篩選特殊單體組成之聚羥基烷酸酯生產菌並分析其PHA合成酶基質專一性

Screening polyhydroxyalkanoate producer with unique composition and analyzing substrate specificity of the PHA synthase

黃筵嘉(Yan-Jia Huang)

指導教授 ： 李佳音

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02439

摘要

本研究利用MSM培養基培養及Nile red染色法，從活性污泥菌株中初步篩選與分離，再經氣相層析儀分析後，可從PHA生產菌株中篩選出，TO7、PO6、PO21、TS18及TF1-1五株具高的PHA生產率。PO6及PO21菌體內的PHA包含短鏈及中鏈的PHA單體組成，且中鏈PHA單體組成的莫耳百分比(mol%)可經由培養基中不同的碳氮比(C/N ratio)調控，當C/N ratio越高則中鏈PHA單體組成的mol%則越高，然而經丙酮劃分及氣相層析儀分析後確定PO6及PO21菌體內所生產的PHA是屬於混合的PHA (blend PHA)，而不是短鏈及中鏈單體隨機合成在同一條高分子鏈上的雜合的PHA (hybrid PHA)。分離株TS18及TF1-1的PHA單體組成為3-HB，可利用廉價的蔗糖做為碳源累積PHA，並且在使用果糖及propionic acid複合式碳源時，其累積的PHA單體有3-HB及3-HV兩種。菌株TS18在使用1.5%果糖為碳源時，其PHA產率更可高達75.7%的菌體乾重，且使用果糖及propionic acid複合式碳源時，其累積的PHA (3HB-co-3HV)產率高達60%以上。TF1-1的3-HV單體mol%可經由不同的碳源比值1.5%/0.025% ~ 1.5%/0.1% (果糖/propionic acid)可調控範圍為17 mol% ~ 42 mol%。篩選菌株TO7是中鏈
PHA生產菌，當提供0.5% octanoate為唯一碳源時，PHA產率為56.4%，而其中3-HO單體組成比率占PHA之93.7%。此5株PHA生產菌株經16S rDNA系統演化分析後，鑑定出TO7、PO6及PO21分別為Pseudomonas mosselii、Pseudomonas nitroreducens及Pseudomonas citronellolis，TS18及TF1-1為Duganella zoogloeoides。將中鏈PHA生產菌TO7之PHA合成酶PhaC1及PhaC2選殖，再分別送入PHA合成酶缺陷株Pseudomonas putida GPp104 PHA-中表現其基因產物。當使用0.5% octanoate為碳源時，TO7的PHA合成酶PhaC1及PhaC2在Pseudomonas putida GPp104 PHA-中表現分析後，其PHA的組成也是以3-HO單體為主的PHA。

關鍵字

聚羥基烷酸酯

並列摘要

We screened the poly(3-hydroxyalkanoates) (PHAs) producers by cultivated in MSM containing 0.5 µg/ml Nile red from environment. Nine PHA producers had been isolated and verified for their PHA accumulation ability by flask fermentation and gas chromatography analysis. Among the wild type strains, TO7, PO6, PO21, TS18, and TF1-1 possess high PHA accumulation ability. The monomer compositions of PHAs produced by PO6 and PO21 possessed short-chain-length (scl) and medium- chain-length (mcl) monomers. The mcl monomer mol% of PHA produced by PO6 and PO21 could be regulated by the C/N ratio in PHA accumulation medium. The PHAs produced by PO6 and PO21 were further identified as scl-mcl blend PHA not the scl-mcl hybrid PHA by hot-acetone fractionation and GC analysis. The monomer composition of PHA accumulated by strains TS18 and TF1-1 was only 3-hydroxybutyrate (3-HB). Strains TS18 and TF1-1 could use cheaper carbon source such as sucrose to produce PHA. The PHA yield of TS18 could reach up to 75.7% of cell dry weight while fructose was used as the sole carbon source. Strains TS18 and TF1-1 both could incorporate 3-hydroxyvalerate (3-HV) monomer into PHB polymer when propionic acid was supplemented in the medium. 3-HV monomer composition of PHA produced by TF1-1 could also be regulated from 17 mol% to 42 mol% by supplementing various concentration of propionic acid with constant fructose as
carbon sources, concentration ratio of fructose to propionic acid from 60 to 15. Strain TO7 was the mcl-PHA producer and 3-hydroxyoctanoate (3-HO) monomer composition of mcl-PHA was 93.7% of PHA content from octanoate as sole carbon sources. Strains TO7, PO6 and PO21 were considered to belong to Pseudomonas mosselii, Pseudomonas nitroreducens and Pseudomonas citronellolis. TS18 and TF1-1 were the genus Duganella zoogloeoides by 16S rDNA analysis. PHA synthase of Pseudomonas mosselii, TO7, was analyzed by recombinant P. putida GPp104 harboring PHA synthase gene. The 3-HO monomer was the major composition of PHA when P. putida GPp104 PHA- harboring phaC1 and phaC2 accumulated PHA.

並列關鍵字

PHA

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