以生物高分子為載體進行胞內固定化酵素之開發

Development and in vivo immobilization of enzyme on PHA granules

黃騰毅(Teng-Yi Huang)

指導教授 ： 陳姍玗

元智大學/工程學院/生物科技與工程研究所/碩士(2011年)

摘要

D-HPG為非天然之D-amino acid，可作為抗生素中β-lactam系列之合成前驅物，其β-lactam系列之抗生素是目前醫藥市場主要使用之抗生素。藉由生物法生產D-HPG須經由兩個酵素反應步驟，首先由酵素D-hydantoinase (HDT)將 DL-hydroxylphenyl hydantoin (DL-HPH)轉化成N- carbamoyl-D-p-hydroxylphenyl glycine (CpHPG)，接著再經酵素D- carbamoylase (AHL)之反應生成D-HPG，而D-p-hydroxylphenyl glycine (D-HPG)是合成這些抗生素所需要之中間體，是深具商業價值之產物。聚羥基烷酯（Polyhydroxyalkanoates, PHAs）為許多微生物處於營養限制之環境下利用過多之碳源聚合成之高分子聚酯化合物，其在微生物生理扮演著儲存碳源及還原之角色。於前人研究發現當PHA產量提高時會使得固定化D-hydantoinase活性增加，於此我們藉由基因工程將phasin (PhaP)或PHA synthase (PhaC)與D-hydantoinase融合，於能大量累積PHA之宿主Ralstonia eutropha H16中表現此融合蛋白，於胞內產生固定化酵素，於研究結果得知Ralstonia eutropha H16/pBBR1MCS2-phaP-hdt與Ralstonia eutropha H16/pBBR1MCS2-phaC-hdt之最佳IPTG誘導劑添加濃度各別為500 μM與1000 μM，最適化反應溫度為60℃與55℃。於最適化條件下固定化酵素之活性各別為25 U與27 U，於40℃耐受性測試中自由酵素半衰期為50小時而固定化酵素分別為135小時與120小時，固定化後約提升2.7倍與2.4倍，重複使用測試均於重複使用8次後仍保有80%之轉化率，於保存性測試各別於保存43天與40天後仍保有50%以上的活性，且於結果發現Ralstonia eutropha H16/pBBR1MCS2-phaP-hdt酵素穩定性較優於Ralstonia eutropha H16/pBBR1MCS2-phaC-hdt，但Ralstonia eutropha H16/ pBBR1MCS2- phaC-hdt酵素活性均優於Ralstonia eutropha H16/ pBBR1MCS2-phaP- hdt。從SEM觀察中發現，以PHA synthase融合D-hydantoinase之固定化酵素及phasin 融合D-hydantoinase之固定化酵素granules各別大小為0.5~2 μm與0.2~1 μm，相較於PHA granules1~3 μm要來的小，推測融合酵素的鍵結可能會影響PHA granules在合成時的大小，也於文獻中也有指出PhaP能夠調控PHA granules之大小，且於R. eutropha H16 大量表現PhaP會使得PHA granules 變多變小，其結果與文獻結果相符。

關鍵字

Hydantoinase(HYD) ； Polyhydroxyalkanoates (PHAs) ； 胞內固定化 ； phasin ； PHA聚合酶 ； (PHA synthase)

並列摘要

The β-lactam-based antibiotics are widely used in the pharmaceutical market. D-p-hydroxylphenylglycine (D-HPG) is the most important precursor of optically active D-amino acid used for the synthesis of semisynthetic antibiotics. Production of D-HPG with a bioconversion process consists of two hydrolytic steps, which requires D-hydantoinase and amidohydrolase. First, D-hydantoinase (HDT) converts DL-hydroxyphenylhydantoin (DL-HPH) to N-carbamoyl-D-p- hydroxylphenylglycine (CpHPG). Secondly, perform hydrolysis of CpHPG to D-HPG by carbamoylase.
Polyhydroxyalkanoates (PHAs), commonly known as bioplastics, are biodegradable polymers and produced by certain bacteria. They are accumulated as reserve granules in the cytoplasm when the culture conditions are not optimal for cellular growth. It is based on the production of PHA as an immobilization matrix in bacteria. In the previous study, phasin-HDT was overexpressed in Escherichia coli, and the immobilized D-hydnatoinase was utilized to enhance the hydrolysis reaction of DL-HPH by raising the PHA content. In this study, hybrid proteins consisting of D-hydantoinase and PHA granules surface protein (phasin) or PHA synthase (PhaP -HDT and PhaC-HDT) were constructed and expressed in the PHA-producing bacteria Ralstonia eutropha H16. Through the binding of phasin or PHA synthase to PHA, HDase immobilized on the PHA granules in vivo is then generated. The results indicated that the best IPTG (Isopropyl-beta-D-thiogalactoside) concentration for the expression of fusion protein in R. eutropha H16/ pBBR1MCS2-phaP-hdt and R. eutropha H16/pBBR1MCS2-phaC-hdt was 50 ?M and 100 ?M, respectively. To further characterize the immobilized D-hydantoinase, the dependence of enzymatic activity on different temperature was investigated. It was found that the optimal temperature on the immobilized PhaP-HDT and PhaC-HDT was 60oC and 55oC. As compared to its free counterpart, the immobilized PhaP-HDT and PhaC-HDT exhibited higher tolerance to heat and gained a half life of 135 h and 120 h at 40oC, which was 2.7 and 2.4 fold higher than free enzyme. In addition, the shlf life (defined as 50% if initial activity remained) of the immobilized enzyme stored at 4oC was found to reach 43 and 40 days. D-hydantoinase immobilized on PHA granules could be reused for 8 times to achieve the conversion yield exceeding 80%. It is expected that the immobilized enzyme PhaP-HDT or PhaC-HDT can be generally applied to the industrial for various production processes.

並列關鍵字

PHA synthase ； Polyhydroxyalkanoates (PHAs) ； Ralstonia eutropha H16 ； In vivo immobilization ； Hydantoinase (HYD) ； phasin

參考文獻

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