壳聚糖酶高产菌株的筛选、产酶条件的优化及壳聚糖酶的分离纯化

Screening,Optimization of Fermentation Conditions of Chitosanase Producing Strain and Purification of Chitosanase

从采集的土样中分离到3株产壳聚糖酶的菌株,经摇瓶复筛,菌株JH01产酶能力最强。对其发酵产酶条件的研究结果表明其产酶最适培养基组分为:壳聚糖2.0%,NaCl0.05%,MgSO40.05%,KH2PO40.075%,FeSO40.001%,牛肉膏0.3%,pH5.5。最适产酶培养条件为:40℃、180r/min培养48h。在最适产酶培养条件下,48h时菌株JH01发酵液中壳聚糖酶活力可达到26.03U/mL,产酶能力为国内已报道的最高值。经DEAECellulose52柱层析,壳聚糖酶被纯化了11.70倍。经SDS-PAGE鉴定,纯化后酶已达到电泳纯的程度,分子量为30kD。

Three strains producing chitosanase were isolated from soil samples. Shaking cultivation demonstrated that the strain named JH01 possessed the highest chitosanase activity. The optimal composition of medium for JH01 producing chitosanase was as follow： chitosan 2.0%, NaCl 0.05%, MgSO4 0.05%, KHEPO4 0.075%, FeSO4 0.001%, beef extract 0.3%, and pH 5.5. The optimal cultivation conditions for JH01 producing chitosanase were 40℃ incubating temperature and shaking cultivation at 180r/min for 48 hours. The maximal chitosanase activity could reach 26.03U/mL when JH01 was incubated in flask under the optimal cultural conditions. Chitosanase from JH01 was purified about 11.70 fold by DEAE Cellulose 52. The purified enzyme is demonstrated by SDS-PAGE to be a hormogenous protein. The molecular is determined as about 30 kDa by SDS-PAGE.