海洋细菌Pseudoalteromonas flavipulchra HH407产低温碱性蛋白酶的发酵条件和酶学性质研究

Fermentation Conditions for the Production of Low-temperature Alkaline Protease by Pseudoalteromonas flavipulchra HH407 and Its Enzymatic Properties

对一株分离自连云港海域的海洋细菌(Pseudoalteromonas flavipulchra HH407)菌株产蛋白酶的条件及酶学性质进行研究。结果表明,在发酵温度为20℃、培养基pH值为8.0、NaCl质量浓度为20g/L、接种量2.5%和装液量20%时产酶较高。甘露醇、果糖、乳糖、蔗糖、麦芽糖、鱼粉和酵母粉有利于蛋白酶的产生。该酶的最适作用温度为35℃,0℃时相对酶活力达21.7%。45℃条件下保温60min后酶活力仍能保持80%以上。酶的最适作用pH值为10.0,pH值为7.0～11.0范围为时,酶活力相对比较高。酶在pH8.0～11.0范围内稳定,最稳定的pH值为10.0,属于低温碱性蛋白酶,Mn2+、Cu2+、Ca2+对其具有较强的激活作用,Hg2+较强地抑制其酶活力。苯甲基磺酰氟(PMSF)能完全抑制该蛋白酶,EDTA无影响表明该酶属于丝氨酸蛋白酶。

The optimal fermentation conditions for producing low-temperature alkaline protease by a marine bacterium（Pseudoalteromonas flavipulchra HH407） from Lianyungang sea area and some enzymatic properties of this enzyme are reported in this article.The highest enzyme production was achieved at the conditions of fermentation temperature of 20 ℃,pH 8.0,NaCl concentration of 20 g/L,inoculum amount of 2.5% and medium volume in fermentation tank of 20%.Meanwhile,enzyme production was improved by adding mannitol,fructose,lactose,sucrose,maltose,fishmeal and yeast extract.The optimal reaction temperature for this protease was 35 ℃.The relative enzyme activity was 21.7% at 0 ℃ and 80% after incubation at 45 ℃ for 60 min.The optimal pH for this protease was pH 10.0.A high level of relative activity could be remained in the pH range of 7.0 to 11.0.This protease was stable in the range of pH 8.0 to 11.0.Therefore,it is a low-temperature alkaline protease.Moreover,its activity was stimulated in the presence of Mn^2＋,Cu^2＋ and Ca^2＋,and inhibited in the presence of Hg2＋ and PMSF,but was not affected by EDTA.These investigations suggest that this protease might be a serine protease.