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|Title:||Paenibacillus campinasensis BL11 木聚醣酶之特性與突變分析|
Characterization and mutational analysis of a xylanase from Paenibacillus campinasensis BL11
xylanase,mutant,pH stability,temperature stability,substrate specificity,additives,binding,
|Publication Year :||2010|
|Abstract:||比較野生型木聚醣酶 pre-xylX 與兩株變異株（分別是胺基酸取代木聚醣酶 H2 (T44A) 及後段缺失木聚醣酶 L2（胺基酸序列 290 之後鹼基缺失）） 之特性。3 個木聚醣酶在 60oC 經過 8小時後在 pH 5 到 9 仍具有良好活性，另外在 pH 7 耐溫 8 小時試驗中，40oC 到 60oC 均具有良好的活性。在高溫 70oC 下，野生型 pre-xylX 和 突變株 H2 在 2 小時後仍具有 50% 的活性，而突變株 L2 的活性則不到 40%。3 個木聚醣酶對基質具有明顯的專一性，可降解 birchwood 及 oat spelt 木聚醣，但對 CMC, avicel, laminarin, β-D-Glucan, or D-(+)-cellobiose 則沒有良好的降解效果。添加物 Hg2+ 及 N-Bromosuccinimide 對 3 個木聚醣酶具有很強的抑制活性。某些金屬離子及 EDTA 對則對木聚醣酶有部分的抑制活性，其抑制的順序大小為 N- Bromosuccinimide > Hg2+ > Cu2+ > Fe3+ > Zn2+ > Fe2+ > SDS > Pb2+。野生型 pre-xylX、突變株 H2 及 突變株 L2 的 Km 值分別為 6.78 ± 0.59, 3.87 ± 0.39 及 19.23 ± 3.62 mg/mL，而 Vmax 值分別為 4953 ± 73, 11688.02 ± 504.74, 6642.27 ± 961.22 μmol/min mg。野生型 pre-xylX 和突變株 H2 具有吸附能力 (對木聚醣的相對吸附可達80%)，而突變株 L2 則吸附效果不佳 (對木聚醣相對吸附量約 20%)。活性最佳的突變株 H2，能釋出較多還原糖，但吸附效果與還原糖釋出量沒有顯著的關係。另外在飽和低溫吸附試驗中，野生型 pre-xylX 對 3 種紙漿的吸附相似，對原始漿、氧漂漿及全漂漿的最大吸附量分別為115.6、115.9、及115.1 mg/g substrate。但野生型 pre-xylX 對三種漿的親和力大小則為原始漿 (51.8 L/g) > 氧漂漿 (49.3 L/g) > 全漂漿 (43.1 L/g)。在紙漿脫色試驗中，3 種木聚醣酶對釋出發色基團 (OD237 nm and OD465 nm ) 效果則相近。|
Wild type pre-xylX and mutant H2 (amino acid substitution mutant, T44A) and mutant L2 (amino acid sequence deletion mutant after 270) were studied. The pH stability of three xylanases was fully stable from pH5 to 9 after 8 h of incubation. The thermal stability of three xylanases was fully stable from 40 oC to 60 oC after 8 h incubation. At 70oC after 2 h, wild type pre-xylX and mutant H2 retained about 50% of its initial activity while mutant L2 retained lower than 40%. Substrate specificity of three xylanases showed that the xylanases degraded xylans from birchwood and oat spelt, but no delectable activity on CMC, avicel, laminarin, β-D-Glucan, or D-(+)-cellobiose. Additives of Hg2+ and N- Bromosuccinimide strongly inhibited three xylanases activities. Partial inhibition was observed in the some metal ions, and EDTA. Inhibition of the xylanases was in the order of N- Bromosuccinimide > Hg2+ > Cu2+ > Fe3+ > Zn2+ > Fe2+ > Pb2+. On the other hand, 2-ME strongly promoted the xylanases activities, as over 100% residual activity. The Km value of wild type pre-xylX, mutant H2, and mutant L2 were 6.78 ± 0.59, 3.87 ± 0.39, and 19.23 ± 3.62 mg/mL, respectively while Vmax value were 4953 ± 73, 11688.02 ± 504.74, 6642.27 ± 961.22 μmol/min mg, respectively. Relative bound of mutant H2 and wild type pre-xylX were about 80% to insoluble xylan, whereas mutant L2 had no delectable binding ability (relative bound about 20%). Mutant H2 showed the highest activity among three xylanases, and it could release more reducing sugar than the others. There was not specific relationship between binding ability and hydrolysis. In enzyme adsorption study under 4oC, wild type pre-xylX showed similar adsorption ability among three different pulps. Maximum adsorption capacity onto control pulp, oxygen bleached pulp, and fully bleached pulp were 115.6, 115.9, and 115.1 mg/g substrate, respectively. But affinity of wild type pre-xylX was observed in the order of control pulp (51.8 L/g) > oxygen bleached pulp (49.3 L/g) > fully bleached pulp (43.1 L/g). Wild type pre-xylX, mutant H2 and mutant L2 had similar abilities in releasing chromophores (OD237 nm and OD465 nm).
|Appears in Collections:||森林環境暨資源學系|
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