为获得高活性的乳糖酶制剂,根据米曲霉乳糖酶基因序列以及宿主细胞乳酸克鲁维酵母GG799密码子的偏爱性,对米曲霉乳糖酶基因进行优化.将已优化的米曲霉乳糖酶lacA基因片段插入到乳酸克鲁维酵母GG799表达载体pKLAC1中,构建重组载体pKLAC1-lacA,用电脉冲法将SacⅡ酶线性化的重组质粒转入乳酸克鲁维酵母GG799中.经过5 mmol/L酵母碳基础培养基(yeast carbon base,YCB)活性筛选,全细胞聚合酶链式反应(polymerase chain reaction,PCR)鉴定,最后获得了一株多拷贝整合的基因工程菌株lacA-1.经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)和薄层层析分析(thin-layer chromatography,TLC),该重组菌株可胞外分泌乳糖酶,并具有生物活性.摇瓶发酵培养120 h,酶活力最高达到120.65 U/mL.本研究成功地将米曲霉乳糖酶基因在乳酸克鲁维酵母GG799中表达,获得的重组菌株lacA-1可胞外分泌的乳糖酶并有较高的乳糖酶活性,试验结果为利用乳酸克鲁维酵母GG799表达系统规模化生产乳糖酶奠定基础.
对牛凝乳酶的研究比较成熟,而对骆驼凝乳酶的研究报道较少。本研究为获得高效骆驼凝乳酶发酵培养基,在单因素实验的基础上, 采用 Plackett-Burman 试验考察培养基中的 8 个成分对骆驼凝乳酶产量的影响。研究表明葡萄糖、玉米浆、尿素的对产酶影响显著。 采用最陡爬坡试验逼近最大响应区间,利用 Box-Behnken 响应面法对 3 个主要影响因素进行分析,得到高效发酵培养基配方:葡萄 糖 86.6 g/L、玉米浆 56.9 g/L、酵母粉 4 g/L、磷酸二氢钾 3 g/L、尿素 18 g/L、硫酸锌 3 g/L、硫酸氨 1 g/L 和氯化镁 0.3 g/L。在该条件 下凝乳酶活达到 431.25 U/mL,较优化前的基础培养基(207.03 SU/mL)提高了 2.08 倍。再利用 1L 发酵罐进行发酵培养,凝乳酶活 性达到 530 SU/mL,约为摇瓶培养最高酶活性的 1.22 倍。 关键词:骆驼凝乳酶;培养;响应曲面法;培养基优化 文章篇号:1673-9078(2017)12-170-176 DOI: 10.13982/j.mfst.1673-9078.2017.12.026
Chymosin can specifically cleave the peptide bond between κ-casein Phe 105 and Met 106 in milk,cause milk-clotting and widely used in dairy products.Bovine chymosi has been deeply studied,but few research on camel chymosin were reported.According to the single-factor experiments design,Plackett-Burman design was used to evaluate the effects of 8 medium components on the production of chymosin to obtain a high-performance fermentation medium.The results showed that glucose,corn syrup,and urea concentrations had significant effects on the chymosin production.Box-Behnken experimental design was employed to optimize the three medium components furtherly.The optimal fermentation medium for antifungal substance production was composed of glucose 86.6 g/L,com syrup 56.9 g/L,yeast extract 4g/L,KH2PO4 3 g/L,urea 18 g/L,ZnSO43 g/L,(NH4)2SO4 1 g/L and MgCl20.3 g/L.Under the optimal conditions,the activity of chymosin could reach 431.25 SU/mL,which were 2.08 folds higher than the original medium (207.03 SU/mL).The chymosin activity reached 530 SU/mL after fermentation in an 1Lfermentation tank with exact medium components,being 1.22 folds higher than the chymosin activity obtained by shake flask fermentation.
