Beta-disaccharide mixture synthesized by reverse catalysis of glucose through beta-glucosidase and used for growth of trichoderma reesei and induced production of cellulase

Abstract

The invention discloses beta-disaccharide mixture syrup which is mainly prepared from sophorose, cellobiose and gentiobiose through reverse catalytic synthesis of beta-glucosidase by taking glucose as a substrate. The syrup is used as a carbon source and an inducer, and is used for mycelial growth in a Trichoderma reesei engineering strain culture process and induction of lignocellulose biomass hydrolysis sugar preparation special cellulase synthesis. By controlling the flow acceleration rate of syrup in the feeding operation process of the fermentation tank, the appropriate reducing sugar in the fermentation liquid is maintained, the growth of hyphae is promoted, and the continuous production of cellulase is realized.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering and technology; more specifically, the present invention relates to a proprietary technical method for the reverse catalysis of β-glucosidase to synthesize a β-disaccharide mixture from high-concentration glucose (syrup), which is used as a carbon source and inducer for the cultivation of engineered strains of Trichoderma reesei, and promotes mycelial growth and the induced synthesis of dedicated cellulase for the preparation of hydrolysates of lignocellulosic biomass through fed-batch control in a fermenter. Background Art

[0002] Lignocellulosic biomass represented by crop straws and forestry waste is not only rich in reserves but also a renewable natural resource. Bioenergy and bio-based chemicals produced through the biorefinery technology route have the outstanding characteristics of being carbon-neutral and environmentally friendly because the CO 2 released during the use of these bio-based products can be absorbed during the raw material production process of plant photosynthesis without increasing the CO 2 content in the atmospheric environment.

[0003] The main components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. The natural evolution process causes these biological macromolecules to be tightly intertwined to form a complex, which, while providing support and protection for plant growth and development, also increases the difficulty of its degradation and utilization, and requires appropriate pretreatment for component separation. The cellulose and hemicellulose obtained after pretreatment are polysaccharides and must be hydrolyzed into monosaccharides before they can be used as the basic raw materials for microbial fermentation to produce various bio-based products. Although cellulose and hemicellulose can be hydrolyzed by acid or base catalysis, the chemical catalytic technology route has the prominent problems of large consumption of acids and bases, harsh reaction conditions, and many by-products. Using cellulase to catalyze the hydrolysis of cellulose and hemicellulose has the advantages of being environmentally friendly, having mild reaction conditions, and few by-products, and is the main direction for the development of lignocellulosic biomass hydrolysate preparation technology.

[0004] Cellulase is a complex enzyme, mainly composed of endoglucanase, exoglucanase and β-glucosidase, which hydrolyzes cellulose through synergistic action. In addition, cellulase also contains xylanase that can hydrolyze hemicellulose, as well as various auxiliary enzymes and proteins that promote cellulose hydrolysis. These auxiliary enzymes and proteins can enhance the hydrolysis effect of cellulase on cellulose (Trends Biotech 2024, 42: 418-430). It is known that Trichoderma reesei (abbreviated as T. reesei) produces cellulase, but the synthesis of cellulase by T. reesei requires induction, and there is a lack of low-cost and highly efficient inducers suitable for industrial production applications, which has always been one of the greatest challenges in the production of cellulase suitable for the hydrolysis of lignocellulosic biomass to prepare hydrolysates (Bioresour Bioprocess 2021, 8: 107).

[0005] Cellulose is a natural inducer for T. reesei to synthesize cellulase. Therefore, cellulase can be produced by solid-state fermentation using straw and other materials. Li Hongbing et al. invented a method for producing cellulase by solid-state fermentation using rice straw as the main raw material (CN105238704). Although the technical route of the solid-state fermentation process is simple, the investment in production equipment is low, and the production cost of cellulase is low, the solid-state fermentation cannot effectively prevent and control the contamination of miscellaneous bacteria in the production process due to incomplete sterilization of the culture medium and equipment. Therefore, the cellulase produced can only be used in industries with low requirements for hygiene indicators such as feed, and cannot be used to hydrolyze the cellulose and hemicellulose components in lignocellulosic biomass to obtain hydrolysates for the production of bioenergy and biobased chemicals based on liquid deep fermentation of pure microbial cultures.

[0006] Liquid deep fermentation is the only option for producing cellulase that meets the requirements for the preparation of hydrolysates from lignocellulosic biomass. A large amount of research work has been carried out both at home and abroad. Feng Jiaxun et al. applied for a patent technology for liquid deep fermentation of cellulase using microcrystalline cellulose as an inducer (CN201210276891). However, microcrystalline cellulose is a water-insoluble solid and needs to be slowly hydrolyzed into soluble sugars by the strain secreting a very low level of background cellulase before it can be absorbed and utilized (J Biol Chem 1997, 272: 10169-10174). This characteristic makes the time required for liquid deep fermentation of cellulase based on microcrystalline cellulose and other cellulose-based inducers extremely long, significantly increasing the energy consumption of stirring and ventilation during the cellulase fermentation process. Moreover, the price of microcrystalline cellulose with good induction effect for enzyme production is expensive, resulting in a particularly high cost of cellulase produced by liquid deep fermentation and making it impossible to be used for the low-cost preparation of hydrolysates from lignocellulosic biomass. Therefore, the research and development of low-cost soluble inducers is a technical bottleneck that must be solved in the liquid deep fermentation production of cellulase for the preparation of hydrolysates from lignocellulosic biomass.

[0007] The most effective soluble inducer for cellulase synthesis by T. reesei is sophorose. However, sophorose is scarce in resources and expensive in price, making it impossible to be used for large-scale production of cellulase at low cost in liquid deep fermentation. Lactose can also induce T. reesei to synthesize cellulase. Moreover, the lactose-containing whey powder as a by-product in the dairy processing industry has a relatively low cost and is an inducer that can be used in the industrial production of cellulase in liquid deep fermentation. However, the effect of inducing cellulase synthesis using lactose or whey powder is far less than that of sophorose. The low cellulase activity in the fermentation broth and the long fermentation time lead to prominent problems such as low production intensity of the fermenter equipment and high operating energy consumption. The low enzyme activity in the fermentation broth makes this cellulase unable to be directly used for preparing hydrolysates of lignocellulosic biomass and needs to be concentrated before use, significantly increasing the production cost of cellulase.

[0008] Synthetic soluble inducers are an effective strategy to solve the technical and economic problems faced by T. reesei in using cellulose substances such as microcrystalline cellulose as inducers and lactose / whey powder as inducers for cellulase production. Using glucose as a substrate, β-disaccharides such as sophorose can be synthesized by the reverse catalysis of β-glucosidase. However, β-glucosidase is a hydrolase that catalyzes the hydrolysis of cellobiose to release glucose. The reaction equilibrium constant for the reverse catalysis of glucose to synthesize β-disaccharides is very small, resulting in extremely low β-disaccharide content in the reaction product or insufficient effective inducer. It is necessary to increase the dosage of β-glucosidase. However, β-glucosidase is expensive. Even industrial-grade enzyme preparation products cost up to 120 yuan per kilogram. As a result, the inducer synthesized by this technical route cannot be used for the liquid deep fermentation production of cellulase dedicated to the preparation of hydrolysates of lignocellulosic biomass at an acceptable cost. Summary of the Invention

[0009] The purpose of the present invention is to provide a technical method for synthesizing a β-disaccharide mixture by the reverse catalysis of β-glucosidase on high-concentration glucose (syrup), which is used as a carbon source and inducer for the cultivation of Trichoderma reesei (T. reesei) engineering strains, and promoting the growth of mycelia and the induced synthesis of cellulase dedicated to the preparation of hydrolysates of lignocellulosic biomass through the feeding control and feeding of a fermenter.

[0010] In the first aspect of the present invention, a method for preparing a β-disaccharide mixture syrup is provided. The syrup is used for cultivating Trichoderma reesei (T. reesei) engineering strains to induce the synthesis of cellulase dedicated to the preparation of hydrolysates of lignocellulosic biomass. The method includes: under the conditions of a temperature of 60-70°C and a pH of 4.5-5.0, using a β-glucosidase dosage of 5-12 U / g (glucose) to reversely catalyze high-concentration glucose (syrup) to obtain a β-disaccharide mixture syrup; wherein, the high-concentration glucose (syrup) contains 580-700 g / L of glucose; wherein, in the β-disaccharide mixture syrup, the β-disaccharide mixture includes (mainly includes): sophorose, cellobiose, and gentiobiose.

[0011] In a second aspect of the present invention, there is provided a method for culturing an engineered strain of Trichoderma reesei using a β-disaccharide mixture syrup, the method comprising:

[0012] (1) preparing a β-disaccharide mixture syrup by the method described in the first aspect;

[0013] (2) culturing the engineered strain of Trichoderma reesei to allow mycelial growth and production of cellulase; during the culturing process, monitoring the reducing sugar concentration in the fermentation broth, and when the reducing sugar concentration is lower than 1.0 g / L, feeding and adding the β-disaccharide mixture syrup prepared in (1).

[0014] In one or more embodiments, the reducing sugar is defined and analyzed and detected by conventional methods in the fermentation industry.

[0015] In a third aspect of the present invention, there is provided a method for inducing the synthesis of cellulase specific for preparing hydrolysates of lignocellulosic biomass, the method comprising:

[0016] (a) culturing the engineered strain of Trichoderma reesei using the method described above;

[0017] (b) fermentatively culturing the engineered strain obtained in (a), and by feeding, maintaining the reducing sugar concentration in the fermentation broth at 1 - 2 g / L, thereby inducing the synthesis of cellulase specific for preparing hydrolysates of lignocellulosic biomass.

[0018] In one or more embodiments, in the β-disaccharide mixture (by weight / volume ratio), the ratio of sophorose: cellobiose: gentiobiose is (100 - 130):(4 - 6):(35 - 55); preferably, the ratio of sophorose: cellobiose: gentiobiose is 114.4:5.3:43.7, and the proportion of each component can fluctuate up and down by 30%, 20%, 10% or 5%.

[0019] In one or more embodiments, the β-disaccharide mixture further comprises unreacted glucose.

[0020] In one or more embodiments, the reaction system of the reverse catalysis is mixed evenly and reacted for 72 ± 6 h; preferably, the reaction is for 72 ± 4 h (more preferably 72 ± 2 h).

[0021] In one or more embodiments, at a temperature of 65 - 70 °C and a pH of 4.5 - 5.0, using a β-glucosidase dosage of 5 - 10 U / g (glucose) to reversely catalyze high-concentration glucose (syrup) to obtain a β-disaccharide mixture syrup.

[0022] In one or more embodiments, the high-concentration glucose (syrup) contains 600 - 700 g / L of glucose.

[0023] In one or more embodiments, in step (2) or step (b): the temperature for mycelial growth in the initial stage of fermentation is 30 ± 1 °C, preferably 30 ± 0.5 °C (more preferably 30 ± 0.2 °C).

[0024] In one or more embodiments, in step (2) or step (b): the temperature during the enzyme production stage after fed-batch addition of syrup is 28 ± 1 °C, preferably 28 ± 0.5 °C (more preferably 28 ± 0.2 °C).

[0025] In one or more embodiments, in step (2) or step (b): the pH of the fermentation broth during mycelial growth and enzyme production stage is not lower than 4.5; for example, pH 4.5 - 5.0, preferably pH 4.5 - 4.8, more preferably pH 4.5 - 4.6.

[0026] In one or more embodiments, in step (2) or step (b): the dissolved oxygen (DO) is not lower than 15%; for example, dissolved oxygen 15 - 40%, preferably 20 - 35%, more preferably 20 - 30%.

[0027] In one or more embodiments, in step (2) or step (b): the fermentation medium comprises: 4.0 g / L glucose monohydrate, 30.0 g / L lactose, 23.0 g / L corn steep liquor, 0.6 g / L calcium chloride dihydrate, 5.0 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.9 g / L magnesium sulfate heptahydrate, 0.2 mL / L T1, 0.2 mL / L T2, 0.3 g / L antifoaming agent; wherein, T1 comprises: 0.6% ferric citrate, 0.08% zinc acetate dihydrate and 0.08% ethylenediaminetetraacetic acid; T2 comprises: 0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% ferrous sulfate heptahydrate and 1.6% manganese sulfate monohydrate; wherein, the dosage of each component of the fermentation medium can fluctuate up and down by 1 - 50%, 1 - 40%, 1 - 30%, 1 - 20%, 1 - 10%, 1 - 5%, 1 - 3% or 1 - 2%.

[0028] In one or more embodiments, the fermentation medium further comprises an antifoaming agent; preferably, it contains 0.3 g / L antifoaming agent, or its dosage can be according to the conventional operation in the fermentation industry.

[0029] In one or more embodiments, the fermentation time of step (2) or step (b) is 120 - 160 hours; preferably 120 - 140 hours; more preferably 120 - 130 hours.

[0030] In the fourth aspect of the present invention, there is provided the use of a β-disaccharide mixture syrup for culturing an engineered strain of Trichoderma reesei and / or inducing the synthesis of cellulase dedicated to the preparation of hydrolysates of lignocellulosic biomass; wherein, the preparation method of the β-disaccharide mixture syrup is as described in the first aspect.

[0031] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A process schematic diagram of reverse catalytic synthesis of a β-disaccharide mixture mainly containing sophorose, cellobiose, gentiobiose, etc. using a low dose of β-glucosidase with high-concentration glucose as a substrate under high-temperature conditions and using the resulting mixture for enzymatic hydrolysis of a cellulose-containing substrate.

[0033] Figure 2 A schematic diagram of the device for preparing the β-disaccharide mixture syrup.

[0034] In this figure, 1. Bioreactor, 2. Temperature sensor, 3. Temperature indicating and regulating unit, 4. Constant-temperature water inlet regulating valve, 5. Constant-temperature water circulation pump, 6. Constant-temperature water bath, 7. Reactor heating chamber, 8. pH electrode, 9. pH indicating and regulating unit, 10. Peristaltic pump, 11. Acid-base storage tank, 12. Driving motor, 13. Stirrer.

[0035] Figure 3 A schematic diagram of the device for culturing and fermenting Trichoderma reesei to produce cellulase (a) and the enzyme activity and dry mycelial weight during the enzyme production process (b);

[0036] In this figure, 1. Air compressor, 2. Air regulating valve, 3. Air flowmeter, 4. Gas distributor, 5. Fermentation tank, 6. Temperature sensor, 7. Temperature indicating and controlling unit, 8. Circulating cooling water regulating valve, 9. Circulating water pump, 10. Circulating water bath, 11. Cooling chamber, 12. Dissolved oxygen electrode (DO), 13. DO indicating and controlling unit, 14. Stirring system, 15. pH electrode, 16. pH indicating and controlling unit, 17. Ammonia storage tank, 18. Peristaltic pump, 19. MDG storage tank, 20. Peristaltic pump, 21. Antifoaming agent storage tank, 22. Peristaltic pump.

[0037] Figure 4 A device for hydrolyzing corn cob cellulose residue with cellulase to prepare hydrolysates (a) and an analysis of the glucose production during the enzymatic hydrolysis process (b). DETAILED DESCRIPTION OF THE INVENTION

[0038] In the prior art, in the process of culturing Trichoderma reesei and producing cellulase specialized for preparing hydrolysates of lignocellulosic biomass by liquid submerged fermentation, there are prominent problems such as the high cost of using cellulose inducer including microcrystalline cellulose, long fermentation time and difficult process control. Moreover, sophorose is too expensive to be used in the industrial production of this cellulase. Lactose and whey powder as inducers result in low cellulase activity and low production intensity in the fermentation tank during the fermentation process, leading to the inability to directly use the produced cellulase, as well as high energy consumption for stirring and ventilation in the fermentation tank. Through in-depth experimental research and analysis, the inventor provides a method for reverse catalytic synthesis of a β-disaccharide mixture containing sophorose, cellobiose, gentiobiose, etc. based on high-concentration glucose (600 - 700 g / L), at high temperature (60 - 70 °C), appropriate pH value (4.5 - 5.0) and adding a low dose (5 - 12 U / g (glucose)) of β-glucosidase, and controlling the feeding to make this β-disaccharide mixture serve as the carbon source for the mycelial growth of Trichoderma reesei engineering strain and induce the synthesis of cellulase.

[0039] As used in the present invention, "Trichoderma reesei" can be abbreviated as "T. reesei".

[0040] As used in the present invention, the "β-disaccharide mixture" contains at least sophorose, cellobiose, gentiobiose and glucose.

[0041] As used in the present invention, "containing", "having" or "including" encompasses "comprising", "consisting essentially of", "consisting substantially of", and "consisting of"; "consisting essentially of", "consisting substantially of" and "consisting of" are subordinate concepts of "containing", "having" or "including". Unless otherwise specified, the components / constituents listed after the terms "containing", "having" or "including" are all the active components / constituents that play the desired functions / roles.

[0042] As used in the present invention, "enzymolysis" and "hydrolysis" can be used interchangeably.

[0043] As used in the present invention, "slant culture" and "seed culture" refer to the culture for the purpose of obtaining a suitable inoculum. The "fermentation culture" is the fermentation for the purpose of the mycelial growth of Trichoderma reesei strain and the production of cellulase. Unless otherwise specified, the "fermentation" and "culture" mentioned in the claims and the specification of the present invention refer to the fermentation for the purpose of the mycelial growth of Trichoderma reesei strain and the production of cellulase.

[0044] In a preferred embodiment of the present invention, a specific strain type of T. reesei, namely the T. reesei SCB18 engineering strain (Biotechnol Biofuels 2017, 10:272), is used. However, those skilled in the art should understand that given the commonalities among different strains of T. reesei, other types of T. reesei can also be applied to the technical solution of the present invention. According to the technical solution provided by the present invention, those skilled in the art can easily operate and verify the growth and cellulase synthesis effects of the corresponding T. reesei. In addition, derivative strains or recombinant strains obtained by genetic engineering modification of T. reesei strains (such as the strains in the examples) are also included in the present invention.

[0045] As a preferred embodiment of the present invention, a method for efficiently producing cellulase specific for the preparation of lignocellulosic biomass hydrolysate using a T. reesei engineering strain is provided. The method includes the following steps:

[0046] S1. Using glucose or a syrup containing glucose, a β-disaccharide mixture containing sophorose, cellobiose, and gentiobiose is prepared by reverse catalysis with β-glucosidase.

[0047] S2. Using the β-disaccharide mixture, the T. reesei engineering strain is cultured by controlled fed-batch. The untransformed glucose in the reaction process serves as the carbon source for mycelial growth, while the β-disaccharide mixture such as sophorose, cellobiose mixture, and gentiobiose synthesized by the reverse catalysis reaction serves as an inducer to efficiently induce the production of cellulase specific for the preparation of lignocellulosic biomass hydrolysate.

[0048] Preferably, in step S2, during the culture process, by the fed-batch of the β-disaccharide mixture, the reducing sugar concentration in the fermentation broth is controlled at 1.0 - 2.0 g / L, which can better promote the mycelial growth of the T. reesei engineering strain, induce the biosynthesis of cellulase specific for the preparation of lignocellulosic biomass hydrolysate, and achieve efficient production of cellulase.

[0049] Preferably, the specific fed-batch scheme of the β-disaccharide mixture is as follows: when the reducing sugar in the inoculated medium (bottom material) is reduced to 1.0 g / L, the β-disaccharide mixture is started to be fed-batch, and the feeding rate is regulated according to the reducing sugar concentration in the fermentation broth so that the reducing sugar concentration in the fermentation broth is controlled at 1.0 - 2.0 g / L.

[0050] Preferably, in step S2, during the culture process, the spore or mycelial suspension of the T. reesei engineering strain is inoculated into the fermentation medium at 5 - 10% of the fermentation broth volume and cultured at 30 °C, but the temperature is reduced to 28 °C during the fed-batch of the β-disaccharide mixture for enzyme production.

[0051] Preferably, in step S2, during the cultivation of the engineered T. reesei strain, ammonia water is used to control the pH value to 4.0 - 5.0, preferably pH 4.5 - 5.0, and the dissolved oxygen level is controlled to be not less than 15% saturation value by ventilation and stirring.

[0052] Preferably, in step S2, the composition of the medium for inoculating the fermenter is as follows: 4.0 g / L glucose monohydrate, 30.0 g / L lactose, 23.0 g / L corn steep liquor, 0.6 g / L calcium chloride dihydrate, 5.0 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.9 g / L magnesium sulfate heptahydrate, 0.2 mL / L T1 (0.6% iron citrate, 0.08% zinc acetate dihydrate, and 0.08% ethylenediaminetetraacetic acid), 0.2 mL / L T2 (0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% iron sulfate heptahydrate, and 1.6% manganese sulfate monohydrate), and 0.3 g / L antifoaming agent. It should be understood that the components can be fluctuated up and down as long as they can provide sufficient nutrients for the growth of T. reesei and the synthesis of cellulase.

[0053] By controlling the batch feeding flow rate, the reducing sugar in the fermentation broth is controlled at the level of 1.0 - 2.0 g / L. Under the condition that the fermentation time does not exceed 140 h, the filter paper enzyme activity of cellulase in the fermentation broth can reach more than 80 FPU / mL. This cellulase is used for hydrolyzing cellulose and hemicellulose in lignocellulosic biomass represented by crop straws and forestry waste to prepare hydrolyzed sugars, and the technical and economic indicators can meet the needs of fermentative production of cellulosic ethanol and other bulk bio - based products.

[0054] In the present invention, the reaction temperature for the reverse catalytic synthesis of β - glucosidase used is preferably 65 - 70 °C, which is completely different from the reaction temperature of 50 °C for β - glucosidase to catalyze the hydrolysis of β - disaccharides such as cellobiose.

[0055] The fermentation system described in the present invention can be scaled up to cooperate with industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on the general knowledge they have to be beneficial to the growth of the strain or the production of cellulase.

[0056] In the present invention, those skilled in the art can adjust the composition of the fermentation medium according to the common knowledge of cultivating T. reesei strains, such as appropriately increasing or decreasing certain inorganic salts to regulate the growth of mycelia, adjusting the addition amount of Tween 80 according to the growth state of mycelia to change the cell membrane permeability, and promoting the secretion of cellulase, etc.

[0057] In the actual use process, parameters such as the dosage of cellulase, the enzymolysis time, and the sugar yield during the cellulase hydrolysis process can be analyzed technically and economically according to the material situation, the cost of cellulase, and the production equipment, etc., so as to achieve the low-cost preparation of cellulose hydrolyzed sugar for microbial fermentation to produce bioenergy and biobased chemicals.

[0058] Compared with the prior art, the present invention has at least the following beneficial effects:

[0059] 1. In the β-disaccharide mixture syrup prepared by the present invention, the β-disaccharide mixture contains untransformed glucose, sophorose, cellobiose, gentiobiose, etc. catalytically synthesized by β-glucosidase reaction. The content of various sugars is beneficial to achieve the purpose of the present invention, that is, to culture Trichoderma reesei engineering strains and / or induce the synthesis of special cellulase for preparing hydrolyzed sugars from lignocellulosic biomass. Particularly, the β-disaccharide mixture contains a very high amount of sophorose, which is very advantageous.

[0060] 2. By controlling the feeding scheme of the reducing sugar concentration, the present invention can better promote the mycelial growth, induce the synthesis of special cellulase for preparing hydrolyzed sugars from lignocellulosic biomass, and reduce the production cost of cellulase.

[0061] 3. The cellulose (crude) enzyme solution produced by the present invention is suitable for hydrolyzing cellulose and hemicellulose components in lignocellulosic biomass represented by straw to prepare hydrolyzed sugars.

[0062] The present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, appropriate adjustments and improvements can also be made, and these all belong to the protection scope of the present invention.

[0063] Example 1: Reverse catalysis of glucose by β-glucosidase to synthesize β-disaccharide mixture

[0064] Prepare a 600 g / L glucose solution, add 10 IU of commercial β-glucosidase (purchased from Ningxia Xiasheng Industrial Group Co., Ltd.) per gram of glucose, and place it in Figure 2 the reactor 6 shown. The temperature sensor 2, temperature indicating and controlling unit 3, constant temperature water inlet regulating valve 4, constant temperature water circulation pump 5, constant temperature water tank 6 and reactor heating chamber 7 constitute the temperature control system; the pH electrode 8, pH indicating and controlling unit 9, peristaltic pump 10 and acid-base storage tank 11 constitute the pH control system; the drive motor 12 and stirrer 13 constitute the stirring system. React for 72 h under the condition of proper stirring to make the reaction system mix evenly to obtain β-disaccharide mixture syrup. Analyze the components of the β-disaccharide mixture therein.

[0065] It was determined that the β-disaccharide mixture contained 488.5 g / L of untransformed glucose, 114.4 g / L of sophorose, 5.3 g / L of cellobiose, and 43.7 g / L of gentiobiose.

[0066] Example 2: Batch fed-batch culture and fermentation of the engineered strain T. reesei SCB18 based on the β-disaccharide mixture to produce cellulase

[0067] The process flow for producing cellulase specifically for hydrolyzing lignocellulosic biomass by batch fed-batch culturing the engineered strain T. reesei SCB18 is as Figure 3 (a) shown. The air from the air compressor 1 is adjusted by the regulating valve 2 and metered by the flowmeter 3, and then enters the fermenter 5 through the air distributor 4 at a flow rate of 1.0 volume per volume per minute (vvm); the temperature sensor 6 and the indicating control unit 7, together with the circulating water regulating valve 8, the circulating water pump 9, the circulating water tank 10, and the bottom cooling system 11 of the fermenter, form a temperature regulation control system; the dissolved oxygen electrode (DO) 12 and the indicating control unit 13 jointly control the rotation speed of the stirrer 14 to maintain the DO value in the fermentation broth not lower than 20% to meet the requirements for the growth of hyphae and the synthesis of cellulase specifically for hydrolyzing lignocellulosic biomass; the pH electrode 15, the indicating control unit 16, the ammonia water storage tank 17, and the control peristaltic pump 18 constitute a pH regulation control system to control the pH value of 4.5 ± 0.1 during the culture and fermentation process; the β-disaccharide mixture storage tank 19 is linked with the peristaltic pump 20, and the addition of the β-disaccharide mixture is controlled by detecting the reducing sugar concentration in the fermentation broth; the antifoaming agent storage tank 21 is linked with the peristaltic pump 22 to timely eliminate the foam generated during the culture and fermentation process.

[0068] The specific steps of the culture process are as follows:

[0069] 1. Slant culture

[0070] A small amount of spores of the engineered strain T. reesei SCB18 were taken from the original bacteria stored in a -80 °C refrigerator and inoculated on a solid medium containing 46 g / L of potato dextrose agar (supplier: Qingdao Haibo Biotechnology Co., Ltd.), cultured at 30 °C for 7 d, and the spores were washed off with sterile water and stored at 4 °C for standby.

[0071] 2. Seed culture

[0072] The spores washed in Step 1 were inoculated into 250 mL Erlenmeyer flasks with a liquid volume of 50 mL (8 flasks, which can meet the inoculation requirements of Step 3), and cultured in a shaker at 30 °C and a rotation speed of 220 revolutions per minute (rpm) for 24 h to prepare a spore suspension as the seed solution. The medium composition was as follows: 23.3 g / L glucose monohydrate, 10.0 g / L corn steep liquor, 0.7 g / L calcium chloride dihydrate, 10.0 g / L potassium dihydrogen phosphate, 5.0 g / L ammonium sulfate, 1.0 g / L magnesium sulfate heptahydrate, 0.2 g / L T2 (0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% iron sulfate heptahydrate, and 1.6% manganese sulfate monohydrate), and 0.08 g / L antifoaming agent.

[0073] 3. Fermentation process

[0074] Inoculate into a fermenter with a liquid volume of 4 L of the fermentation medium at an inoculation amount of 10%. Control the fermentation temperature at 30 °C, and automatically control the pH to 4.5 ± 0.1 by feeding ammonia water, which also serves to supplement the nitrogen source. By coupling the stirring speed of the fermenter with the DO, ensure that the DO is not lower than 20% (under industrial production conditions, the DO can be reduced to about 5%). Detect the concentration of reducing sugar in the fermentation broth in the fermenter every 4 h. When the concentration of reducing sugar in the fermentation broth is lower than 1.0 g / L, start feeding the β-disaccharide mixture and regulate the feeding rate of the β-disaccharide mixture according to the concentration of reducing sugar to ensure that the concentration of reducing sugar in the fermenter is 1.0 - 2.0 g / L. After starting to feed the β-disaccharide mixture, adjust the fermentation temperature to 28 °C. The method for measuring the filter paper enzyme activity adopts the standard method promulgated by the International Union of Pure and Applied Chemistry (IUPAC) (Pure and applied Chemistry 1987, 59(2): 257 - 268).

[0075] In Step 3, the formula of the fermentation medium was as follows: 4.0 g / L glucose monohydrate, 30.0 g / L lactose, 23.0 g / L corn steep liquor, 0.6 g / L calcium chloride dihydrate, 5.0 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.9 g / L magnesium sulfate heptahydrate, 0.2 mL / L T1 (0.6% iron citrate, 0.08% zinc acetate dihydrate, and 0.08% ethylenediaminetetraacetic acid), 0.2 mL / L T2 (0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% iron sulfate heptahydrate, and 1.6% manganese sulfate monohydrate), and 0.3 g / L antifoaming agent.

[0076] The results of the fermentation process are as Figure 3(as shown in (b)). The main enzyme production phase starts at 60 h of fermentation, with the dry weight of mycelium being 56.6 g / L. After that, the dry weight of mycelium slowly increases while continuously producing enzymes. By 120 h, the dry weight of mycelium reaches 69.7 g / L. Enzyme production starts at 40 h, with the measured enzyme activity being 18.8 FPU / mL. At 100 h, the enzyme production reaches 78.2 FPU / mL. When the fermentation time is extended to 120 h, the enzyme activity slowly increases to 85.5 FPU / mL. At this time, the enzyme production can be ended and the fermentation broth can be discharged for treatment.

[0077] Example 3: Preparation of Hydrolysate Sugar by Hydrolyzing Corncob Cellulose Residue with Cellulase

[0078] Taking the hydrolysis of the cellulose residue remaining after extracting the hemicellulose hydrolysis component xylose from corncob (Jinan Shengquan Group Co., Ltd.) as an example, the hydrolysis performance of the cellulase prepared in Examples 1 - 2 was evaluated. The specific steps are as follows:

[0079] 1. The experimental materials (Jinan Shengquan Group Co., Ltd.) were actually measured to have a moisture content of 67.5% and a cellulose content of 65.0% in the cellulose residue.

[0080] 2. It was carried out in an enzymatic hydrolysis reactor with a total volume of 5 L and a working volume of 3 L as shown in (a). The temperature of the enzymatic hydrolysis reaction was controlled at 50 ± 0.5 °C by electric heating. The pH was adjusted to 5.0 ± 0.5 using 20% NaOH solution, and proper stirring was carried out to make the solid materials evenly suspended. Figure 4 (as shown in (a)). The temperature of the enzymatic hydrolysis reaction was controlled at 50 ± 0.5 °C by electric heating. The pH was adjusted to 5.0 ± 0.5 using 20% NaOH solution, and proper stirring was carried out to make the solid materials evenly suspended.

[0081] 3. The dosage of cellulase was 10 FPU per gram of dry - based material, which was added to the enzymatic hydrolysis reactor along with the materials. The initial dry matter concentration of the materials was 10 - 12%, and make - up feeding was carried out as the viscosity of the materials decreased until the dry matter concentration reached 25%. The glucose produced during the enzymatic hydrolysis process is as shown in (b). Figure 4 (as shown in (b)).

[0082] The results showed that after 72 h of enzymatic hydrolysis, the glucose concentration was 127.6 g / L, and the cellulase hydrolysis yield was 67.2%; after 94 h of enzymatic hydrolysis, the glucose concentration was 143.1 g / L, and the cellulase hydrolysis yield was 75.3%; after 120 h of enzymatic hydrolysis, the glucose concentration was 152.5 g / L, and the cellulase hydrolysis yield was 80.3%.

[0083] According to the above, the self - produced cellulase of the present invention has excellent performance in hydrolyzing cellulose and can efficiently obtain an enzymatic hydrolysis product containing hydrolysate sugar.

[0084] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A method for preparing a β-disaccharide mixture syrup, wherein the syrup is used to cultivate an engineered strain of Trichoderma reesei to induce synthesis of lignocellulosic biomass to hydrolyze sugars and prepare special cellulase, characterized in that: The method comprises: under the conditions of temperature of 60-70°C and pH of 4.5-5.0, using a β-glucosidase dosage of 5-12 U / g (glucose) to reversely catalyze high-concentration glucose (syrup) to obtain β-disaccharide mixture syrup; Wherein, the high concentration glucose (syrup) contains 580-700 g / L of glucose; Wherein, in the β-disaccharide mixture syrup, the β-disaccharide mixture includes: sophorose, cellobiose and gentiobiose.

2. A method for culturing an engineered strain of Trichoderma reesei using a β-disaccharide mixture syrup, characterized in that: The method comprises: (1) preparing a β-disaccharide mixture syrup by the method according to claim 1; (2) Cultivating the engineered strain of Trichoderma reesei to allow mycelium to grow and produce cellulase; during the cultivation process, monitoring the reducing sugar concentration in the fermentation broth, and when the reducing sugar concentration is lower than 1.0 g / L, adding the β-disaccharide mixture syrup of (1) as a feed stream.

3. A method for inducing the synthesis of lignocellulosic biomass to hydrolyze sugars to prepare special cellulase, characterized in that: The method comprises: (a) culturing an engineered strain of Trichoderma reesei using the method of claim 2; (b) fermenting and culturing the engineered strain of (a), maintaining the reducing sugar concentration in the fermentation broth at 1-2 g / L by feeding, thereby inducing the synthesis of special cellulase for preparing sugar by hydrolyzing lignocellulosic biomass.

4. The method according to any one of claims 1 to 3, characterized in that: In the β-disaccharide mixture, the ratio of sophorose: cellobiose: gentiobiose is (100-130): (4-6): (35-55); preferably, the ratio of sophorose: cellobiose: gentiobiose is 114.4: 5.3: 43.7, and the ratio of each component can fluctuate up and down by 30%, 20%, 10% or 5%.

5. The method according to claim 1, characterized in that The reverse catalytic reaction system is mixed evenly, and the reaction time is 72±6h; preferably, the reaction time is 72±2h.

6. The method according to claim 1, characterized in that Under the conditions of temperature of 65-70°C and pH of 4.5-5.0, a high concentration of glucose (syrup) is reversely catalyzed by a β-glucosidase dosage of 5-10 U / g (glucose) to obtain a β-disaccharide mixture syrup.

7. The method according to claim 1, characterized in that The high-concentration glucose (syrup) contains 600-700 g / L of glucose.

8. The method according to claim 2 or 3, characterized in that: In step (2) or step (b): The mycelium growth temperature at the initial stage of fermentation is 30±1°C, preferably 30±0.5°C; and / or After the feed stream is added with syrup, the temperature of the feed stream entering the enzyme production phase is 28±1°C, preferably 28±0.5°C; and / or The pH of the fermentation liquid during mycelial growth and enzyme production period is not less than 4.5; and / or The dissolved oxygen is not less than 15%.

9. The method according to claim 2 or 3, characterized in that: In step (2) or step (b), the fermentation medium comprises: 4.0 g / L glucose monohydrate, 30.0 g / L lactose, 23.0 g / L corn steep liquor, 0.6 g / L calcium chloride dihydrate, 5.0 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.9 g / L magnesium sulfate heptahydrate, 0.2 mL / L T1, 0.2 mL / L T2, and 0.3 g / L defoamer; Wherein, T1 comprises: 0.6% ferric citrate, 0.08% zinc acetate dihydrate and 0.08% ethylenediaminetetraacetic acid; T2 comprises: 0.2% boric acid, 0.3% zinc sulfate heptahydrate, 0.7% copper sulfate pentahydrate, 2.0% cobalt chloride hexahydrate, 1.2% sodium molybdate dihydrate, 9.2% ferrous sulfate heptahydrate and 1.6% manganese sulfate monohydrate; The dosage of each component of the fermentation medium may fluctuate by 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 1-3% or 1-2%.

10. Application of β-disaccharide mixture syrup for culturing engineered strains of Trichoderma reesei and / or inducing synthesis of lignocellulosic biomass hydrolyzing sugars to prepare special cellulase; wherein, The preparation method of the β-disaccharide mixture syrup is as described in claim 1.

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