Studies On Strains Producing Thermostable Isoamylase From Hot Spring In Yunnan And The Enzyme Properties

Isoamylase is a debranching enzyme which specifically cleaves a-1,6 glycosidic linkages in amylopectin, starch, glycogen and some dextin. The enzyme is widely used in the multiple processing industry of starch, because it can promote the utility efficiency and productivity of starch. The most important industrial application of isoamylase is used in combination with glucoamylase, a-amylase or β-amylase, respectively, in the saccharification process. Isoamylase widely exists in microbial. The microbe which can produce isoamylase includes bacteria and fungi. It's full-blown application is used for the production of dextrose, high-maltose syrups and beer from hydrolysates.In this thesis, we had isolated 48 strains without endospore from the environmental samples, which were collected from the hot springs of Tengchong, Eryuan and Lianghe County, Yunnan province. 16 strains were screened which can produce debranching enzyme, and 2 strains were selected for further study for the relatively high enzyme activity. After the UV mutagenetic and substrate inducing, the strain ZZC6-6 was finally chosen for the highest special activity and thermostablity in a wide temperature range. The enzyme activity was increased from 4.14 u/mL to 18.54 u/mL. Wang et al isolated a strain from the soil of the starch factory in Lanzhou. After many UV and UV+DES mutagenetic treatments and selections from a lot of mutants, finally they obtained a high yield, stable and mid— temperature strain which can yield isoamylase. The enzyme activity was increased from 3.35U/mLto 7.37U/mL.The bacteria ZZC6-6 was identified as Thermus sp. according to the morphological, physiological, biochemical characteristics. We also find the optimum condition of the fermentation is: amylopectin, 2.83g/L, Yeast extract, 0.86g/L, Tryptone, 0.73g/L.Following the steps of (NH₄)₂SO₄ treatment, ion exchange chromotograph and gel chromotograph, the fermentation broth was purified for 9.9-fold and the purified enzyme special activity was 91.38u/mg. The molecular weight of enzyme is 41KDa.The properties of the isoamylase were studied. The optimum temperature and pH are 75℃, 8.0. The enzyme is thermostable for it can conserve higher activity after 30minheated by 75 °C. Na+,Mg2+,Ca2+,Cr2+are the activator, and Cu2+,Zn2+,Co2+ inhibit the activity obviously.