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1、毕 业 设 计（论 文）外 文 参 考 资 料 及 译 文潜在的饲料添加剂在瘤胃液中的稳定性学生姓名： 学号： 专业： 动物科学 所在学院： 动物科学与技术学院 指导教师： 职称： 副教授 Stability and stabilization of potential feed additive enzymes in rumen fluidDiego P. Morgavia, 1, C.James Newbolda，David E. Beeverb ，R.John WallaceAbstractSome activities, including the -1,4-endoglucanase
2、 and xylanase from the extract derived from Aspergillus niger, were stable for at least 6 h in rumen fluid. The same activities in the other extracts also retained substantial activity for several hours. -Glucosidase and -xylosidase activities were much more labile. most being almost completely dest
3、royed after 1. and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most proteins in the extracts were digested extensively after up to 7 h of incubation. Adding bovine serum albumin (0.5 g/l) to the incubation increased the half-life of Trichoderma viride -glucosidase activi
4、ty from less than 0.5 h to 3 h. Proteins extracted from plant materials, particularly the soybean 7S globulin fraction, also conferred protection from proteolytic breakdown，but none was as effective as bovine serum albumin. It was concluded that the stability of most fibrolytic enzymes in rumen flui
5、d, is not likely to be a limiting factor in the use of enzymes as feed additives for ruminants. but if the enzymes are not stable, means can be found for their stabilization.KeywordsEnzymes; Ruminants; Cellulase; Xylanase; Stability; Proteolysis1. IntroductionIndustrially produced enzymes are increa
6、singly being used as feed additives to improve the nutritional efficiency of farm animals. Their application in ruminant diets is still under development. Early results show promise, although many factors such as the precise nature of beneficial enzymes and the diet specificity of the response, rema
7、in to be resolved. A potential problem, which was identified previously by Kopecny et al. is that protein in the diet, particularly soluble protein, is usually degraded rapidly in the rumen. Thus, if dietary enzymes are to be effective as modifiers of rumen fermentation, the enzymes must resist prot
8、eolysis by rumen microorganisms for a time sufficiently long to affect digestion.The rate and extent of hydrolysis of individual proteins are affected by their chemical structure: Most commercial enzyme preparations are the product of fungal fermentation, predominantly by Trichoderma and Aspergillus
9、 species, and they consist of a mixture of hydrolytic enzymes. As some of these are used as feed additives in nonruminants, they presumably resist degradation by gastric and pancreatic proteinases and may have structures that are also resistant to rumen microbial proteases.The objective of this stud
10、y was to determine the resistance of the enzyme activities in potential feed additives to rumen microbial proteolytic activity, and to investigate means of enhancing their resistance to proteolytic breakdown.2. Materials and methods2.1. Enzyme preparationsThe enzymes used were powdered commercial pr
11、eparations from fungal extracts that contained a variety of plant polysaccharide hydrolases. 2.2. Incubations with rumen fluidRumen inocula were obtained from four rumen-fistulated sheep receiving a mixed diet consisting of hay, barley, molasses, fish meal, and minerals and vitamins fed in equal mea
12、ls of 500 g at 8 a.m. and 4 p.m. Rumen fluid was removed before the morning feeding and strained through four layers of muslin. Enzyme preparations, calculated to have similar carboxymethylcellulase (CMCase) activities, were added to Hungate tubes and dissolved in 1 ml of 0.1 M anaerobic sodium phos
13、phate buffer, pH 6.5. Then 9 ml of strained rumen fluid were added, the tubes were incubated at 39C under O2-free CO2 and samples were removed periodically into microcentrifuge tubes on ice. Protein concentration in the incubation mixture ranged from 0.15 to 0.30 g/l. Particulate material was remove
14、d by centrifugation at 15 000 g for 15 min. The supernatant was stored at 80C until analyzed.2.3. Stabilization of enzyme activityThe influence of bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) or plant proteins on enzyme stability was examined by using the T. viride preparation. Incubations
15、were performed as described above, with the experimental proteins being added to the enzyme solution before addition of rumen fluid. Plant proteins used were maize zein (Sigma) and those obtained from soybean and rice flours. Soluble soybean proteins were extracted with 15 volumes (w/v) of 0.03 M Tr
16、is-HCl buffer, pH 8, containing 10 mM2-mercaptoethanol for 1 h. The slurry was mixed thoroughly at 5-min intervals and centrifuged at 45 000 g for 20 min to obtain the soluble protein fraction in the supernatant.2.4. Enzyme assaysEndocellulase, xylanase, and amylase activities were assayed by using
17、medium viscosity carboxymethyl cellulose (CMC), oat spelt xylan, and soluble wheat starch as substrates, respectively, and all were obtained from Sigma. Assays were carried out by adding 50 l of sample to a tube containing 100 l of 0.1 M sodium citrate/phosphate buffer, pH 5, and 50 l of 2% CMC, 2%
18、oat spelt xylan, or 1% wheat starch, respectively. The mixture was incubated at 39C for 1 h for CMCase, 30 min for xylanase or 4 h for amylase. The reaction was stopped by adding Somogyi reagent, and reducing sugars generated were measured by the NelsonSomogyi method. Incubations were also carried o
19、ut with sample in the absence of added substrate and with enzyme solutions alone plus buffer in place of rumen fluid. Each sample was incubated in duplicate and results are expressed as means of samples from four sheep.Glycosidase activities were measured in 96-well plates. Each well contained 10 1
20、of sample, 80 l of 0.1 M sodium citrate/phosphate buffer, pH 5, and 10 l of 50 M 4-methylumbelliferyl-d-glucopyranoside(Sigma) for -glucosidase and -xylosidase, respectively. Substrates were prepared by diluting 20 l of 25 mM stock dimethylformamide solution in 10 ml of water. The plates were incuba
21、ted at 39C for 5 min for -glucosidase or 60 min for -xylosidase, then the reaction was stopped by adding 100 l of 1 M glycine-NaOH buffer, pH 10.6. Release of 4-methylumbelliferone was measured fluorometrically at 365-nm excitation and 450-nm emission. Each assay was carried out in triplicate. Resul
22、ts are expressed as means samples from four sheep.The proteolytic activity of strained rumen fluid was measured by using digesta taken immediately before feeding, and also using digesta taken 2 h after feeding. Proteolytic activity was measured by the rate of breakdown of casein, which had been labe
23、led with 14C by reductive methylation。 The results are means of duplicate incubations with each sheep.2.5. Electrophoretic analysis of enzymic propertiesElectrophoresis was performed with a Multiphor II electrophoresis system and ExcelGel SDS 818% precast gradient polyacrylamide gels (Pharmacia, St.
24、 Albans, Herts AL1 3AW, UK). Samples were treated with SDS under reduced (in the presence of DL-dithiothreitol) or nonreduced conditions as described by Pharmacia. Proteins were stained with Coomassie Blue or silver staining and glycoproteins were detected with a Glycoprotein Detection Kit (Sigma) b
25、ased on the periodic acid-Schiff reagent method. Samples were obtained after incubation of the enzyme preparations with rumen fluid. Rumen fluid from three animals obtained as described above was pooled and incubated with the enzyme preparations under CO2 for 7 h in a shaking water bath at 39C. Afte
26、r incubation, particulate material was removed by centrifugation and the supernatant was dialyzed for 24 h against distilled water. Sample aliquots were stored at 80C until electrophoresis. For 0 h incubations, chilled rumen fluid was added to the enzymes and kept on ice for 1 to 2 h to allow enzyme
27、 solubilization.3. Results3.1. Addition of enzymes to rumen fluidThe enzyme mixtures were added to rumen fluid in quantities sufficient to give a significant increase in the total extracellular CMCase activity of rumen fluid. As a consequence, xylanase, amylase, -glucosidase, and -xylosidase activit
28、ies also increased. All of these enzyme activities were measured against a background of similar activities already present in rumen fluid. However, because the assays were carried out by using supernatants after centrifugation, from which microorganisms had been removed, the interference in measuri
29、ng the added enzymes was not great. Furthermore, all enzyme activities present in extracellular rumen fluid remained relatively constant throughout the incubations 。3.2. Proteinase activity of rumen fluidThe proteinase activity of strained rumen fluid was measured by using 14C-labeled casein. Proteo
30、lytic activity in the four sheep was 2.10, SD 0.54 and 2.19, SD 0.57 mg casein hydrolyzed/h/ml strained rumen fluid immediately before and 2 h after feeding, respectively.3.3. Stability of enzyme activity in rumen fluid-1,4-Endoglucanase activity in the A. niger preparation, measured by the release
31、of reducing sugars from CMC, was particularly stable when added to rumen fluid in vitro, with no apparent decrease in activity after 6 h of incubation in rumen fluid. The other enzyme preparations had a half-life of 2 h for T. viride and about 4 h for I. lacteus and preparation M. Activity against x
32、ylan was more stable than the CMCase activities, except for I. lacteus, which lost 60% of its xylanase after 2 h of incubation. A. niger xylanase was not affected by the incubation, whereas T. viride and preparation M retained nearly 75% of the original activity at the end of the incubation period.
33、Amylase was not a predominant activity in any the preparations. The highest amylase activities were detected in preparation M and T. viride, but they were not stable. The A. nigerenzyme was again the most stable 。3.4. Relation between enzyme stability and chemical structureThe electrophoretic mobili
34、ty of proteins present in the enzyme extracts was determined in SDS-PAGE, with and without reduction, and before and after incubation with rumen fluid. Multiple protein bands were present in all of the extracts, particularly I. lacteus, and most of these bands disappeared after incubation in rumen f
35、luid. However, certain bands appeared to survive after 7 h of incubation. The bands that survived this incubation were different in the presence and absence of dithiothreitol, indicating that they were cross-linked by disulfide bonds. In general, however, the banding patterns in the presence and abs
36、ence of dithiothreitol were completely different, and it was not possible to correlate the banding patterns. An attempt was made to associate this characteristic with the presence of cystine in the protein molecule, by analysis of bands excised from the gels, but no conclusive results were found. Ge
37、ls were stained for glycosylated proteins: glycosidation was a common feature in all the enzyme preparations (results not shown). However, it appeared that there was no direct relation between resistance to proteolysis and the presence of sugar moieties in proteins.4. DiscussionThe effectiveness of
38、dietary enzymes in nonruminants is assumed to stem partly from their marked resistance to proteolytic degradation If enzymes are to be effective in diets fed to ruminants, it is reasonable to assume that similar resistance will have to be among their properties. Indications from an earlier study wer
39、e that cellulases from T. viride were not stable when incubated with rumen digesta. The rumen microbial population is generally perceived, because of the significance of rumen proteolysis in depriving the host animal of much of the protein it consumes, as being highly proteolytic. In fact, in microb
40、ial terms the activity is not particularly high; it is the high microbial biomass and long residence time of protein in the digesta, which causes the high extent of breakdown of susceptible proteins. Not all proteins are susceptible to rapid breakdown, depending on their solubility and structure. Th
41、e susceptibility to ruminal breakdown of the proteins and enzyme activities present in several commercial enzyme preparations was therefore measured in order to assess whether fibrolytic enzymes were likely to be sensitive to ruminal proteolysis, and if this was likely to be an obstacle to their pot
42、ential application as feed additives for ruminants.The experiments were done by adding enzyme mixtures to rumen fluid taken from sheep receiving a grass hay/concentrate diet. The rumen fluid was strained through muslin to remove large particulate material and therefore to aid pipetting. The rumen fl
43、uid was also taken before the morning feeding in order to minimize problems associated with soluble sugars being present in the rumen fluid after feeding, which would affect the subsequent measurement of glycanase activities. It was established that the proteolytic activity of rumen fluid immediatel
44、y before and 2 h after feeding was similar, suggesting that variation in proteinase according to the time of sampling would not have a large bearing on the results; however, the straining process would remove many of the solids-associated microorganisms, and the proteolytic activity of the strained
45、rumen fluid would inevitably be lower than whole digesta. The extent of this underestimate was not determined; however, another advantage of taking prefeeding samples would be that the solids-associated microbial population would presumably be minimal at this time.After incubation of the enzyme prep
46、arations with the strained rumen fluid, small particulate material, principally microorganisms, was removed by centrifugation. The background cell-free activity due to rumen fluid was usually much lower than the activity of enzymes added, and it was in any case fairly stable. It should be noted that
47、 enzyme activity determinations were performed at pH 5, closer to the optimal pH of most fungal enzymes than the usual pH of rumen fluid, which is 66.5.It is concluded that the stability of fibrolytic enzymes in rumen fluid is unlikely to be a limiting factor in their application as feed additives f
48、or ruminants. Particular enzyme activities may, however, be more labile than others. If such an activity is a key component of the mode of action of feed-additive enzymes for ruminants, it would appear likely that enzyme activity might be protected by simple means. The emphasis should now be placed on identifying which, if any, fibrolytic activity is limiting in the rumen and on devising means of amplifying that activity. Applying enzymes as feed supplements promises to be the simplest technology for achieving such an amplification in the immediate future.潜在的
