Pseudomonas sp. RT-1低温脂肪酶的纯化及酶学特性

Pseudomonas sp. RT-1是从低温环境下分离的低温脂肪酶产生菌,对该菌产生的胞外脂肪酶(PL-1)进行纯化,并对其酶学特性进行初步研究。Pseudomonas sp. RT-1的发酵上清液经60%(NH4)2SO4沉淀、12~14000截留相对分子质量(MWCO)透析袋透析、Sephadex G75分子筛和超滤浓缩后,得到了电泳纯的P-L1。SDS-PAGE电泳估算其表观相对分子质量为4.43×104。对其酶学特性研究表明:PL-1是低温碱性脂肪酶且对有机溶剂的耐受性较好。10~40℃内有较好的催化活性,最适作用温度为18℃;0~50℃该酶的稳定性较好,当温度超过50℃时则容易失活;最适作用pH为10.2,且pH在9~11时较稳定;该酶对有机溶剂的耐受性较好,10mmol/L的Ca2+、K+、Na+和Fe3+对PL1的酶活力有促进作用,其中Ca2+促进作用最大,提高了146.07%,而10mmol/L的Cu2+、Co2+、Mn2+、Mg2+、Zn2+、Ba2+和Al3+对酶活力具有不同程度的抑制作用,其中Al3+抑制作用最强,抑制了98.55%;PL-1对C链长度小于或等于12的短链脂肪酸形成的甘油三酯具有较强的水解能力;1mmol/L的去氧胆酸盐(desoxycholate)和0.01%的Triton X100对酶活力具有提高作用,分别提高了30.74%和11.83%;0.01%的SDS和Tween-80、1mmol/L的EDTA和尿素对酶活都有抑制作用,其中EDTA的抑制作用最大,抑制了80%。

Purification and characteristics of cold-active lipase secreted by Pseudomonas sp.RT-1

Pseudomonas sp.RT-1 for secreting cold-active lipase was separated in a low temperature environment.The lipase（PL-1）secrete by the strain was purified and characterized.The lipase was purified from culture supernatant to gel electrophoretic homogeneity by ammonium sulfate precipitation,dialysis,Sephadex G-75 gel filtration,column chromatography,and ultrafiltration.The apparent molecular mass of PL-1 determined by SDS-PAGE was 4.43×104.PL-1 was a cold-active alkaline lipase with high activity and stable under the conditions of low temperature of 10-40 ℃ and high pH of 9-11.The optimum temperature and pH were 18 ℃and 10.2,respectively.The organic solvent-tolerant of PL-1 was good.10 mmol/L of Ca2＋,K＋,Na＋ and Fe3＋could enhance the activity of PL-1,the promotion effect of Ca2＋ was the most strong,the rate was 146.07%.The activity of PL-1 was inhibited by 10 mmol/L of Cu2＋,Co2＋,Mn2＋,Mg2＋,Zn2＋,Ba2＋,and Al3＋.The inhibition effect of Al3＋ was the most strong and the rate was 98.5%.The enzyme exhibited high activity with short-chain（C≤12）fatty acid glycerides.1 mmol/L of desoxycholate and 0.01% of Triton X-100 could enhance the activity of PL-1 by 30.74% and 11.83%.0.01% of SDS,Tween-80 and 1 mmol/L of EDTA,urea could inhibit the activity of PL-1,the inhibition effect of EDTA was the most strong,and the rate was 80%.