CAJ | 학술논문

根据灰盖鬼伞漆酶基因 LCC 1的蛋白序列和毕赤酵母的密码子偏爱性设计并合成 CCLCC 1 I 基因片段，将其连接到经过改造的 pPIC9K 质粒上构建分泌表达载体 pYN2395；构建好的载体经 Bgl Ⅱ酶切线性化后电激转化到毕赤酵母 GS115中，通过 ABTS 底物显色反应筛选获得阳性转化子。从培养基、发酵温度、铜离子浓度和甲醇添加量等方面对重组酵母的发酵条件进行了研究。结果显示：添加0．6％的丙氨酸能极大地提高灰盖鬼伞漆酶的表达量，并确定最终优化组合为：培养基使用 MMA，发酵温度为20℃，铜离子体积浓度为0．5 mmol？L，甲醇添加量为1．0％？24 h。
근거회개귀산칠매기인 LCC 1적단백서렬화필적효모적밀마자편애성설계병합성 CCLCC 1 I 기인편단，장기련접도경과개조적 pPIC9K 질립상구건분비표체재체 pYN2395；구건호적재체경 Bgl Ⅱ매절선성화후전격전화도필적효모 GS115중，통과 ABTS 저물현색반응사선획득양성전화자。종배양기、발효온도、동리자농도화갑순첨가량등방면대중조효모적발효조건진행료연구。결과현시：첨가0．6％적병안산능겁대지제고회개귀산칠매적표체량，병학정최종우화조합위：배양기사용 MMA，발효온도위20℃，동리자체적농도위0．5 mmol？L，갑순첨가량위1．0％？24 h。
The laccase gene LCC 1 of C .cinereus was synthesized chemically according to the protein se-quence of Coprinus cinereus and bias codons of Pichia pastoris .The synthesized laccase gene CCLCC 1I was subcloned into the vector pPIC9K and integrated into the genome of Pichia pastoris GS115 by electropora-tion.Laccase-secreting transformations were selected by their ability to oxidize the substrate ABTS.The fermentation conditions were studied with the culture medium,temperature,copper concentration and addition amount of methanol.The results showed that the expression of Coprinus cinereus laccase improved greatly by adding 0.6% alanine and the final optimazation was:MMA culture medium,20 ℃,copper concentration 0.5 mmol?L and 1.0%?24 h addition amount of methanol.