【摘要】 为实现南极假丝酵母脂肪酶B（Candida antarctica lipase B,CALB）在黑曲霉（Aspergillus niger）中的表达，研究选用黑曲霉高效分泌信号肽SglaA、SpepB和融合蛋白XynB分别构建3个CALB的表达载体，并转入黑曲霉TH-2（ΔasaA::pyrG）、TH-2（ΔasaA::scerv29ΔprtT::pyrG）中，得到6株黑曲霉重组菌株。摇瓶发酵重组菌株并进行酶活力检测，发现以SglaA为信号肽的CALB酶活是以SpepB为信号肽酶活的105%和108%；以XynB为融合蛋白的CALB酶活是未融合XynB的114%～123%；敲除prtT的CALB重组菌株酶活是未敲除菌株酶活的102%～105%。以SglaA为信号肽，XynB为融合蛋白并转入TH-2（ΔasaA::scerv29ΔprtT::pyrG）的重组菌株酶活力较高，发酵至第12天为21.78±0.77 U·mL^-1。对重组CALB进行酶学性质分析，其最适温度为50℃，在30～50℃范围内温度能保持70%以上的相对酶活力；最适pH为8.0，在p H 6.0～8.0时较为稳定并且能保持80%左右的相对酶活力，Na⁺、K⁺和甘氨酸对CALB的酶活性产生了增强效果。更多还原

【Abstract】 To achieve the expression of Candida antarctica lipase B（CALB） in Aspergillus niger, in this study,three CALB expression vectors were constructed using the highly efficient secretion signal peptides SglaA and SpepB and the fusion protein XynB of A. niger, and then were transformed into A. niger TH-2（ΔasaA::pyrG） and TH-2（ΔasaA::scerv29 ΔprtT::pyrG）, resulting in six recombinant A. niger strains. The recombinant strains were shaken in flasks for fermentation and enzyme activity was detected. It was found that the CALB activity with Sgla A as the signal peptide was 105% and 108% of that with Spep B as the signal peptide; the CALB activity with Xyn B as the fusion protein was from 114% to 123% of that without Xyn B fusion; the CALB activity of the recombinant strains with prt T knockout was from 102% to 105% of that of the strains without knockout. The recombinant strain using Sgla A as the signal peptide and Xyn B as the fusion protein, when transformed into TH-2（ΔasaA::scerv29 ΔprtT::pyrG） showed the highest enzyme activity, reaching 21.78±0.77 U·mL^-1after 12 days of fermentation. The enzymatic properties of the recombinant CALB were analyzed. The optimal temperature was 50 ℃, and the relative enzyme activity remained above 70% within the range of 30-50 ℃. The optimal p H was 8.0, and the enzyme activity was relatively stable at pH6.0-8.0, maintaining about 80% of the relative enzyme activity. Na⁺, K⁺and glycine enhanced the enzyme activity of CALB.更多还原