摘要
糖化酶是目前重要的工业酶制剂之一,广泛应用于淀粉加工业和发酵工业中。然而,在目前的糖化酶制剂生产过程中常会伴随有一种水解酶副产物—α-葡萄糖苷酶的生成,它可以从低聚糖类底物的非还原末端切开α-1,4糖苷键,释放出葡萄糖或将游离出的葡萄糖残基转移到另一糖类底物形成α-1,6糖苷键,得到非发酵性寡聚糖。α-葡萄糖苷酶的存在影响了糖化酶制剂的质量和纯度,给工业生产带来不便,因此它越来越受到研究者的重视。
CICIM F0410是生产糖化酶的工业菌株,本文考察了该菌株产α-葡萄糖苷酶的变化进程与酶学性质,发现该菌株在发酵周期96 h时的α-葡萄糖苷酶产量最高;该酶的最适pH值5.0,最适反应温度55℃,0.5 mM的Mg2+对该酶活有较显著的激活作用,并对其热稳定性有一定的保护作用。
对菌株的培养基和发酵条件进行了优化,在保证糖化酶产量的前提下,最大限度的降低α-葡萄糖苷酶的活力。最佳发酵培养基组成(g/L):葡萄糖109,豆饼粉25(添加终浓度0.01 M的NH4Cl),玉米浆31。摇瓶发酵最佳培养条件:培养基初始pH值5.5,种子培养96 h,接种量9%,旋转式摇床34℃培养,摇床转速220 r/min,发酵周期为144 h。通过发酵条件的优化,每1000U糖化酶中α-葡萄糖苷酶的含量降低了53.16%。
利用PCR方法扩增到大小为3124 bp的α-葡萄糖苷酶基因,其中编码序列长度为2973 bp,含有三段内含子,推衍得到该段序列编码990个氨基酸残基,其中包括一条含有57个氨基酸残基的信号肽,与GenBank上公布的黑曲霉CBS513.88的α-葡萄糖苷酶基因相似性达到99.94%。构建了用于敲除该基因片段的重组质粒pMD19-aglU::G418,为下一步在基因水平上改造菌株奠定了基础。
Glucoamylase is one of the important industrial enzymes. It has been widely applied in fermentation industry that need glycosylated starch as a raw material. However, when microorganism produces glucoamylase, a kind of hydrolase—α-glucosidase is produced as a by-product, which would decrease glucoamylase efficiency in fermentation process. Theα-glucosidase can catalyze gulcose from the non-reducing ends of oligosaccharides or transfer the glucose residue to another carbohydrate substrate to formα-1, 6 glycosidic bonds which belongs to the oligosaccharides of non-fermentation. Therefore, theα-glucosidase is paid more attention by many researchers.
CICIM F0410 is an industrial producer of glucoamylase. The time course of the enzyme production and the properties were investigated in this study. The maximum glucosidase activity was obtained in 96 h of the fermentation, at 55℃and pH 5.0. Furthermore, 0.5 mM Mg2+ promoted the enzyme activity and increased the heat stability of glucosidase.
The optimization of medium and fermentation conditions for F0410 was conducted. We tried to decrease the relativeα-glucosidase activity per 1000 U glucoamylase as much as possible Get the optimal fermentation medium (g/L): glucose 109, soybean powder 25 (add a final concentration of 0.01 M of NH4Cl), corn steep liquor 31; The optimal fermentation conditions were 50 mL broth with initial pH 5.5 in 250 mL flask, inoculated 96 h at 34℃on rotary shaker, the rotational speed was 220 r/min, inoculum concentration was 9%, fermentation period was 144 h. Under the optimized condition, the content ofα-glucosidase per 1000U glucoamylase decreased to 51.36%.
A 3124 bp nucleic acid fragment containingα-glucosidase gene was amplified by PCR. The coding sequences were 2973 bp, containing three introns. The deduced protein was 990 amino acids containing a signal peptide consisting of 57 amino acid residues. Sequence alignment with theα-glucosidase sequence of another A. niger.(GenBank accession number XP_001402053) showed that the two nucleotide sequences' similarity reached to 99.94%. Meanwhile, a recombinant plasmid pMD19-aglU::G418 was constructed which would be used to knockout theα-glucosidase gene in the further study.
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