Abstract: To investigate the properties of different malic enzymes from M6-22, MIME1 gene was cloned from Mortierella isabellina M6-22 and expressed in E. coli BL21, enzymatic activity was analyzed after purification by nickel affinity chromatography. The recombinant expression of MIME1 protein showed the property of malic enzyme which could catalyze the dehydrogenation of malic acid and produce pyruvate and NADPH. The enzymatic property of MIME1 was compared with MIME2 from the previous study. The results showed that both MIME1 and MIME2 had the property of malic enzyme; The optimum temperature and pH of MIME1 and MIME2 were 28 ℃ and 7.5, 30 ℃ and 5.8, respectively; The activities of purified MIME1 and MIME2 were 292.84 U / mg and 235.14 U / mg; Mg²⁺, Co²⁺, Cu²⁺ and Zn²⁺ increased the activity of MIME1 with Cu²⁺ showing the strongest effect, while EDTA, Ca²⁺ and Mn²⁺ inhibited the activity of MIME1. Mn²⁺, Mg²⁺, Co²⁺ and Ca²⁺ showed different degrees of activation on MIME2 with Mn²⁺ showing the strongest effect, while EDTA, Cu²⁺ and Zn²⁺ significantly inhibited the activity of MIME2; Different malic enzymes in the same cell had different enzymatic properties, which may be related to their different biological functions. By purification, isolation and heterologous expression, some characteristics of enzymatic properties of two Mortierella isabellina M6-22 malic enzymes are showed. A theoretical basis and reference for the structure and function of the malic enzymes and the relationship with the lipid accumulation in M6-22 strain is provided.