植酸酶和甘露聚糖酶双功能毕赤酵母工程菌的构建和产酶分析

Construction of a double functional recombinant strainof Pichia pastoris co-expressing phytase and mannanase and the enzymatic analyses

摘要:

根据已发表的植酸酶基因和甘露聚糖酶基因序列设计并合成引物,应用PCR技术,分别以土曲霉总DNA和质粒pHBM1201为模板,扩增出均不含假定信号肽序列的植酸酶基因phyA和甘露聚糖酶基因man,将它们各自克隆到毕赤酵母表达载体pHBM907C上,分别得到重组质粒pHBM907C-phyA和pHBM907C-man。将质粒pHBM907C-phyA上由乙醇氧化酶(AOX1)启动子和终止子引导表达、酿酒酵母α信号肽序列引导分泌的phyA表达盒式结构插入到质粒pHBM907C-man中,构成双基因表达分泌质粒pHBM907C-phyA-man。pHBM907C-phyA-man经SalⅠ酶切线性后转化毕赤酵母(Pichiapastoris)GS115,获得了同时分泌表达植酸酶和甘露聚糖酶的双功能酵母工程菌。研究了该酵母工程菌所分泌表达的重组植酸酶和甘露聚糖酶的相关酶学性质,并进行了双功能酵母工程菌的稳定性测试。

Abstract:

Co-expression of phytase and mannanase in Pichia pastoris is a useful way to reduce the production cost in feedstuff industry.Based on the published DNA sequences of phytase gene and mannanase gene, primers were designed and genes phyA and man were cloned by PCR from Aspergillus terreus and the plasmid pHBM1201,respectively. Then the two fragments were treated and inserted into the same expression vector pHBM907C, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pHBM907C-phyA and the plasmid pHBM907C-man. The phyA expression cassette was combined to the expression vector pHBM907C-man which contains the expression cassette of mannanase, so pHBM907C-phyA-man was obtained. The recombinant expression plasmid pHBM907C-phyA-man was digested by SalⅠ and introduced into the chromosomes of Pichia pastoris GS115 by using the LiCl/PEG method. Following transformation, several parameters that demonstrated the expression of phytase and mannanase were measured. Firstly, the two different Petri dishes that contain enzymatic substrates such as calcium phytate and mannose were screened simultaneously, thus 100 clones were found to be positive on both of these plates. Secondly, 6 clones among them were chosen for induced expression at shakeflasks showing the probability of high expression. After that, some relative enzymatic properties were measured. At 72 hours′ induction in the condition of shake cultivation, the enzyme activity of phytase in supernatant was 120.6U/mL, while the enzyme activity of mannanase in supernatant was 39.7U/mL. The expression product phytase was active under pH2.0～6.5, and the activity was up to the highest under pH5.5. And the other expression product mannanase was active under pH5.5～10.5,and the activity was up to the highest under pH7.5. The optimal temperatures for the two enzyme were both around 52℃:the optimal temperature for phytase activity was 50℃,and that for mannanase was 55℃.At last, stability test of the engineered yeast was taken, and the engineered yeast still showed an excellent stability even after 10 generation growth in the absence of selective pressure. The stable double functional engineered yeast simultaneously expressing extracellular phytase and mannanase is obtained. It will satisfy the demand for industrialized production in some degree.