Cellulase（β-1,4-glucan-4-glucan hydrolase) is the general name of a group of enzymes that degrade cellulose to produce glucose. It is not a monomer enzyme, but a multi-component enzyme system that plays a synergistic role. It is aComplex enzyme, mainly byCircumscribeβ-Glucanase, inscribedβ-Glucanase andβ-Glucosidase and other components, as well as highly activeXylanase。It acts on cellulose and products derived from cellulose.Microbial cellulase plays a very important role in transforming insoluble cellulose into glucose and destroying cell wall in fruit and vegetable juice to improve juice yield.
celluloseEnzyme (English: cellulase)It is a kind of enzyme, which can be used to decompose celluloseCatalysis。It is a protein that can decompose cellulose into oligosaccharides or monosaccharides.
Cellulase widely exists innatureIn the organism.Bacteria、fungusCellulase can be produced in animals.Cellulase commonly used for production comes from fungi, such as Trichoderma, Aspergillus and Penicillium（
The strain producing cellulase is easy to degenerate, leading to the reduction of enzyme production capacity.
The production of cellulase by bacteria is low, mainly glucan endonuclease, most of which have no degradation activity to crystalline cellulose, and the enzymes produced are mostly intracellular enzymes or adsorbed on the cell wall, not secreted into the culture medium, increasing the difficulty of extraction and purification, so there is less research on bacteria.However, the optimal pH of cellulase produced by bacteria is generally neutral to alkaline.In the past 20 years, with the successful application of neutral cellulase and alkaline cellulase in cotton fabric washing finishing process and detergent industry, bacterial cellulase preparation has shown good application prospects.
cellulase
Cellulase is widely used in food industry and environmental industry.In progressalcohol fermentationThe addition of cellulase can increase the utilization rate of raw materials and improve the quality of wine.
Because cellulase is difficult to purify, it usually containsHemicellulaseAnd other related enzymes, such asamylase（amylase）、protease(Protect), etc.
Cellulase has a wide range of sources.Cellulases from different sources have different structures and functions.Due to the high yield and high activity of fungal cellulase, fungal cellulase is mainly used in animal husbandry and feed industry.
English name Cellular[2]
English alias Cellular [USAN];Ku-zyme; Kutrase; Cellulase, aspergillus niger; Cellulase, trichoderma viride; Fungal cellulase
EINECS 232-734-4
classification
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By composition and function
Molecular Structure Diagram of Cellulose and Chitin
Cellulase according to itscatalytic reactionDifferent functions can be divided into internal tangencyGlucanase（1,4-β-D-glucan glucanohydrolase or endo-1,4-β-D-glucanase, EC3.2.1.4), from fungus (EG for short), from bacteria (CenExoglucanase（1,4-β-D-glucan cellobilhydrolase or exo-1,4-β-D-glucannase, EC. 3.2.1.91), CBH from fungi, Cex from bacteria) andβ-Glucosidase（β-1,4 glucosidase, EC. 3.2.1.21) is called BG for short.EndoglucanaseRandomly cut the amorphous region inside the cellulose polysaccharide chain to produce oligosaccharides of different lengths and the end of a new chain.Exoglucanase acts on theseReducibilityAnd the end of the non reducing cellulose polysaccharide chain, releasingglucoseorCellobiose。β-GlucosidasehydrolysisCellobioseProduces two molecules of glucose.Fungal cellulase has high yield and high activity. It is mainly used in animal husbandry and feed work.
According to degradation mechanism
celluloseEnzyme reactionDifferent from ordinary enzyme reactions, the main difference is that cellulase is a multi-component enzyme system, and the substrate structure is extremely complex.becausesubstrateThe adsorption of cellulase replaces the process of ES complex formed by enzyme and substrate.Cellulase first adsorbs specifically on the substrate cellulose, and then decomposes cellulose into glucose under the synergistic effect of several components.
In 1950, Reese et al. put forward the C1 Cx hypothesis, which believed that different enzymesSynergyTo completely hydrolyze cellulose into glucose.SynergyIt is generally believed that (C1 enzyme) first attacks the amorphous region of cellulose to form a new free terminal required for Cx, and then CX enzymePolysaccharideThe reducing end or non reducing end of the chain is cut offCellobioseUnits, finally byβ-Glucosidase hydrolyzes cellobiose into two glucose.However, the synergistic order of cellulase is not absolute, and subsequent studies found that C1 Cx andβ-Glucosidase must exist simultaneously to hydrolyze natural cellulose.If the crystalline cellulose is first treated with C1 enzyme, then C1 enzyme is removed, and then Cx enzyme is added, the crystalline cellulose cannot be hydrolyzed by such sequential action.[1]
Factors affecting the action of cellulase
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The optimal pH of cellulase is generally 4.5~6.5.Gluconolactone can effectively inhibit cellulase, and heavy metal ions such as copper and mercury can also inhibit cellulase, but cysteine can eliminate their inhibition, and even further activate cellulase.There are natural cellulase inhibitors in plant tissues;It can protect plants from the decay of mold. These inhibitors are phenolic compounds.If there is high oxidase activity in plant tissue, it can oxidize phenolic compounds into quinones, which can inhibit cellulase[1]。
Strain breeding
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Strain breeding is the basic work of cellulase production. Many experts at home and abroad have conducted a lot of research. In order to produce high-quality cellulase products, Wang Jialin et altrichoderma Wood 10, Trichoderma viride Sn-91014, Trichoderma koningii NT-15Aspergillus nigerXX-15A, on this basis, adopts ultraviolet ray, specific electromagnetic wave radiation, linear accelerator,NitrosoguanidineHigh yield strains NT15-H, NT15-H1, XT-15H, XT-15H1 were obtained by physical and chemical mutagenesis.Trichoderma NT-15HSolid cultureThe vitality of the filter paper is 3670u/g, C1, according to the detection of the Nanjing Station of the Food Quality Supervision and Testing Center of the Ministry of Light Industry-enzyme activity24460u/g, Cx enzyme activity 1800u/g, which has reached the international advanced level.This strain is stable in industrial production.Zhang Linghua et al. (1998) used Trichoderma koningii W-925, J-931, with a concentration of 2%Diethyl sulfateAfter combined mutagenesis with ultraviolet ray (15W, 30cm, 2min), Wu-932 strain with high enzyme activity was obtained, and the strain CMCSaccharificationThe activity of filter paper glucoamylase was 531, which was 100% and 81% higher than that of the original strain W-925, respectively.Ministry of Chemical Industryfeed additive Technical Service CenterWang ChengshuEt al. (1997)trichoderma reesei A3 Carry out UV andNitrosoguanidineAfter compound mutagenesis, inoculate the treated spores on the fiber double-layer plate, culture them at 30 ℃ for 5-8 days, place them at 15 ℃ for 7-10 days, and select the diameter andcolony of bacteriaLarger diameterSingle colonyCarry out triangular bottleSolid state fermentationAfter re screening, Trichoderma reesei 91-3 strain with high cellulase activity was obtained.
Cellulase strain is easy to degenerate, and its enzyme production capacity is significantly reduced after degradation, which may be caused by three aspects:
① The strain screened by mutagenesis had reverse mutation.
② Natural negative mutation.
③ If the strain is preserved at low temperature for a long time, secondary hyphae will grow on the conidia, and the conidia formed by the secondary hyphae have weak vitality, which may be the main reason for the degradation of the strain.In order to avoid the degradation of cellulase strains, Zhang Linghua et al. (1998) reported that the strains were stored in sand tubes.That is to say, the sieved and washed sand and soil are mixed and sub packed in test tubes at a ratio of 3:2, sterilized under pressure of 1kg/cm2 for 30 minutes for three times, and the slant bacteria to be preserved are prepared into 1000ml spore suspension. Each sand tube is filled with 0.5ml, shaken up, and stored in a vacuum dryer containing anhydrous CaCl2.After testing, the enzyme activity was basically unchanged within 121 days;The time for enzyme activity to decrease by 50% was extended from 60 days to 160 days by conventional methods, which significantly slowed down the rate of strain degradation.
fermentation process
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There are two main production processes of cellulase, namelySolid fermentationAnd liquid fermentation, the process is as follows:
Factors affecting enzyme production and activity
There are many factors affecting the yield and activity of cellulase, including culture temperature, pH, moisture, substrate, culture time, etc.These factors are not isolated, but interrelated.Zhang ZhongliangUsing uniform design Cl12 (1210) and T. VirilePers. expr as the strain, et al. (1997) studied the effects of five factors affecting cellulase production on the enzyme production and activity. They believed that the maximum enzyme production of 26 mg/g and CMC enzyme activity of 20 mg/g · h could be obtained when the crude fiber content of the substrate was 40%, the initial pH was 7.5, the addition of water was 4 times, and the condition was incubated at 26-31 ℃ for 45 h.Wang ChenghuaEt al. (1997) also studied the enzyme production conditions of its mutant screened Trichoderma reesei 91-3. The results showed that the strain used 7:3 straw powder and wheat bran, and added 4% ammonium sulfate, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate as the best medium, 28-32 ℃ as the appropriate culture temperature, 30 ℃ as the best temperature, 4% as the best inoculation amount, and reached the fermentation peak in 96h.Zhang Linghua et al. (1998) studied the optimal fermentation conditions of Wu-932 cellulase producing strain obtained by mutagenesis with Trichoderma koningii W-925 as the starting strain.The results showed that the optimum fermentation conditions were as follows: 1: 2 wheat bran and rice straw powder as the medium, 5% inoculation amount, average length of rice straw crushing 3-5 mm, initial pH 4-5, temperature at 28-35 ℃, and fermentation time of 72 h.
Control of contaminating bacteria
There is a common known "white hair fungus" pollution in feed cellulase.After the pollution, the enzyme activity of the light ones decreased, and the fermentation of the heavy ones failed.Therefore, it is of great significance to study and control fermentation pollution.Zhang Linghua et al. (1998) studied the colony characteristics, source, growth, physiological characteristics and control methods of "white hairy fungus", and found a symbiotic relationship with Trichoderma koningii Wu-932, but a competitive inhibition relationship with "white hairy fungus"Candida tropicalisBacteria J-931.Using this strain for mixed fermentation can effectively control the pollution of "white hairy fungus".
Agricultural application
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Cellulase inLivestock and poultry productionApplications in:
Common livestock and poultry feed, such as grain, beans, wheat and processing by-products, all contain a large amount of cellulose.In addition to ruminantsRumen microorganismSome animals, such as pigs and chickens, can be usedMonogastric animalCellulose cannot be used.
The following is the application of cellulase in cattle, chicken and pig diets:
Cattle ration
Jiao Pinglin et al. (1996) conducted an experiment with steers, adding 40 g cellulase per head per day to the diet for 60 days. The results showed that the daily gain of the enzyme added group was 892.78 g, while that of the control group was 746.8 g, with a significant difference (P<0.01).Jiao Pinglin also tested 30 Holstein cows. The test group added 50g cellulase per day. The results showed that the total milk yield of 15 cows in the test group was 2916kg in 68 days, while the total milk yield of 15 cows in the control group was 2689kg in 68 days, with a significant difference (P<0.05).Fu Liansheng et al. (1998) reported that under the normal rumen function, after feeding cellulase for 5 days, the dry matter in the feces of adult cows and bred cows decreased by 30% compared with that before feeding. After one week, the ammonia content in the closed cowshed decreased by about 70%, the coarse feed intake increased by 8-10%, and the urineureaIt decreased by 58.9%. Pregnant cows were fed cellulase 30 days before delivery and did not produce cellulase after deliveryPhysiological dyspepsiaSymptoms: the fetal weight can be increased by 1.5-3kg, and there is no abnormality or weak fetus.The physical fitness of cows recovered quickly, and the peak milk production lasted for a long time (until the fourth lactation month).Zhao Changyou et al. (1998) reviewed the application of cellulase in the diet of herbivores and achieved remarkable results.
Chicken ration
The diet for broilers is generally high in fish meal, corn and soybean meal.In order to reduce the use of these conventional raw materials and widely use cheap feed materials, Qin Jiangfan et al. (1996) increased the proportion of wheat bran rich in fiber in broiler diets by adding 0, 0.05%, 0.1% celluloseEnzyme preparationThe results showed that the daily weight gain of the group added 0.1% cellulase increased by 4.31%, 4.54%, 4.13%, and the material consumption ratio decreased by 1.56%, 4.50%, and 4.3%, respectively, compared with the control group in the growth stages of 1-2, 3-6, and 7-8.Xu Qiyou (1998) added 0.1%, 0.15% and 0.5% cellulase to the diet of laying hens. The results showed that during the laying period from January to October, the egg production rate increased by 0.53%, 1.25% and 2.88% respectively, the egg breaking rate of 0.15% and 0.5% enzyme levels decreased by 34.49% and 16.19%, and the eggshell strength increased by 14.71% and 8.41%, respectively.
Pig ration
according toYin QingqiangEt al. (1992) reported that 0.6% and 1.2% cellulose were added to the basic dietComplex enzymeResults The weight gain of growing finishing pigs was 16.84% and 21.86% higher than that of the control group, respectively.Wank et al. (1993) reported that the addition of cellulase increased the digestibility of neutral detergent fiber from 30.3% to 34.1%, acid detergent fiber from 68.8% to 73.9%, and energy digestibility from 69.3% to 71.8%.
expectation
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China is a country with very tight feed resources. With less land and a large population, the contradiction between people and livestock competing for food is very prominent.In order to maintain the sustainable development of China's feed industry and animal husbandry, we must solve the feed problem, otherwise it will seriously restrict its development.Cellulose is a very rich resource in nature, which is polymerized from 800-1200 glucose molecules.Therefore, you can use theMicrobial fermentationMaking full use of agricultural byproduct leftovers, straw and bran to produce cellulase additives, which can be used to improve the production performance of livestock and poultry, improve feed utilization, improve the nutritional value of feed, reduce feed costs and improve economic benefits, has broad prospects for development. In the future, research and development of cellulase should be further strengthened.It mainly includes the following aspects:
Further strengthen the research on the action mechanism of cellulase
Cellulase is applied to feed and animalsdigestive tractThe mechanism is not yet clear.Theoretically, it is difficult to determine the amount of its addition. There are many factors affecting it, and the effect is often not ideal.There are few studies on the optimal addition amount of cellulase using multiple raw materials alone, which will seriously restrict the popularization and application of cellulase.
The yield and activity of enzyme are not high, and the cost is high
In the future, basic research work such as strain selection and fermentation process should be strengthened to improve its yield and activity, especially the application of DNA gene recombination technology to select strains with high activity and large enzyme production.
Strengthen the research of cellulase detection method
Although there are many detection methods for cellulase, there is no detection method that is really suitable for feed, which brings difficulties to the practical application. For example, it is difficult to compare the product quality of different manufacturers and determine the amount of cellulase added. Relevant forces should be organized to develop a unified detection method standard for use in production.
