Production and utilization of cellulytic enzyme and protease from rumen bacteria
Date Issued
2004
Date
2004
Author(s)
Wang, Han-Tsung
DOI
zh-TW
Abstract
Part I
The best growth performance and cellulase production were obtained as the ruminal cellulolytic bacterium Fibrobacteria succinogenes S85 was incubated in ATCC medium 1715E with 0.2% cellobiose for 48 to 96 hr at 39℃. However, higher level of cellobiose (1.0%) had no benefit on bacterial cellulase production. The use of filter paper as a carbon source in enzyme production medium without rumen fluid resulted in the best enzyme production of F. succinogenes S85 after 72 hr of incubation. After passing through ion exchange and gel filtration columns, the extra cellular fraction (ECF) of F. succinogenes S85 yielded two groups of cellulase with molecular weights of 60 and 120 kDa. Sucrose wash incurred higher cellulase activity and more protein than Triton X-100 extraction for recovering cell surface associated enzymes from F. succinogenes S85. Groups of proteins of 40 to 50 kDa molecular weight with cellulase activity was purified from cell surface sample of F. succinogenes S85. Xylanase of 62 kDa molecular weight was purified from Ruminococcus albus 7 ECF by using ion-exchange and gel filtration column, and the specifity activity of the xylanase was 94 μg xylose/min/mg. Cellulose binding protein (CBP) from F. succinogenes S85 was collected by adherence to Avicel PH 101 followed by 5% SDS buffer elution. Specific activities of cellulase from F. succinogenes S85 and xylanase from R. albus 7 were increased by 1.1 to 8.3 times in the presence of CBP. The result showed that CBP can effectively support non-cellulase fibrolytic enzyme from other rumen fibrolytic bacterium.
Part 2
In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used to examine the optimal condition for protease production. The carbon and nitrogen sources for the maximum growth of bacteria were sucrose and peptone. Both sucrose and glucose could stimulate protease production. Casein and peptone were the better nitrogen sources for protease production. The best enzyme production condition was 18 to 20 hours incubation at 39℃in the broth of 5% glucose or sucrose as carbon source with 0.1% ammonium chloride plus 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and 18% on cell surface, protease activity was low in periplasmic and intracellular portions. The optimal condition for protease reaction were 40℃and pH 6.8. The crude extract maintained 50% of its protease activity at 30 and 50℃, and its protease activity was stable between pH 6 to 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause the inhibition of proteolysis. Protein feedstuff degradation test showed that protease in crude extract had high degradation ability on fish meal, whey, and feather meal ( 2.39 , 2.60 and 1.76μmol amino acid/mg/hr ) in comparison with soybean meal and blood meal ( 1.11 and 1.09μmol amino acid/mg/hr ) .
Part 3
Cellulase from Aspergillus niger
Subjects
瘤胃細菌
纖維分解酵素
蛋白質分解酵素
酵素保護
rumen bacteria
cellulytic enzyme
protease
enzyme protection
Type
thesis
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