耐高温微生物β淀粉酶的制备及其酶学性质的研究

Preparation of high-temperature-resistant β-amylase and its enzymological characteristics

目的筛选高产微生物β淀粉酶的优良菌株,通过微生物发酵法制备微生物β淀粉酶。方法从南宁明阳生化淀粉厂附近的土壤分离出枯草芽孢杆菌(Bacillus subtilis)原始菌株,取10g土样制成稀释10-2～10-4的倍,平面涂布,单菌落平面纯培养后,纯化选得产β淀粉酶原始菌株,鉴定为枯草芽孢杆菌(命名为NKJ-00),采用NTG诱变法对枯草芽孢杆菌诱变得突变株,过滤、离心得β淀粉酶粗酶液,采用硫酸铵沉淀法纯化;用蒸馏水代替酶液作为对照组。测定β淀粉酶的酶活力,且对其最适温度、热稳定性、pH的稳定性和金属离子对其的影响等酶学性质进行了测定;最后对β淀粉酶转化可溶性淀粉溶液制备麦芽糖的能力进行了测定。结果枯草芽孢杆菌原始菌株产酶能力为220～230U/ml,得到的4株突变株产酶活力远高于原始菌株,其中NJK-05产酶能力及稳定性最佳,传6代后发酵产酶能力稳定在1500U/ml以上。用NKJ-05菌株发酵制得耐高温微生物β淀粉酶,酶最适温度60～65℃,酶活力为2.2万U/ml,50～65℃是稳定的,pH5～8时相对稳定。pH>8相对酶活下降较明显,pH<5时,相对酶活迅速下降;金属离子对β淀粉酶活性有一定的抑制。提取纯化后的β淀粉酶作用于22%～25%的可溶性淀粉溶液得到60%～70%的麦芽糖浆。结论β淀粉酶可通过微生物发酵法生产,微生物β淀粉酶的稳定性、耐高温等指标均超过植物β淀粉酶,纯度基本上达到工业生产的要求。

Objective To screened the strains highly yielding β-amylase, and then to produce β-amylase by microbial fermentation. Methods Original strains of Bacillus subtilis were separated from the soil collected from the areas around the Nanning Biochemical Strain Plant. 10g of the soil were dissolved with water and then diluted by 10^-2 - 10^-4 times. The dilution was smeared onto a cultured plate to obtain purified original strains by using inoculation single colony. After been identified, the Bacillus subtilis （NKJ-00） was mutated with NTG （150 μg/ml）, infiltrated and centrifugated using crude β-amylase dilution, and then purified by sulfate precipitation method. In control group, the distilled water was employed instead of the enzyme dilution to determine the activity of β-amylase, its enzymotogical characteristics, as well as its ability to being transformed into soluble amylum dilution for preparation of maltose. Results The yield of β-amylase from the original strains of Bacillus subtilis ranged from 220 to 230 U/ml, which was significantly lower than that of the four mutant strains. Among the four mutant strains, NJK-05 had the highest amylase-yielding ability and stability, the yield of β-amylase reached above 1500 U/ml after 6 passages. For the high-temperature-resistant β-amylase produced using NKJ-05 strain, the proper temperature was 60 - 65 ℃, the activity of the enzyme was 2.2×10^4 U/ml, and enzyme was stable at 50 - 60 ℃ and pH 5-8. Its activity decreased significantly when pH 〉 8, and decreased rapidly when pH 〈 5. The activity could be inhibited by metal ions. By using the purified β-amylase, 22% - 25% soluble amylum dilution could be obtained to prepare 60% - 70% maltose. Conclusions β-amylase can be produced by microbial fermentation. The stability and high-temperature resistance of the product are better than plant-β-amylase, and its purity is good enough.