What Is Cellulase?
Cellulase (-1,4-glucan-4-glucan hydrolase) is a collective name for a group of enzymes that degrade cellulose to produce glucose. It is not a monomeric enzyme, but a multi-component synergist. The enzyme system is a complex enzyme mainly composed of exo-beta-glucanase, endo-beta-glucanase and beta-glucosidase, etc., as well as a highly active xylanase. Acts on cellulose and products derived from cellulose. Microbial cellulase is of great significance in transforming insoluble cellulose into glucose and destroying cell walls in fruit and vegetable juice to improve fruit juice yield.
- Chinese name
- Cellulase
- Nature
- Complex enzyme
- Application area
- Feed, alcohol, textile and food, etc.
- CAS
- NO.9012-54-8
- Cellulase (-1,4-glucan-4-glucan hydrolase) is a collective name for a group of enzymes that degrade cellulose to produce glucose. It is not a monomeric enzyme, but a multi-component synergist. The enzyme system is a complex enzyme mainly composed of exo-beta-glucanase, endo-beta-glucanase and beta-glucosidase, etc., as well as a highly active xylanase. Acts on cellulose and products derived from cellulose. Microbial cellulase is of great significance in transforming insoluble cellulose into glucose and destroying cell walls in fruit and vegetable juice to improve fruit juice yield.
Cellulase Basic Information
- Cellulase (English: cellulase) is a kind of enzyme that plays a role in biocatalysis when breaking down cellulose.
- hengno-CA type neutral cellulase (powder)
- Cellulases are widely found in organisms in nature. Cellulase can be produced by bacteria, fungi, and animals. The cellulases commonly used for production come from fungi, and the typical ones are Trichoderma, Aspergillus, and Penicillium.
- Cellulase-producing bacteria are easily degraded, resulting in reduced enzyme-producing ability.
- Bacterial cellulase production is less, mainly dextran endonucleases, most of which have no degrading activity on crystalline cellulose, and the enzymes produced are mostly intracellular enzymes or adsorbed on cell walls and are not secreted to the culture fluid In addition, the difficulty of extraction and purification is increased, so there is less research on bacteria. However, the optimum pH of cellulase produced by bacteria is neutral to alkaline. In the past 20 years, with the successful application of neutral cellulase and alkaline cellulase in cotton fabric washing process and detergent industry, bacterial cellulase preparations have shown good application prospects.
- Cellulase is widely used in the food industry and the environmental industry. In alcohol fermentation, the addition of cellulase can increase the utilization of raw materials and improve the quality of wine.
- Because cellulase is difficult to purify, it generally contains hemicellulase and other related enzymes, such as amylase and protease.
- There are many types of cellulases and a wide range of sources. Cellulase from different sources varies greatly in structure and function. Due to the high yield and activity of fungal cellulase, the cellulase used in animal husbandry and feed industry is mainly fungal cellulase.
- English name Cellulase
- English alias Cellulase [USAN]; Ku-zyme; Kutrase; Cellulase, aspergillus niger; Cellulase, trichoderma viride; Fungal cellulase
- EINECS 232-734-4
Cellulase classification
Cellulase by composition and function
- Cellulases can be divided into endoglucanases (1,4--D-glucan glucanohydrolase or
- Molecular structure of cellulose and chitin
Cellulase
- The cellulase reaction is different from the general enzyme reaction. The main difference is that the cellulase is a multi-component enzyme system and the substrate structure is extremely complicated. Due to the water insolubility of the substrate, the adsorption of cellulase replaced the ES complex process formed by the enzyme and the substrate. Cellulase first adsorbs specifically on the substrate cellulose, and then breaks down cellulose into glucose with the synergy of several components.
- In 1950, Reese et al. Proposed the C1-Cx hypothesis, which believed that different enzymes must work together to completely hydrolyze cellulose to glucose. The synergy is generally considered to be that the (C1 enzyme) first attacks the non-crystalline region of cellulose to form a new free end required for Cx, and then the CX enzyme cuts the cellobiose unit from the reducing or non-reducing end of the polysaccharide chain, and finally Cellobiose is hydrolyzed to two glucose by -glucanase. However, the order of cellulase synergy is not absolute, and subsequent research found that C1-Cx and -glucanase must coexist in order to hydrolyze natural cellulose. If the crystalline cellulose is first treated with the C1 enzyme, then the C1 enzyme is removed, and then the Cx enzyme is added, the crystalline cellulose cannot be hydrolyzed in this order. [1]
Factors affecting the action of cellulase
- The optimum pH of cellulase is generally 4.5 ~ 6.5. Gluconolactone can effectively inhibit cellulase. Heavy metal ions such as copper and mercury ions can also inhibit cellulase, but cysteine can eliminate their inhibitory effect and even activate cellulase. Plant tissue contains natural cellulase inhibitors; it protects plants from the effects of mold decay. These inhibitors are phenolic compounds. If high oxidase activity exists in plant tissue, it can oxidize phenolic compounds to quinones, which can inhibit cellulase [1] .
Cellulase strain selection
- Bacterial breeding is the basic work of cellulase production. Many experts at home and abroad have carried out a lot of research. In order to produce high-quality cellulase products, Wang Jialin et al. (1996) have successively introduced Green Trichoderma spp. 10, Trichoderma spp. Sn-91014, Trichoderma koningii NT-15, Aspergillus niger XX-15A, on the basis of which, UV, specific electromagnetic wave radiation, linear accelerator, nitrosguanidine and other physics, High-yield strains NT15-H, NT15-H1, XT-15H, and XT-15H1 were obtained by chemical mutagenesis. Among them, the solid culture vigor of Trichoderma NT-15H was tested by Nanjing Station of Food Quality Supervision and Testing Center of the Ministry of Light Industry. The filter paper viability was 3670u / g, C1-enzyme activity was 24460u / g, and Cx-enzyme activity was 1800u / g, which has reached international advanced Level. This strain is stable in factory production. Zhang Linghua et al. (1998) used Trichoderma koningii W-925, J-931, and after compound mutagenesis with 2% diethyl sulfate and ultraviolet (15W, 30cm, 2min), Wu- with high enzyme production activity was obtained. For 932 strains, the CMC saccharification power of this strain reached 2975, and the filterase saccharase activity was 531, which was 100% and 81% higher than the starting bacteria W-925, respectively. Wang Chengshu, etc. of the Feed Additive Technical Service Center of the Ministry of Chemical Industry (1997) used the center's Trichoderma reesei A3 to perform mutagenesis by ultraviolet and nitrosoguanidine, and then inoculated the treated spores on a fiber double-layer plate, 30 ° C. After 5-8 days of cultivation, and 7-10 days at 15 ° C, single colonies with larger transparent circle diameter and colony diameter were selected for solid-state fermentation and re-screening. Trichoderma reesei 91- 3 strains.
- Cellulase strains are susceptible to degradation, and their enzyme production is significantly reduced after degradation. There may be three reasons for this:
- The mutants screened by mutagenesis were back-mutated.
- Natural negative mutation.
- The long-term low-temperature oblique storage of the bacteria will produce secondary hyphae on the conidia, and the conidia formed by the secondary hyphae has weak vitality, which may be the main reason for the degradation of the bacteria. In order to avoid degradation of cellulase strains, Zhang Linghua et al. (1998) reported that the soil strain was used to preserve the strains. The sand and soil to be sieved and washed are mixed into a test tube at a ratio of 3: 2, and then sterilized under pressure of 1 kg / cm2 for 30 minutes for three times. Inject 0.5ml into the soil tube, shake it, and put it into a vacuum desiccator containing anhydrous CaCl2 to store it. It was determined that the enzyme activity remained basically unchanged in the 121 days measured; the time for the 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.
Cellulase fermentation process
- There are two main cellulase production processes, solid fermentation and liquid fermentation. The processes are as follows:
- Factors affecting enzyme production and vitality
- There are many factors that affect cellulase production and viability. In addition to bacteria species, there are also culture temperature, pH, moisture, substrate, and culture time. These factors are not isolated but interconnected. Zhang Zhongliang et al. (1997) used the uniform design Cl12 (1210), and took T. VirionPers.expr as the strain to study the effect of five factors affecting cellulase production on enzyme production and activity. The fiber content was 40%, the initial pH was 7.5, water was added four times, and the maximum enzyme yield was 26 mg / g and the CMC enzyme activity was 20 mg / g · h when cultured at 26-31 ° C for 45 hours. Wang Chenghua et al. (1997) also studied the mutagenesis screening conditions of Trichoderma reesei 91-3, and the results showed that the strain used 7: 3 straw powder and wheat bran, and added 4% ammonium sulfate, 0.4 % Potassium dihydrogen phosphate and 0.1% magnesium sulfate are the best medium, 28-32 ° C is the optimum culture temperature, 30 ° C is the best temperature, 4% is the best inoculation amount, and the fermentation peak is reached at 96h. Zhang Linghua et al. (1998) studied the optimal fermentation conditions of Wu-932 cellulase-producing bacteria obtained by mutagenesis using Trichoderma koningii W-925 as the starting bacteria. The results showed that using 1: 2 wheat bran and straw powder as the medium, 5% inoculation amount, the average length of straw crushing was 3-5mm, the initial pH was 4-5, the temperature was 28-35 ° C, and the fermentation time was 72h. condition.
- Control of contaminated bacteria
- Commonly known as "white wool" contamination in feed cellulases. After contamination, the enzyme activity of the lighter decreased, and the fermentation of the severer failed. For this reason, it is of great significance to study the control of fermentation pollution. Zhang Linghua et al. (1998) studied the colony characteristics, origin, growth and physiological characteristics and control methods of "White Hair Fungus", and found a symbiotic relationship with Trichoderma Wu-932 and competed with "White Hair Fungus" Sexual inhibition of Candida tropicalis J-931. Using this bacteria for mixed fermentation can effectively control the pollution of "White Hair Fungus".
Cellulase agricultural applications
- Application of cellulase in livestock and poultry production :
- Common livestock and poultry feeds such as grains, beans, wheat and processed by-products contain a lot of cellulose. Except for ruminants, which can be used by rumen microorganisms, other animals, such as pigs and chickens, cannot use cellulose.
- The following are the applications of cellulase in cattle, chicken and pig diets:
Cellulase Cattle Diet
- Jiao Pinglin et al. (1996) used a yak test to add 40g of cellulase per head per day to the diet for 60 days. The results showed that the daily weight gain of the enzyme-added group was 892.78g and that of the control group was 746.8g. The difference was extremely significant (P <0.01). Jiao Pinglin used 30 Holstein dairy cows for the test. The test group added 50g of cellulase per head per day. The results showed that the total milk yield of 15 cows in the test group in 68 days was 2916 kg, while 15 cows in the control group were in The total milk yield for 68 days was 2689 kg, a significant difference (P <0.05). Fu Liansheng et al. (1998) reported that under normal rumen function, adult cows and mature cows were fed with cellulase for 5 days, and their fecal dry matter was reduced by 30% compared with before feeding. One week later, closed cattle The ammonia content decreased by about 70%, the feed intake of coarse feed increased by 8-10%, and urea in urine decreased by 58.9%. Pregnant cows were fed cellulase starting 30 days before delivery, and there was no physical indigestion after delivery. Fetal weight can be increased by 1.5-3kg without deformities and weak fetuses. Cattle recovers quickly and milk production peaks last long (until the fourth lactation month). Zhao Changyou et al. (1998) reviewed the application of cellulase in herbivore diets, and achieved significant results.
Cellulase Chicken Diet
- The diet of broilers is generally based on high fish meal, high corn, and high soybean meal. In order to reduce the use of these conventional ingredients and widely use cheap feed ingredients, Qin Jiangfan et al. (1996) increased the proportion of fiber-rich wheat bran in broiler diets by adding 0, 0.05%, and 0.1% cellulase preparations for testing The results showed that the 0.1% cellulase group increased daily weight gain by 4.31%, 4.54%, and 4.13% in the three growth stages of 1-2, 3-6, and 7-8 weeks compared with the control group, and the consumption ratio decreased by 1.56. %, 4.50%, 4.3%. Xu Qiyou (1998) added 0.1%, 0.15%, and 0.5% cellulase to the diet of laying hens. The results showed that the egg production rate increased by 0.53%, 1.25%, and 2.88% respectively during the laying period from January to October. The broken egg rates of the enzyme levels of 0.15% and 0.5% decreased by 34.49% and 16.19%, and the egg shell strength increased by 14.71% and 8.41%, respectively.
Cellulase swine diet
- According to Yin Qingqiang et al. (1992), adding 0.6% and 1.2% cellulose complex enzymes to the basic diet resulted in a 16.84% and 21.86% increase in weight gain in growing and finishing pigs compared to the control group. Wank et al. (1993) reported that the addition of cellulase increased the neutral detergent fiber digestibility from 30.3% to 34.1%, the acid detergent fiber digestibility from 68.8% to 73.9%, and the energy digestibility from 69.3% to 71.8%. .
Cellulase Outlook
- China is a country with very tight feed resources. With less land and a large population, the conflict between people and livestock for food is very prominent. In order to maintain the sustainable development of China's feed industry and animal husbandry, the feed problem must be solved, or its development will be severely restricted. Cellulose is a very rich resource in nature. It is the aggregation of 800-1200 glucose molecules. Therefore, microbial fermentation can be used to make full use of agricultural byproducts, straw, and bran to produce cellulase additives, which can be used to improve the performance of livestock and poultry, improve feed utilization, improve the nutritional value of feed, reduce feed costs and increase economic benefits. Broad development prospects, the research and development of cellulase should be further strengthened in the future. The main aspects are as follows:
- Further Strengthening the Mechanism of Cellulase
- Cellulase is applied to feed and acts on the digestive tract of animals. The mechanism is not clear. It is still difficult to determine the amount of addition in theory, there are many affected factors, and the effect is often not ideal. There is not much research on the optimal addition amount of cellulase using multiple raw materials alone, which will seriously restrict the promotion and application of cellulase.
- The enzyme yield and activity are not high, and the cost is high
- In the future, basic research such as strain selection and fermentation processes should be strengthened to improve its yield and activity. In particular, attention should be paid to the use of DNA gene recombination technology to select strains with high activity and high enzyme production.
- Study on Strengthening Cellulase Detection Methods
- Although there are many detection methods for cellulase, there is no detection method that is suitable for feed. This brings difficulties to practical applications. For example, it is difficult to compare the product quality of different manufacturers and determine the amount of cellulase added. With regard to forces, a unified standard for testing methods was developed for application in production.