第七章－4+纤维素酶.ppt

1、纤维素酶 （CELLULASE ）,Cellulose is the most abundant carbohydrate in nature (annual production of about 4 1010tons) Cellulose is chemically a very simple polymer. It is a homopolymer consisting of up to 1000 -1,4-linked anhydroglucopyranoside units.,Because of the -configuration around C(1), cellulose c

2、hains interact with each other by hydrogen bonding to form oriented crystalline fibrillar micelles. crystalline fibrillar micelles have little water solubility Due to this inertness to water, cellulose is relatively inert to action of hydrolytic enzymes,Animals do not possess digestive enzymes able

3、to degrade crystalline cellulose. Several microorganisms, including filamentous fungi, yeast, and bacteria are able to hydrolyze cellulose to oligosaccharides and eventually to glucose, which they can then use as a carbon source. Ruminants（反刍动物） are able to fully degrade cellulose in their rumen by

4、the wide spectrum of microorganisms present.,纤维素酶分成三类； 纤维二糖水解酶(CBH)：它对于纤维素具有最高的亲和力，也能降解结晶的纤维素； 1，4葡聚糖酶，它又包括两种酶： 外切1，4葡聚糖酶：从纤维素链的非还原性末端逐个地将葡萄糖水解下来 内切1，4葡聚糖酶(EG)：以随机的方式从纤维素链的内部将它裂开 葡萄糖苷酶(BGL)，作用于小分子量底物时最高活力 。 大多数外切1，4葡聚糖酶将水解下来的葡萄糖的构型从型转变成a-型，而内切1，4葡聚糖酶并不改变产物的构型；,(a) 、Endoglucanases（EG） The endoglucana

5、ses (1,4-D-glucan-4-glucanohydrolase, EC 3.2.1.4) catalyse random cuts in cellulose chains, thereby producing shorter and shorter cello-oligomers, which can be further degraded by exoglucanases.,Endoglucanase are inactive against crystalline celluloses such as cotton and Avicel（微晶纤维）, but they hydro

6、lyze amorphous celluloses (including amorphous regions of crystalline celluloses) and soluble substrates such as carboxymethyl cellulose and hydroxyethyl cellulose,Endoglucanase activity is characterized by random hydrolysis of -glucosidic bond cleavage, resulting in a rapid decrease in viscosity re

7、lative to the rate of increase in reducing groups. The products include glucose, cellobiose, and cellodextrins of various sizes.,（b)、cellobiohydrolases (CBH) The cellobiohydrolases (1,4-D-glucan cellobiohydrolase, EC 3.2.1.91) also act on cellulose and are actually the main enzymes responsible for t

8、he degradation of crystalline cellulose,CBH degrade amorphous cellulose by consecutive removal of cellobiose from the nonreducing ends of the substrate. When pure, they usually have little activity on cotton, but can degrade Avicel（ a microcrystalline cellulose）, by as much as 40%. The rate of incre

9、ase in reducing groups in relation to decrease in viscosity is much higher than for the endo-glucanases. Endoglucanases and cellobiohydrolases act synergistically in the hydrolysis of crystalline cellulose.,The trivial name given by the IUB for endoglucanases is cellulases, which is very misleading

10、as cellobiohydrolases (CBH) The name cellulase covers all enzymes that are able to degrade polymeric celluloses, i.e., both EGs and CBHs.,The final degradation of cello-oligomers produced by EGs and CBHs to glucose is accomplished by -glucosidases (BGL) (EC 3.2.1.21) BGL are often also able to hydro

11、lyze other -glucosidic linkages, and although they are not able to hydrolyze polymeric cellulose . Owing to their action on cello-oligosaccharides, BGL usually considered as part of cellulolytic enzyme system.,（c)、 Exoglucohydrolases (l.4-D-glucan glucobiohydrolase: EC 3.2.1.74). Exoglucohydrolases

12、hydrolyze consecutively the removal of glucose units from the nonreducing end of cellodextrins; the rate of hydrolysis decreases as the chain length of the substrate decreases.,（d) 、-glucosidases(BGL) -D-glucoside glucohydrolase, EC 3.2.1.21), which cleave cellobiose to glucose and remove glucose fr

13、om the nonreducing end of small cellodextrins. Unlike the exoglucohydrolases, the rate of -glucosidase increases as the size of the substrate decreases, with cellobiose being hydrolyzed the fastest.,The -glucosidases and exoglucohydrolases have in common the substrates cellobiose to cellohexaose (si

14、x g1ucose units).,They may readily be distinguished on the basis of their relative activity on the two substrates cellobiose and cellohexaose： The -glucosidases hydrolyze cellobiose much more rapidly than cellohexaose,The determination of cellulase activities is not a very easy task, as the degradat

15、ion of the water-insoluble, highly crystalline cellulose is not linear with time and different enzyme dosages. Therefore, Several nonnatural substrates are normally used.,纤维素酶活力的测定,The international standard assay for measuring EGs is based on the hydrolysis of HEC（羟乙基纤维素） Soluble cereal, 1,3-1,4-gl

16、ucans can also be used as substrates,Amorphous cellulose has been used as a substrate mainly for EGs, but in many cases the enzymatic reaction is not linear or is linear only at the short initial reaction stage.,There are several ways to analyze the degree of degradation of the substrate. the reduct

17、ion in viscosity, the formation of new reducing groups, the liberation of dyed oligosaccharides from chromophoric polymers.,The viscosity method is the most sensitive one and can be used to measure very low levels of EGs The action of EGs and CBHs is difficult to differentiate in the methods based o

18、n the formation of reducing groups. The activity of CBHs is often measured by using small fiuorogenic substrates： methylumbelliferyl cellobioside methylumbelliferyl lactoside.,The activity of -glucosidases is in most cases determined using p-nitrophenyl-D-glucopyranoside as a substrate and measuring

19、 the color formed by the liberated p-nitrophenol.,在测定纤维素酶活力的反应体系中含有各种纤维素酶的混合物，这些酶不仅催化水解相同的底物，而且协同作用因此，纤维素酶活力的测定在很大程度上受酶制剂中各种不同的纤维素酶的比例的影响 为了避免因纤维素的溶解度和结晶性质所造成的困难，在测定纤维素酶活力时，常采用纤维素的衍生物作为底物，例如羧甲基纤维素(CMC)或羟乙基纤维素(HEC),APPLICATION OF CELLULASES IN FOOD PROCESSING,Cellulases are not among the major enzyme

20、s used in food production. In many food applications ，cellulases are not used alone but are added to boost the action of other enzymes, mainly pectinases and/or xylanases,1,3-1,4-Glucans and arabinoxylans are the main components in cereal cell walls and 1,3-1,4-glucans exist in high quantities espec

21、ially in barley and oat. Thus, cellulases are useful enzymes in the processing of cereals.,A. Brewing,The main separation problems during brewing processes are connected to the water-soluble cell wall polysaccharides, i.e., -glucan and xylan. They both may increase viscosity, form gels, and cause se

22、rious problems including poor filtration of the wort, slow run-off times, low extract yields, or the development of haze in the final product,Plant enzymes are not very heat stable and are mostly inactivated in kilning after germination, whereas most microbial enzymes can tolerate much higher temper

23、atures. Thus, exogenous microbial enzymes are often used in brewing, - Glucanases (cellulases) are mainly added in the mashing, but they can also be used during malting or after mashing in primary fermentation,The other option to increase the level of -glucanases is to enhance the level of endogenou

24、s plant enzymes by genetic engineering or by cloning a microbial enzyme to barley.,B. Wine and Juice Production,Even though the amount of -glucans in grapes and fruits is signifcantly lower than in cereals, cellulases have, also found applications in the production of wines and fruit juices where th

25、ey are used in combination with pectinases and hemicellulases.,In wine making, the enzymes are generally used for better skin maceration（软化果皮）, improved color extraction, easier must clarification and filtration, and increased wine quality and stability The use of -glucosidases for aroma development

26、 from naturally present glucosylated precursors,The use of these enzymes offers advantages in the treatment of by products of the fruit industry by improving the extraction yield and the overall process efficiency. Cellulases and hemicellulases can be used for the isolation of pectin from citrus fru

27、its to break down the cell walls, increasing the pectin yield. Cellulases can be used alone or in combination with other cell wall degrading enzymes in all processes in which valuable compounds such as juice, oil, polysaccharides, protein, etc., are extracted from the plant material.,C. Baking,Added

28、 enzymes are used increasingly in baking for improved processing and product quality. Traditionally, the main enzymes used in wheat baking are amylases and xylanases, -Glucanases (cellulases) have gained less attention.,Enzymes acting on cell walls are used especially in the manufacture of high-fibe

29、r bread to overcome processing problems caused by the high content of cell wall material in the dough. Addition of cell wall components decreases the bread volume, and the breadcrumb loses its elasticity.,Soluble, nondigestable polysaccharides, which are often called dietary fiber, are believed to b

30、e beneficial for human health by： lowering cholesterol levels (especially soluble -glucan), preventing colon cancer, acting as specific growth substrates (prebiotics) for beneficial intestinal bacteria (probiotics, mainly lactic acid bacteria and bifidobacteria). Thus, breads with a high fiber conte

31、nt are gaining growing interest.,D. Isolation of Starch and Other Polysaccharides,The major application of food enzymes is in starch processing The starch is mainly isolated from wheat or corn by milling and extraction. The isolation procedure can be further improved by cell wall degrading enzymes,

32、i.e, by -glucanases (cellulases), xylanases, or their mixture,Several food hydrocolloids, mainly carrageenan, alginate, and agar, are isolated from algae. After initial isolation steps, the products often contain some contaminating cellulose from algae cell walls that impairs the functional properti

33、es of these polysaccharides. Cellulases are in some cases used in further purification steps to degrade the residual cellulose, after which the in target polysaccharide can be further isolated.,Cellulases can also be used for controlled degradation of some water-adsorbing polysaccharides used in foo

34、d processing, e.g., CMC, konjac glucomannanl some bacterial exopolysaccharides, such as xanthan, which contain-1,4-glucosidic linkages.,E. Animal Feed Production,A large quantity of cereals or various side fractions of grains from food processing are used as animal feed. In many cereals, part of the

35、ir energy content is locked up in the form of nonstarch polysaccharides, undigestible for several animals. Therefore, selected enzymes, such as cellulases and hemicellulases, can be added to break down the cell walls leading to increased metabolizable energy,The viscous arabinoxylan and 1,3-1,4-gluc

36、an also creates difficulties in the adsorption and digestion of nutrients, especially by monogastric animals such as chicken and pig. The viscosity is considered to be an important constraint to animal digestion by interfering with the diffusion of pancreatic enzymes, substrates, and reaction products. As a consequence, the presence of high levels of these components in feed causes poor feed conversion rates, slow weight gain, and wet droppings, particularl