一株α-淀粉酶生产菌的分离鉴定及其产酶条件优化

平板水解圈法从土壤中分离产淀粉酶菌株。通过碘比色法测定产淀粉酶分离菌株的酶活,筛选出一株产酶量较高的菌株,鉴定其种类,并对其产酶条件优化及酶学性质进行研究。通过革兰氏染色、生化鉴定和16SrDNA序列比对鉴定该菌的种类;从pH、温度、碳源、氮源等方面进行产酶条件优化。菌种经16S rDNA PCR序列分析比对,为枯草芽孢杆菌,因此将分离到的菌株命名为Bacillus subtislis-Y9;菌株最佳培养基配方为：淀粉6g,酵母膏13g,氯化钠5g,添加1.0%的吐温于1000mL蒸馏水中;最佳培养条件为：初始pH值为7.5;培养温度为37℃,培养时间36h。在上述培养基和优化培养条件下菌株发酵液的α-淀粉酶酶活达到7.1U/mL,约为出发菌种的5.5倍。酶学研究显示,α-淀粉酶的最适反应温度为40～60℃,反应体系pH值为6.6,并需添加0.5%CaCl2。

Isolation and Identification of An α-Amylase-Producing Bacteria Strain and Optimization of Its Fermentation Conditions

In this study,amylase-producing strains were screened from the soil,and optimization of fermentation conditions was undertaken to improve the enzyme activity of wild strains,as well as to study its enzymatic properties.Experiments were undertaken to determinate the optimal carbon and nitrogen sources for the amylase producing strain Y9;the effect addition of Tween 80 in the culture media,the optimal culture temperature and culture time period was detected.At different temperatures,dissolved with buffer at various pH values,enzyme activity was measured to determine its enzymatic properties.To identify the strain Y9,the sequence of 16Sr DNA was amplified and sequenced,morphological property was observed after Gram staining;biochemical and physical characteristics were tested with microscale reaction tubes.The optimal medium formula was as follows： starch 6g,yeast extract 13g,calcium chloride 5g,10mL Tween 80,dissolved in 1000 mL distilled water;the optimal culture conditions were as follows： the initial pH Value 7.5;incubation temperature 37 ℃,incubation time 36h.Under optimal culture conditions,α-amylase enzyme activity in the fermentation fluid reached 7.1U/g.The optimal reaction temperature of the enzyme was 40 ~ 60℃,the pH value of reaction system 6.6,and added with 0.5% CaCl2.BlastN results showed the 16Sr DNA sequence of strain Y9 shared the highest identity of 97% with that of Bacillus subtilis strains,and based on the morphological properties,biological and physical characteristics,the strain was identified as B.subtilis.