阅读说明：本技术 一种植物纤维原料酶水解糖化的方法 (Method for enzymatic hydrolysis and saccharification of plant fiber raw material ) 是由 戴红旗 周雪莲 卞辉洋 王瑞彬 徐婷婷 刘祝兰 杨益琴 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种植物纤维原料酶水解糖化的方法,属于生物质分离与转化技术领域。本发明方法包括：将生长发育早期的新鲜植物纤维原料经过机械处理获得碎解的植物纤维原料；以碎解的植物纤维原料为底物,在柠檬酸钠或醋酸钠的缓冲液中,加入纤维素酶等进行生物酶水解；由于生长早期的新鲜植物纤维的木质化程度低,木质素聚合程度也低,酶对纤维的可及性较高,加上合理的机械处理,使得本发明提供的方法可在不使用大量化学试剂的情况下促进植物纤维原料的酶水解糖化效率高达95.5％,同时得到富含原生木质素组分的固体渣可进一步进行高值化利用,绿色高效,应用推广价值高。(The invention discloses a method for enzymatic hydrolysis and saccharification of plant fiber raw materials, and belongs to the technical field of biomass separation and conversion. The method comprises the following steps: mechanically treating fresh plant fiber raw materials at the early growth and development stage to obtain disintegrated plant fiber raw materials; adding cellulase and the like into a buffer solution of sodium citrate or sodium acetate by taking the crushed plant fiber raw material as a substrate to carry out biological enzyme hydrolysis; due to the fact that the lignification degree of the fresh plant fibers in the early growth stage is low, the lignin polymerization degree is also low, the accessibility of the enzymes to the fibers is high, and reasonable mechanical treatment is added, the method provided by the invention can promote the enzymatic hydrolysis saccharification efficiency of the plant fiber raw materials to be as high as 95.5% under the condition that a large number of chemical reagents are not used, meanwhile, the obtained solid residues rich in the primary lignin components can be further utilized in a high-value mode, and the method is green, efficient and high in application and popularization values.)

1. A method for enzymatic hydrolysis and saccharification of plant fiber raw materials is characterized in that fresh tender plants with rich cellulose content are used as raw materials, after deactivation of enzymes, mechanical crushing and moisture balancing, buffer solution and enzyme are added for hydrolysis, and enzymatic hydrolysate is obtained through solid-liquid separation.

2. The method for enzymatic hydrolysis and saccharification of a plant fiber raw material according to claim 1, characterized by the specific steps of:

1) obtaining fresh tender plants with rich cellulose content, immediately deactivating enzyme, completely removing leaves and sheaths, and reserving stem parts;

2) adding water into the stalks and then mechanically crushing the stalks to obtain crushed stalks;

3) balancing water content of the crushed stalks, and adding a buffer solution and enzyme for hydrolysis;

4) and (4) performing solid-liquid separation on the hydrolysate to obtain enzyme hydrolysate.

3. The process of enzymatic hydrolysis saccharification of plant fiber feedstock as claimed in claim 1 or 2, wherein said fresh tender plant rich in cellulose is one or more of miscanthus, switchgrass, bamboo, corn stover and wheat straw.

4. The process for enzymatic saccharification of plant fiber feedstock as claimed in claim 1 or 2, wherein the fresh young plants rich in cellulose are grown for a period of 10 days to 6 months.

5. The process for enzymatic saccharification of plant fiber raw material as claimed in claim 1 or 2, wherein the specific conditions for deactivation of enzyme are: 105 ℃ for 20 min.

6. The process of enzymatic hydrolysis saccharification of plant fiber raw material as claimed in claim 1 or 2, characterized in that, the mechanical pulverization is one or more of breaking wall machine pulverization, pulping by Bulboro, ultramicron pulverization, disc mill, PFI mill, ball mill, twin screw extrusion.

7. The process according to claim 1 or 2, wherein the enzyme is a cellulase, xylanase, pectinase, arabinosidase or a combination thereof.

Technical Field

The invention belongs to the technical field of biomass separation and conversion, and particularly relates to a method for enzymatic hydrolysis and saccharification of a plant fiber raw material.

Background

With the rapid development of economy in China, the contradiction between energy supply and demand and the environmental problem will become increasingly serious. Therefore, the full, effective and scientific development and utilization of biomass energy is one of the ways for relieving the energy contradiction in China in the future. The development of biomass energy not only relieves the energy shortage and environmental pressure of China, but also can promote rural economy, promote the income increase of farmers and promote new rural construction, and meanwhile, the whole field of biomass energy is listed in an encouragement catalog by national development and improvement committee in this year.

Cellulose and hemicellulose in the lignocellulose raw material can be degraded into monosaccharide under the catalysis of biological enzyme, and the monosaccharide can be fermented by microorganisms to produce biological energy sources such as ethanol and butanol and high-valued platform compounds such as lactic acid and succinic acid. And the plant forms a complex cell wall structure in the growth and development process, the lignification degree of the fiber cell wall is increased and the fiber crystallinity is gradually improved along with the growth and development of the plant, the accessibility of the biological enzyme is reduced, so that the cell wall is difficult to be degraded by the enzyme, namely a 'biomass anti-degradation barrier', and the biorefinery difficulty is increased. Therefore, the biorefinery of the lignocellulose raw material needs to break the biomass degradation-resistant barrier through a pretreatment link, and the mainly adopted pretreatment technologies comprise dilute acid treatment, steam explosion, Ammonia Fiber Explosion (AFEX), low-temperature ammonia leaching treatment, high-temperature liquid water treatment, an organic solvent method, an alkaline hydrogen peroxide method and the like, wherein the AFEX is one of the most effective methods for breaking the biomass degradation-resistant barrier and is also the most economical and feasible physical-chemical pretreatment technology, but the AFEX pretreatment has a limited effect on the plant fiber raw material with high lignin content. In addition, most pretreatment methods hydrolyze cell wall polymers to a certain extent, the generated oligosaccharides or monosaccharides are dissolved in pretreatment liquid or washing liquid and run off along with the treatment process, and in addition, byproducts such as acetic acid, furan, phenols and furfural are generated from hemicellulose and lignin in the process, so that the metabolic activity of microorganisms is inhibited. Therefore, the search and development of pretreatment methods with low cost, high efficiency, less sugar loss and less inhibitors are still the direction of further industrialization of the bio-refining technology of plant fiber resources.

Because the pretreatment means reported at present usually has the defects of harsh conditions, use of toxic and harmful reagents and the like, the energy consumption is high, the environmental pollution is serious, and the subsequent microbial fermentation is inhibited, experts in the field of biology find that the enzymolysis sugar production efficiency of the cell wall is determined structurally by researching the relation between key structural factors of the cell wall and the efficient degradation of biomass, so that cell wall genetic modification engineering technologies are provided, such as strategies of reducing the lignin content of the cell wall, changing the composition and the structure of lignin monomers, modifying the lignin monomers, increasing the low-crystallinity cellulose content of the cell wall, reducing the high-crystallinity cellulose content of the cell wall and the like. However, by utilizing transgenosis and mutants, the cell wall structure and composition of the mutants are changed systematically, so that the normal growth and development of plants are influenced, the yield of seeds and biomass is greatly changed, the period is long, the cost is high, and no breakthrough progress is obtained.

Research and analysis suggest that along with the growth and development of plants, the lignification degree of fibers is continuously increased, the polymerization degree of lignin is higher and higher, and the accessibility of enzymes to the fibers is reduced; the cohesion of the microfibril layer of the fiber structure is stronger and higher, the crystallinity is higher and higher, and the enzymatic hydrolysis and saccharification are more and more difficult; in addition, after the fresh plants are air-dried, the cohesion of the intercellular layer of the fiber cells is enhanced and the tissues are keratinized due to dehydration, so that the accessibility of the cellulase is weakened; the method has the advantages that the biological refining must adopt a pretreatment means or a genetic modification engineering technology to reduce the lignin content, change the lignin structure and reduce the cellulose crystallinity, thereby increasing the accessibility of the biological enzyme and improving the enzymatic hydrolysis saccharification efficiency, not only the process is complex and the biological refining cost is increased, but also a large amount of waste water is generated and a new environmental problem is formed.

Therefore, there is a need for a green and efficient enzymatic hydrolysis and saccharification method for fresh plant fiber raw material, which can promote the enzymatic hydrolysis and saccharification efficiency of lignocellulose raw material without using a large amount of chemical reagents, and can further utilize the separated native lignin in high value.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for enzymatic hydrolysis and saccharification of plant fiber raw materials, which solves the problems that the existing cellulose enzymatic hydrolysis method is complex in process, uses a large amount of chemical reagents, generates a large amount of waste water and affects the environment and the like.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for enzymatic hydrolysis saccharification of plant fiber raw materials, comprising: fresh tender plants with rich cellulose content are used as raw materials, after deactivation of enzymes, mechanical crushing and moisture balancing, buffer solution and enzyme are added for hydrolysis, and enzyme hydrolysate is obtained through solid-liquid separation.

Further, the method for enzymatic hydrolysis and saccharification of the plant fiber raw material comprises the following specific steps:

1) obtaining fresh tender plants with rich cellulose content, immediately deactivating enzyme, completely removing leaves and sheaths, and reserving stem parts;

2) adding water into the stalks and then mechanically crushing the stalks to obtain crushed stalks;

3) balancing water content of the crushed stalks, and adding a buffer solution and enzyme for hydrolysis;

4) and (4) performing solid-liquid separation on the hydrolysate to obtain enzyme hydrolysate.

Further, the fresh tender plant rich in cellulose is one or more of miscanthus, switchgrass, bamboo, corn stalks and wheat stalks.

Furthermore, the fresh tender plant rich in cellulose has a growth time of 10 days to 6 months.

Further, the specific conditions of the fixation are as follows: 105 ℃ for 20 min.

Further, the mechanical crushing is one or a combination of more of wall breaking machine crushing, pulping by Bulboli, superfine particle crushing, disc milling, PFI milling, ball milling and double-screw extrusion.

Further, the enzyme is one or more of cellulase, xylanase, pectinase and arabinosidase.

Drawings

FIG. 1 is a diagram of fresh miscanthus after the leaves and sheaths are removed; in the figure, the left figure is the miscanthus comprising leaves and sheaths, the middle figure is the miscanthus with leaves and sheaths removed, and the right figure is the miscanthus with leaves and sheaths removed simultaneously;

FIG. 2 is a graph showing the comparison of the enzyme hydrolysis yield of fresh miscanthus sinensis at 96h before and after the fresh miscanthus sinensis is air-dried for 2, 4 and 6 months after growth and development; in the figure, FG: air-drying Chinese silvergrass, XX: fresh miscanthus;

FIG. 3 is a graph showing the results of producing gluconic acid and xylonic acid by using a whole-cell catalytic enzymatic hydrolysate of gluconobacter oxydans.

Detailed Description

The invention is further described with reference to specific examples.

The cellulase is Novoxil second-generation commercial enzyme (CTec2, Novozymes), is an enzyme mixture comprising cellulase, β -glucosidase and hemicellulase xylanase (CAS9025-57-4), pectinase (CAS9032-75-1), derived from aspergillus niger, available from michelin (shanghai) inc.