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Trigonopsis variabilis 的 D-amino acid oxidase (DAO, EC
1,4,3,3)是工業上用於生產頭孢菌素類抗生素前驅物
7-aminocephalosporanic acid 的重要酵素。Isoamylase 是
Pseudomonas amyloderamosa SB15 的胞外酵素，能水解支鏈澱粉中
a-1,6-glycosidic bond，並且對生澱粉有極高的 親和力。為了促進
DAO 回收，利用 PCR 法及酵素的剪接，將 T. variabilis 的 dao 基因
與 P. amyloderamosa SB15 isoamylase 基因構築成五種不同型式的融合
基因，並以 T7 啟動子控制，使基因可以在 Escherichia coli BL21
(DE3) 中表現，測試融合蛋白質中 DAO 活性及對生澱粉的吸附能力。五
種融合蛋白質在E.coli BL21 (DE3) 菌體內表現時皆沒有 isoamylase的
活性。融合蛋白質中以 pEA-DI-2 的 DAO 比活性較佳 (0.357 DU/mg)。
當 DAO 位於融合蛋白質的 C 端時活性均不高 (0.012~0.108 DU/mg)。
以 alpha-lactose 或 IPTG 誘導融合基因表現時，於單位體積的培養液
中，以 alpha-lactose 誘導可以得到較高的 DAO 的活性。融合蛋白質
在 pH8.0 時對生澱粉的吸附力在 56% ~ 72% 之間，但是以 10 % 麥芽糖
緩衝液溶析時，僅有 9% 的吸附融合蛋白質被析出。而在測試的融合蛋白
質中以 DI-7 吸附在生澱粉上的 DAO 活性最高，每克生澱粉具有 0.519
DU 。
Trigonopsis variabilis D-amino acid oxidase (DAO, E.C. 1.4.3.3)
has been used in the enzymatic conversion of cephalosporin C to
7-aminocephalosporanic acid. To facilitate the isolation of DAO
from yeast cells, the Pseudomonas amyloderamosa isoamylase
(ISO, E.C. 3.2.1.68) which can be adsorption-elution on raw
starch was fused to DAO in various types. The fusion gene was
expressed in E. coli BL21(DE3) under the control of T7
promoter. The specific activity of DAO was 0.357 DU/mg in DAO:
ISO fusion type. When DAO was fused to the C-terminal of ISO,
the DAO activity of the usion protein was very low (0.012~0.108
DU/mg). When alpha- actose was used as inducer, the DAO
production was higher than hat of induction by IPTG. Western
blotting analysis revealed that most the fusion proteins were
inclusion body and unmature form. PTG and a-lactose can induce
gene expression. About 56~72% of usion proteins could be
adsorbed on raw starch in 100 mM hosphate buffer (pH 8.0),
whereas only 9% of adsorbed fusion roteins could be eluted by
10% maltose buffer (pH8.0). The DAO activity of fusion protein
DI-7 adsorbed on raw starch was 0.519 DU/ gram raw starch.
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