μM / L, and labeled in the dark for 2.0 h at a constant temperature of 15°C.

[0069] (2) The measurement method is as described in step (2) in Example 1. The distribution of the total enzyme binding sites is shown in Figure 1(d), with an average fluorescence value of 31.8; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E fa 0.90μM / L, E a is 1.10 μM / L (calculated using the Langmuir equation), and the adsorption amount of the cellulosomal fluorescent protein probe per unit area of ​​the sample is E ua 4.58×10 4 μM / m 2 ;

[0070] (3) Add 0.5...

Embodiment 3

[0074] (1) Take a 5mm×5mm×100μm small fragment of pretreated straw in a low-protein adsorption tube (pretreated straw components: cellulose 69.30, hemicellulose 21.24, lignin 1.52, ash and others 7.80), add pH The value was 7.0, 200 μL of phosphate buffer solution with a concentration of 200 mM, and the initial concentration Et of cellulosomal fluorescent protein probe YFP-CBM was 8.0 μM / L, and labeled in the dark for 0.5 h at a constant temperature of 60 °C.

[0075] (2) In situ observation and analysis of dewaxed rice straw samples using laser confocal microscopy (LCSM, Leica TCS SP5) and fluorescent microplate reader, to obtain the total enzyme binding site distribution and cellulosomal fluorescent protein probe adsorption amount of the sample ; The distribution of the total enzyme-binding sites is shown in Fig. 1(f), with an average fluorescence value of 142.4; the concentration of free cellulosomal fluorescent protein probes in the supernatant of the labeling solution E f...

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