利用原生質體融合技術構建植酸酶\\纖維素酶枯草芽孢桿菌工程菌的研究

摘要:以產植酸酶枯草芽孢桿菌(A5)復合誘變的再生突變株Z56和產纖維素酶枯草芽孢桿菌(B6)復合誘變的再生突變株X57為親本，利用雙親滅活原生質體融合技術進行種內融合，構建可同時產植酸酶、纖維素酶的工程菌。結果表明，從構建的385個融合子中篩選到6株兩種酶活性相對較高的工程菌，其中R4、R5的纖維素酶產量高于親本，植酸酶產量也相對較高;粗酶液用90 ℃處理10 min后，纖維素酶剩余酶活分別為對照的62%和58%，植酸酶剩余酶活分別為對照的73%和71%。

關鍵詞:枯草芽孢桿菌;植酸酶;纖維素酶;原生質體融合;工程菌

中圖分類號:Q814文獻標識碼:A DOI編碼:10.3969/j.issn.1006-6500.2010.05.007

Study on the Application of Intraspecific Protoplast Fusion Technique to Construct Bacillus subtilis Engineering Bacteria with Phytase and Cellulase Activities

XIE Feng-xing， ZHANG Feng-feng， ZHOU Ke， ZHAO Yu-jie

(Tianjin Research Center of Agricultural Biotechnology，Tianjin 300192，China)

Abstract:In order to construct phytase and ellulose-producing engineering bacteria， intraspecific protoplast fusion between Bacillus subtilis Z56， phytase-producing strain obtained from protoplast combination mutagenesis， and Bacillus subtilis X57， ellulose-producing strain obtained from protoplast combination mutagenesis， was performed by inactivated protoplast fusion technique. The results showed that 6 fusants with high phytase and cellulose activities were selected from 385 regeneration colonies. The productions of ellulose of R4 and R5 were higher than the parental strain and phytase activities were relatively high. The enzyme thermal stability results showed that the remained ellulose activities of R4 and R5 kept 62% and 58%， while the remained phytase activities kept 73% and 71% when the crude enzymes were treated with 90℃ for 10 min.

Key words: Bacillus subtilis;phytase;ellulose;protoplast fusion;engineering bacteria

植酸酶是催化植酸及植酸鹽水解產生磷酸和肌醇的一類酶的統稱[1]。植酸酶作為飼料添加劑，一方面可以有效的提高動物對飼料中磷的利用率，從而使飼料中添加的無機磷減少70%[2]，降低飼料成本的同時節約磷礦資源，另一方面可將動物糞便中排磷量減少30%～50%[3]，減輕環境污染，有利于生態農業的發展。纖維素酶是一類將纖維素水解成葡萄糖的一組酶的統稱。它可破解富含纖維的細胞壁，使其包含的蛋白質、淀粉等營養物質釋放出來并加以利用，同時又可將纖維降解為可被畜禽機體消化吸收的還原糖，從而提高飼料利用率[4]。雖然目前利用微生物發酵生產酶制劑已達到工業化水平，但外源酶制劑的熱穩定性問題一直沒有得到很好的解決，導致其在飼料加工的高溫(70～90 ℃)制粒工藝中，酶的活性大幅度喪失[5]。枯草芽孢桿菌是一種能產內生芽孢的革蘭氏陽性菌，有較強的熱穩定性，同時也是農業部允許作為飼料添加劑的安全菌種之一，可直接添加到飼料當中[6]。……