CN114703164A

CN114703164A - Efficient cellulase inducer and preparation and application methods thereof

Info

Publication number: CN114703164A
Application number: CN202210201654.5A
Authority: CN
Inventors: 刘晨光; 邓林; 白凤武
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-07-05

Abstract

The invention discloses a high-efficiency cellulase inducer and preparation and application methods thereof, and relates to the field of bioengineering and biotechnology. The high-efficiency cellulase inducer is a mixture ACM of disaccharide and poly-oligosaccharide, wherein the disaccharide comprises sophorose and/or gentiobiose and/or cellobiose. Preparing the high-efficiency cellulase inducer by taking glucose or syrup containing the glucose as a substrate and catalyzing the chemical reaction of the glucose through acid; the high-efficiency cellulase inducer is applied to high-efficiency production of cellulase. The preparation of the ACM only needs sulfuric acid as a catalyst, has short preparation time and simple process, greatly reduces the investment of production equipment and reduces the production cost of the cellulase. And the cellulase with high enzyme activity can be directly used for hydrolyzing cellulose components in the refining process of the lignocellulose biomass. Thereby further reducing the utilization cost of the lignocellulose.

Description

Efficient cellulase inducer and preparation and application methods thereof

Technical Field

The invention relates to the field of bioengineering and biotechnology, in particular to a high-efficiency cellulase inducer and a preparation and application method thereof.

Background

The development and utilization of traditional fossil energy provides possibility for the rapid development of modern society and industry. However, under the rapid development of society, the resource shortage and environmental pollution problems caused by fossil energy are becoming more serious. Therefore, in order to solve the problems of environmental pollution and resource shortage, human beings must develop new sustainable energy. Bioenergy is a new type of energy that is considered to replace fossil energy. The biological energy source is renewable and environment-friendly, and simultaneously, the carbon neutralization macro target can be realized in the production process of the biological energy source. Therefore, the method has great potential for producing biological energy by using renewable raw materials such as lignocellulose and the like, such as corn straws and the like. The corn straws are cheap and have huge yield, and if the energy substances such as bioethanol and the like can be produced by using the corn straws as raw materials, the problems caused by fossil resources can be relieved to a great extent or even solved.

Unfortunately, the production of bioenergy or other bio-based products from lignocellulosic feedstock has not been industrialized to date, due to the many steps involved in converting the lignocellulosic feedstock into fermentable sugars. Firstly, the lignocellulose raw material with a compact structure needs to be subjected to pretreatment to loose the structure, then the pretreated material needs to be subjected to enzymolysis by cellulase to form fermentable sugar, and finally the fermentable sugar is used for producing biofuel or other biological base products. The numerous steps result in the production of lignocellulosic ethanol at a cost that is difficult to achieve industrially. In total cost, the enzymatic hydrolysis step accounts for about 30%, so the high cost of the enzymatic hydrolysis process has been a bottleneck for the industrial utilization of lignocellulose. Therefore, only the development of highly efficient cellulase preparations can reduce the cost of the enzymatic hydrolysis process, thereby enabling the production of cellulosic ethanol.

At present, there is a long history of producing cellulase by microbial fermentation, among which Trichoderma reesei (Trichoderma reesei), Penicillium oxalicum (Penicillium oxalicum) Neurospora crassa (Neurospora crassa), and the like are microorganisms which have been proven to produce cellulase so far, and among them, Trichoderma reesei has been studied most widely because it produces cellulase with high yield and complete enzyme system. Unfortunately, the current enzyme production efficiency of trichoderma reesei is not sufficient to reduce the cost of the enzymatic hydrolysis step, and there is a need to improve the efficiency of the production of cellulase by trichoderma reesei. A plurality of bottlenecks are met in the process of improving the efficiency of producing the cellulase by the trichoderma reesei, wherein the most important problem is that the inducer is needed for producing the cellulase by the trichoderma reesei, and the cellulase can be secreted by the trichoderma reesei only under the induction action of the inducer. The additional addition of an inducer, which is low in inducing effect and expensive, is the most important reason for the high cost of cellulase production. Therefore, in order to solve the problems, the research core here is to develop a cheap and efficient cellulase inducer, reduce the production cost of cellulase, further reduce the utilization cost of lignocellulose and provide possibility for the industrial utilization of lignocellulose.

At present, whey is mainly used as an inducer for producing cellulase of trichoderma reesei in industry, but lactose is expensive and has poor induction effect on trichoderma reesei, and the inducer research in other researches mainly focuses on microcrystalline cellulose, treated lignocellulose raw materials and the like, but the microcrystalline cellulose is more expensive and the induction effect of the lignocellulose materials is very poor, so that the raw materials can not meet the industrial requirements. In recent years, a great deal of work has shown that oligosaccharides such as cellobiose, gentiobiose, sophorose, and the like, which are linked by β -glycosidic bonds, can efficiently induce trichoderma reesei to produce cellulase. However, the cost is high due to factors such as separation and purification, which makes the price very expensive. Therefore, if the above-mentioned oligosaccharide substances can be produced efficiently at low cost, cellulase can be produced efficiently. Thereby reducing the production cost of the cellulase and further reducing the utilization cost of the lignocellulose.

Accordingly, those skilled in the art have been devoted to the development of a cost-effective and efficient cellulase inducer and methods for its preparation and use.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a cellulase inducer with low cost and high efficiency, and a preparation method and an application method thereof.

In order to achieve the purpose, the invention provides a high-efficiency cellulase inducer, which is a mixture ACM (Acid-catalyzed mixture) of disaccharide and poly-oligosaccharide, wherein disaccharide comprises sophorose, and/or gentiobiose, and/or cellobiose.

The invention also provides a preparation method of the high-efficiency cellulase inducer, which takes glucose or glucose-containing syrup as a substrate and prepares the high-efficiency cellulase inducer through the chemical reaction of catalyzing the glucose by acid; in the process, the concentration of reducing sugar in the fermentation reaction is controlled to be below 1g/L by an ACM fed-batch strategy.

Further, the acid includes a chemical agent capable of providing H + for catalysis.

Further, chemical agents capable of providing H + for catalysis include liquid acids comprising sulfuric, phosphoric, or hydrochloric acid, or the like, or solid acids comprising amberlyst15, amberlyst35, or the like.

Further, the acid used is sulfuric acid, and the concentration of the acid is not less than 40 mM.

Further, the concentration of glucose in the substrate is greater than or equal to 300 g/L.

Further, the concentration of glucose in the substrate is not less than 700 g/L.

Further, the concentration of the liquid acid is greater than 1mM and the concentration of the solid acid is greater than 0.01% (w/w).

Further, the catalysis method is to add acid into the substrate, and then incubate, wherein the incubation temperature is 50-250 ℃, and the incubation time is 1 min-100 h, wherein the higher the concentration of the added acid, the lower the incubation temperature, and the shorter the incubation time.

Furthermore, the adopted temperature is not lower than 120 ℃, and the heating time is not lower than 1 h.

Further, after preparing 700g/L of a glucose solution, 20mL of the glucose solution was added to a 50mL bomb, 127.6. mu.L of concentrated sulfuric acid was added so that the concentration of sulfuric acid was 120mM, and the resultant was heated in an oil bath at 140 ℃ for 1 hour.

The invention also provides application of the high-efficiency cellulase inducer in high-efficiency production of cellulase.

Further, the application method comprises the following steps: the ACM is used as a carbon source and an inducer, and the microorganisms capable of producing the cellulase and the components thereof are cultured to efficiently produce the cellulase, wherein the microorganisms comprise natural microorganisms or microorganisms obtained by artificial mutagenesis and gene modification.

Further, the microorganism capable of producing cellulase and its components comprises Trichoderma reesei (Trichoderma reesei) or Penicillium oxalicum (Penicillium oxalicum), spores of Trichoderma reesei (Trichoderma reesei) or Penicillium oxalicum (Penicillium oxalicum) are inoculated into a solid medium (PDA), cultured at 28 ℃ for 7 days, washed with sterile water to obtain spore liquid; inoculating the spore liquid into a liquid culture medium, and performing shake culture for 24h at 28 deg.C and 150rpm to obtain mycelium liquid; and (3) using the ACM as a unique carbon source of an enzyme production culture medium to induce hypha liquid to produce the cellulase.

Further, when t.reesei was cultured with ACM as a carbon source and an inducer, the glucose concentration in the fermentor was less than 1 g/L.

Further, the cellulase producing strain is from t.reesei, american type culture collection No. ATCC56765 and chinese industrial microorganism collection No. cic 13052, t.reesei RUT C30.

In the present invention, the composition of the fermentation medium can be adjusted by a skilled person based on the general knowledge of t.

In the preferred embodiment 1 of the present invention, the preparation of ACM by using sulfuric acid to catalyze the reaction of glucose is explained in detail;

in another preferred embodiment 2 of the present invention, the preparation of ACM by the reaction of glucose catalyzed by amberlyst15 as a solid acid is described in detail;

in another preferred embodiment 3 of the present invention, it is specified that the cellulase is efficiently produced by culturing t.reesei RUT C30 in a shake flask using ACM prepared using sulfuric acid as a catalyst as a carbon source and an inducer;

in another preferred embodiment 4 of the present invention, the induction of T.reesei RUT C30 cellulase production in a fermentor by using ACM as a carbon source and an inducer is described in detail.

Compared with the prior art, the invention has the following outstanding advantages:

the ACM is a mixture containing glucose, sophorose, gentiobiose, cellobiose and other poly-oligosaccharides, the glucose concentration is controlled to be below 1g/L, the growth of hyphae is facilitated without inhibiting the production of cellulase, the beta-disaccharide mainly containing sophorose can efficiently induce and synthesize the cellulase, the fermentation time is shortened, the energy consumption of the process is saved, and the cost of the cellulase is reduced.

The preparation of the ACM only needs sulfuric acid as a catalyst, has short preparation time and simple preparation process, greatly reduces the investment of production equipment and reduces the cost of the cellulase.

The pH value of the ACM after reaction is close to 2, so that the ACM is easy to store, and the ACM does not change the pH value of a fermentation system when added into a fermentation enzyme production culture medium.

The high-enzyme-activity cellulase in the invention can be directly used for hydrolysis of cellulose components in the refining process of lignocellulose biomass.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram of the process for preparing ACM by the sulfuric acid-catalyzed glucose reaction according to a preferred embodiment of the present invention 1;

FIG. 2 is a chromatogram for measuring the components of the reaction of glucose catalyzed by sulfuric acid to prepare ACM according to a preferred embodiment of the present invention;

FIG. 3 is a bar graph showing the inducing effects of the T.reesei RUT C30 cultured in a shake flask for efficient production of cellulase and other common inducers using ACM prepared using sulfuric acid as a catalyst as a carbon source and inducers according to a preferred embodiment 3 of the present invention;

FIG. 4 is a flow chart of the process of inducing T.reesei RUT C30 enzyme production in a fermentor using ACM as a carbon source and an inducer according to a preferred embodiment 4 of the present invention;

the system comprises a 1-regulating valve, a 2-humidifier for humidification, a 3-fermentation tank, a 4-Dissolved Oxygen (DO) detection system, a 5-detection system, a 6-water circulation system, a 7-pH system, an 8-ammonia water storage tank, a 9-first peristaltic pump, a 10-ACM storage tank, a 11-second peristaltic pump, a 12-defoaming agent storage tank and a 13-third peristaltic pump, wherein the 1-regulating valve is connected with the 2-humidifier for humidification;

FIG. 5 is a graph showing the results of inducing T.reesei RUT C30 enzyme production in a fermentor over fermentation time using ACM as a carbon source and an inducer in accordance with a preferred embodiment 4 of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

In the drawings, structurally identical elements are represented by like reference numerals, and structurally or functionally similar elements are represented by like reference numerals throughout the several views. The size and thickness of each component shown in the drawings are arbitrarily illustrated, and the present invention is not limited to the size and thickness of each component. The thickness of the components may be exaggerated where appropriate in the figures to improve clarity.

EXAMPLE 1 preparation of ACM by sulfuric acid catalyzed glucose reaction

As shown in figure 1, the process for preparing ACM by the reaction of catalyzing glucose by sulfuric acid is to prepare 700g/L glucose solution, add 6.39mL of concentrated sulfuric acid into 1L of glucose solution to make the final concentration of the concentrated sulfuric acid be 120mM, and heat the solution for 1h at 140 ℃ to prepare the soluble inducer ACM; FIG. 2 shows the components of ACM prepared by the reaction of glucose catalyzed by sulfuric acid, which contains 390g/L glucose, 4.6g/L sophorose, 46g/L gentiobiose and 4g/L cellobiose.

EXAMPLE 2 preparation of ACM by solid acid amberlyst15 catalysis of glucose reaction

Preparing 700g/L glucose solution, adding solid acid with concentration of 3% (mass fraction) of glucose, heating at 140 deg.C for 3h to obtain soluble inducer ACM, and determining that ACM contains glucose 390g/L, sophorose 12.3g/L, gentiobiose 30g/L, and cellobiose 12 g/L.

Example 3 cultivation of t.reesei RUT C30 in shake flasks with ACM prepared with sulfuric acid as catalyst as carbon source and inducer produces cellulase efficiently.

Measuring the concentration of reducing sugar in the ACM, after the T.reesei RUT C30 culture medium is sterilized, adding 1% of reducing sugar (mass fraction) of the ACM as a carbon source and an inducer to induce the T.reesei RUT C30 to produce cellulase, and culturing the T.reesei RUT C30 in a shake flask by using the ACM prepared by using sulfuric acid as a carbon source and an inducer to efficiently produce the cellulase, wherein the induction effect of the ACM is not greatly different from that of microcrystalline cellulose, but the induction effect of the ACM is far greater than that of lactose and is 1.5 times of that of the lactose, as shown in FIG. 3.

Example 4 Induction of T.reesei RUT C30 enzyme production in fermentors with ACM as carbon Source and inducer

The process flow of batch fed-batch culture of T.reesei RUT-C30 for producing cellulase is shown in figure 4, air of an air compressor enters an air humidifier 2 through an adjusting valve 1 for humidification, then enters a fermentation tank 3, and is linked with a Dissolved Oxygen (DO) detection system 4 to control DO in fermentation liquor to be not less than 20% so as to meet requirements of hypha growth and cellulase biosynthesis. The temperature detection system 5 and the water circulation system 6 adjust the temperature of the system, and the pH system 7 is linked with the ammonia water storage tank 8 and the first peristaltic pump 9 to control the pH value to be not lower than 4.2 in the fermentation process. The ACM storage tank 10 is linked with the second peristaltic pump 11 to detect the concentration of glucose in the fermentation liquid, the fed-batch of the ACM is controlled, and the defoaming agent storage tank 12 is linked with the third peristaltic pump 13 to eliminate foam generated in the culture process in time.

The method specifically comprises the following steps:

1. slant culture: a small amount of T.reesei RUT C30 spores were inoculated from a freezer at-80 ℃ to PDA medium, cultured at 28 ℃ for 7 days, and the spores were washed with sterile water.

2. Seed culture: inoculating the spores washed in the step 1 into a corn steep liquor culture medium containing 4g/L glucose and 10g/L, and culturing at 28 ℃ and 150rpm for 24h to prepare a hypha liquid as a seed solution.

3. Fermentation culture: inoculating into a 3L fermentation culture medium fermentation tank according to the inoculation amount of 10%, taking the first 12h-36h of fermentation as the main stage of thallus growth, controlling the fermentation temperature at 28 ℃, feeding ammonia water to make the pH not lower than 4.2, and simultaneously supplementing nitrogen source. And detecting the concentration of glucose in the fermentation liquor, reducing the temperature to 25 ℃ when the glucose in the fermentation liquor is exhausted, starting to add ACM, detecting the concentration of the glucose in the fermentation tank every 4h, and ensuring that the concentration of the glucose in the fermentation tank is less than 1 g/L. The enzyme activity of the filter paper adopts a standard method issued by IUPAC of International theory of pure chemistry, the fermentation result is shown in figure 5, the activity unit of the cellulase in the fermentation liquor after 96h of culture reaches 20FPU/mL

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. A high-efficiency cellulase inducer, which is characterized in that the inducer is a mixture ACM (Acid-catalyzed mixture) of disaccharide and poly-oligosaccharide, wherein the disaccharide comprises sophorose, and \ or gentiobiose, and \ or cellobiose.

2. The method for producing a potent cellulase inducer according to claim 1, wherein the method comprises preparing the potent cellulase inducer by acid-catalyzing a chemical reaction of glucose using glucose or a syrup containing the glucose as a substrate; and inducing enzyme production by the ACM fed-batch feeding strategy, and controlling the concentration of reducing sugar in the enzyme production reaction to be below 1 g/L.

3. The method of claim 2, wherein the acid comprises a chemical reagent capable of providing H + for catalysis.

4. The method of claim 3, wherein the chemical reagent capable of providing H + for catalysis comprises a liquid acid comprising sulfuric acid, phosphoric acid, or hydrochloric acid, or a solid acid comprising amberlyst15 or amberlyst 35.

5. The method of claim 2, wherein the concentration of glucose in the substrate is greater than or equal to 300 g/L.

6. The method of claim 4, wherein the liquid acid is added at a concentration of greater than 1mM and the solid acid is added at a concentration of greater than 0.01% (w/w).

7. The use according to claim 2, wherein the method comprises adding the acid to the substrate followed by incubation at a temperature of 50-250 ℃ for a period of 1 min-100 h, wherein the higher the concentration of the acid added, the lower the incubation temperature and the shorter the incubation period.

8. Use of a potent cellulase inducer according to claim 1 for the efficient production of cellulase.

9. The application of claim 8, wherein the application method comprises the steps of: and culturing microorganisms capable of producing the cellulase and components thereof by using the ACM as a carbon source and an inducer, wherein the microorganisms comprise natural microorganisms or microorganisms subjected to artificial mutagenesis and genetic modification.

10. The use according to claim 9, wherein the microorganism comprises Trichoderma reesei (Trichoderma reesei) or Penicillium oxalicum (Penicillium oxalicum), spores of the Trichoderma reesei (Trichoderma reesei) or the Penicillium oxalicum) are inoculated into a solid medium (PDA), cultured at 28 ℃ for 7 days, washed with sterile water to obtain the spore liquid; inoculating the spore liquid into a liquid culture medium, and performing shake culture for 24h at the temperature of 28 ℃ and the rotation speed of 150rpm to obtain a hypha liquid; and (3) inducing the hyphal liquid to produce the cellulase by using the ACM as the only carbon source of the enzyme production culture medium.