Cholesterol oxidase (ChOx) is a flavoprotein enzyme that catalyzes the oxidation of cholesterol to 4-cholesten-3-one with the concomitant reduction of oxygen to hydrogen peroxide [1], [2], [3], [4]. This enzyme is naturally produced by several microorganisms, such as Rhodococcus sp. [5], [6], [7], Streptomyces sp. [8], [9], Rhodococcus erythropolis [10], [11] and Bacillus subtilis [12], [13], [14], [15]. ChOx has been widely utilized in the food [16], [17], pharmaceutical [18], and clinical diagnostic industries [12], [19], [20], [21], [22] owing to its ability to oxidize cholesterol selectively. Nevertheless, large-scale applications remain limited due to difficulties in producing the enzyme, high costs, and stability issues [8].

Although Streptomyces and Rhodococcus species naturally produce ChOx, their yields are low and downstream purification is labor-intensive [23], [24], [25]. Recombinant expression has therefore been pursued to address these challenges. Most prior studies have focused on ChOx from Streptomyces or Chromobacterium species, with only limited exploration of R. erythropolis as a recombinant source [11], [26]. For instance, Fazaeli et al. (2018) reported recombinant ChOx in Escherichia coli (E. coli) with a specific activity of 7.07 U/mg [27], while ChOx from Nocardioides simplex has been tested for diagnostic applications [28]. Alternative hosts such as B. subtilis and Pichia pastoris have also been evaluated, but E. coli remains the most practical host for achieving higher yields and simplified purification [12], [29], [30].

Beyond cholesterol oxidation, ChOx has also been widely explored in diagnostic biosensors because it produces hydrogen peroxide as an electroactive signal [31], [32], [33], [34]. Although several studies have reported recombinant ChOx from other microorganisms [8], [35], [36], [37], the enzyme from R. erythropolis has not yet been developed in recombinant form. In fact, this strain is known to produce a stable and active enzyme in its native form [38]. However, when expressed recombinantly, the yield is usually low and the enzyme becomes less soluble. These issues limit its practical application.

Therefore, a simple expression and refolding approach is needed to recover more active enzyme and make it applicable for cholesterol oxidation and biosensor applications [39]. Our previous work showed that crude ChOx still exhibited limited efficiency due to impurities [40]. Given these challenges, this study focuses on cloning, expressing, and purifying recombinant ChOx from R. erythropolis in E. coli BL21(DE3), using a His-tag-assisted refolding strategy to improve enzyme activity and stability for future diagnostic and industrial applications.