植酸酶phyA基因在解脂耶氏酵母po1h中的表達

陳云，鄒由，王一丁，馬立新

1 四川師范大學生命科學學院，成都 610101

2 湖北大學生命科學學院，武漢 430062

工業生物技術

植酸酶phyA基因在解脂耶氏酵母po1h中的表達

陳云1，鄒由2，王一丁1，馬立新2

1 四川師范大學生命科學學院，成都 610101

2 湖北大學生命科學學院，武漢 430062

本實驗通過 PCR方法從畢赤酵母 GS115-phyA中擴增出不含有信號肽及內含子的黑曲霉 NRRL3135植酸酶phyA基因，并將其克隆到表達載體pINA1297中，得到表達載體pINA1297-phyA，利用醋酸鋰轉化法將線性化載體轉化到解脂耶氏酵母po1h中，通過YNBcasa和PPB平板篩選出陽性表達菌株，陽性菌株在YM培養基中28℃培養6 d后酶活達到最大為636.23 U/mL。表達上清經SDS-PAGE分析得到表達植酸酶分子量約為130 kDa，但通過去糖基化處理后其分子量變為51 kDa，與理論值相符。經過酶學性質分析表明重組植酸酶最適pH為5.5，最適溫度為55℃，該酶在pH 2.0～8.0處理1 h后仍有較高酶活，并且90℃處理10 min后還有86.08%的殘留酶活，其抵抗胃蛋白酶和胰蛋白酶能力也較強。

黑曲霉，植酸酶，phyA基因，解脂耶氏酵母po1h

Abstract:Using the polymmerse chain reaction (PCR), we amplified the phytase genephyAfromPichia pastorisGS115-phyAinAspergillus nigerNRRL3135 without the signal peptide sequence and intron sequence,. Then, it was cloned into pINA1297 vector to generate a recombinant vector of pINA1297-phyA. pINA1297-phyAwas linearized and transformed intoYarrowia lipolyticapo1h by the lithium acetate method. The positive transformants were obtained by YNBcasaand PPB plates, after induced in YM medium at 28°C for 6 day. The activity of the expressed phytasephyAreached 636.23 U/mL. The molecular weight of the enzyme was 130 kDa measured with SDS-PAGE analysis, whereas its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytasephyArevealed its optimal pH and temperature was 5.5 and 55°C, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h. Moreover, its activity remained 86.08% after exposure to 90°C for 10 min. It also was resistant to pepsin or trypsin treatment.

Keywords:Aspergillus niger, phytase,phyAgene,Yarrowia lipolyticapo1h

植酸酶是一種新型的環保型“綠色”飼料添加劑，它能分解飼料中的植酸和植酸鹽形成禽畜可利用的磷酸和肌醇，提高了飼料中有機磷的利用率，減少了添加無機磷的量，降低飼料成本，減輕磷污染，并可抑制植酸對礦物質和蛋白質的親和力，解除植酸的抗營養作用[1]。而商品化植酸酶大多為來源于曲霉的植酸酶，其中由黑曲霉Aspergillus nigerNRRL3135菌株phyA基因編碼的植酸酶應用最早最廣泛[2]。

目前黑曲霉Aspergillus nigerNRRL3135菌株phyA基因已經在釀酒酵母、畢赤酵母GS115、KM71和X-33中得到表達，但還未見其在非常規酵母解脂耶氏酵母中進行表達研究，而解脂耶氏酵母被認為是安全的 (Generally regarded as safe)，因此，能用于食品和藥物生產上。……