固定化酵素生產果寡糖與半乳寡糖

Production of fructooligosaccharides and galactooligosaccharides by immobilized enzymes

鄭慈千(Tzu-Chien Cheng)

指導教授 ： 段國仁

大同大學/工程學院/生物工程學系所/博士(2005年)

摘要

中文摘要

本研究以Tris(hydroxymethyl)phosphine (THP) 為交聯劑與幾丁聚醣共交聯分別與β–夫喃果糖苷酶和β–半乳糖苷酶固定生產果寡糖(FOS)與半乳寡糖(GOS)。結果顯示使用THP為交聯劑與幾丁聚醣共交聯固定酵素後，酵素之熱穩定性、重複使用性與產率良好。
從黴菌Aspergillus japonicus (BCRC 930007)純化得到β–夫喃果糖苷酶，分別以Tris(hydroxymethyl)phosphine (THP)與戊二醛為交聯劑與幾丁聚醣共交聯反應以固定β–夫喃果糖苷酶生產果寡糖。以2.5 mg/ml THP或5 % (w/v)戊二醛分別與0.1 g幾丁聚醣及4.2 μg/ml的酵素進行固定化反應，結果顯示自由酵素與固定化酵素的最適pH皆為5.5，最適溫度為60 oC。自由酵素保存於37 oC下，第二天只剩下60 %活性，但是若將THP-固定化酵素保存於37 oC下，11天後仍然有80 %活性，顯然使用THP為交聯劑固定後酵素的熱穩定性良好。THP-固定化酵素在重複使用實驗中，於37 oC下經過11天後仍然有75 %的活性。自由酵素，THP-固定化酵素與戊二醛-固定化酵素的酵素動力參數Km，分別為0.03 g/l (s.d.=0.03), 0.06 g/l (s.d.=0.05) 與0.05 g/l (s.d.=0.03) ，顯示固定化酵素在反應過程中有立體障礙與擴散阻力效應。以50 %蔗糖的基質溶液分別加入固定化酵素與自由酵素(1 unit/g)，於pH 5.5，50 oC下反應，果寡糖產率分別為48 %與58 %。
利用Tris(hydroxymethyl) phosphine(THP)與幾丁聚醣共交聯反應以固定β–半乳糖苷酶生產半乳寡糖(GOS)。以2.5 mg /ml THP活化0.5 g幾丁顆粒及1 mg/ml的酵素進行固定化反應。固定化酵素與自由酵素最適反應溫度為55 oC，最適反應pH為5.0。固定化酵素在重複使用實驗中，於55 oC下經過13天後仍然有75 %的活性，顯示使用THP為交聯劑固定化酵素的熱穩定性良好。自由酵素在pH 5.0，55 oC，36 %乳糖溶液下反應，GOS的產率為43 %，而固定化酵素在相同條件下GOS的產率則為41 %兩者約略相同。

關鍵字

交聯劑 ； 固定化 ； β–半乳糖苷酶 ； 幾丁聚醣 ； β–夫喃果糖苷酶

並列摘要

ABSTRACT

We present the results of this study in the production of fructooligosaccharides (FOS) and galactooligosaccharides (GOS) by immobilized enzymes from sucrose and lactose solution. The β-frutofuranosidase and β-galactosidase were immobilized onto chitosan using tris (hydroxymethyl) phosphine (THP) as a coupling agent to produce fructooligosaccharides and galactooligosaccharides, respectively. Our results demonstrated the possible industrial application of the THP-immobilized β–frutofuranosidase and -galactosidase for continuous production of fructooligosaccharides and galactooligosaccharides at longer duration and higher temperature tolerance at 55 oC. Our study show the THP-immobilization has better thermal stability then the glutaraldehyde as a coupling agent.
For the production of fructooligosaccharides, β–frutofuranosidase from Aspergillus japonicus (BCRC 930007) was immobilized onto chitosan using tris (hydroxymethyl) phosphine (THP) and glutaraldehyde as coupling agents to produce FOS. The optimal pH was 5.5 and the optimal temperature was 60oC for both free and two immobilized enzymes. The THP-immobilized β–frutofuranosidase retained more than 75 % activity after 11 batches (or days) of FOS production at 37oC. The THP-immobilized enzyme had higher reusability than that immobilized by glutaraldehyde. The Km for the free, THP-immobilized and glutaraldehyde-immobilized enzyme were 0.03 g/l (s.d.= 0.03), 0.06 g/l (s.d.= 0.05) and 0.05 g/l (s.d.= 0.03), respectively. The yield of FOS with the free enzyme from the sucrose solution (50 %, w/v) at 50 oC was 58 % and the THP-immobilized enzyme (48 %) is less than the free enzyme.
For the production of galactooligosaccharides, the β-galactosidase was immobilized on to chitosan using tris (hydroxymethyl) phosphine (THP) as a coupling agent to produce galactooligosaccharides from lactose solution. Both the THP-immobilized and the free enzymes were maximally achieved at pH 5.0 and the optimal temperature was 55 oC. The THP-immobilized enzyme still can remain 70 % residual activity from 36 % (w/v) of lactose after 13 batches (or days) at 55oC. This show the THP-immobilized has better thermal stability. The yield of GOS with the free enzyme from the lactose solution (36 %, w/v) at 55 oC is 43 %, which is about the same as that with the THP-immobilized counterpart with 41 % yield.

並列關鍵字

chitosan ； immobilized ； tris (hydroxymethyl) phosphine (THP) ； β-frutofuranosidase ； β-galactosidase

參考文獻

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