Enzyme and Microbial Technology 39 (2006) 1051–1059 Production by Clonostachys compactiuscula of a lovastatin esterase that converts lovastatin to monacolin J Lin-Cheng Chen a , Yiu-Kay Lai a , Suh-Chin Wu a , Chih-Chien Lin a , Jia-Hsin Guo b,∗ a Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinshu 30013, Taiwan, ROC b Department of Food Science, National Pingtung University of Science and Technology, No. 1, Shuehfu Road, Neipu, Pingtung 91201, Taiwan, ROC Received 16 November 2005; received in revised form 2 February 2006; accepted 7 February 2006 Abstract The conversion of monacolin K (lovastatin) to monacolin J, a core structure in the synthesis of other statins, was achieved using the fungus Clonostachys compactiuscula and optimized with response surface methodology. To study the proposed second-order polynomial model, a central composite experimental design with multiple linear regression was used to estimate the model coefficients of five selected factors believed to influence the conversion process. The experimental results indicated that the optimal conditions for growth of C. compactiuscula mycelium were as follows: 2.0 g glucose/L, initial pH of the medium 8.5, and incubation of the mycelium for 4 days. These conditions yielded an optimal concentration of the substrate lovastatin of 1 mg/mL, and a conversion time of 15 h. A lovastatin esterase was isolated and purified from the mycelium of C. compactiuscula by ammonium sulfate precipitation, size-exclusion chromatography, and ion-exchange chromatography. Following SDS-PAGE, the purified enzyme appeared as a single band with an apparent molecular mass of 28 kDa. The converted product, monacolin J, was isolated and purified, and its structure was analyzed by NMR. © 2006 Elsevier Inc. All rights reserved. Keywords: Clonostachys compactiuscula; Hypercholesterolemia; Lovastatin; Monacolin esterase; Response surface methodology 1. Introduction The endogenous synthesis of cholesterol is carried out by the mevalonate pathway, in which the rate-limiting reaction, the conversion of (S)-HMG-CoA to (R)-mevalonate, is catalyzed by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reduc- tase (EC. 1.1.1.34). The series of drugs referred to as statins are often prescribed to control hypercholesterolemia. All statins function similarly, i.e., by binding to the active site of HMG- CoA reductase, thereby inhibiting cholesterol biosynthesis and causing a marked reduction of serum cholesterol levels [1,2]. The statin lovastatin [3–6], known as monacolin K, is admin- istered as the pharmacologically active lactone form, which has the chemical structure 1 ′ ,2 ′ ,6 ′ ,7 ′ ,8a ′ -hexahydro-3,5-dihydroxy- 2 ′ ,6 ′ -dimethyl-8 ′ -2 ′′ -methyl-1 ′′ -oxobutoxy)-1-naphtalene hep- tanoic acid-5-lactone [7]. Schimmel and Borneman [8] reported that an enzyme produced by the fungus Clonostachys com- pactiuscula selectively hydrolyzes the 2-methylbutyryloxy side- ∗ Corresponding author. Tel.: +886 8 770 3202x7449; fax: +886 8 7740378. E-mail address: jhguo@mail.npust.edu.tw (J.-H. Guo). chain of lovastatin to generate monacolin J and methyl butyrate. This enzyme was named lovastatin esterase, and its occurrence in nature was also described by Komagata et al. [9], who purified the enzyme from dried mycelia of the fungus Emericella unguis. Monacolin J [8] has also been shown to inhibit HMG-CoA reductase [8–10] and thus may also be useful as a cholesterol- lowering agent [10]. The compound is also potentially valuable as an intermediate in the synthesis of other, semi-synthetic HMG-CoA reductase inhibitors [8,11–13]. For example, mona- colin J could serve as a core molecule that could be variously modified to generate different, and possibly novel, HMG-CoA reductase inhibitors [8,11,14]. Thus, the enzymatic hydrolysis of lovastatin, and the subsequent production of monacolin J may have a large number of important therapeutic applications. In a preliminary study, four strains of mold capable of hydrolyzing lovastatin to form monacolin J were examined. Of these, C. compactiuscula strain BCRC 33575, had the highest hydrolytic activity and was therefore chosen for further study. The growth conditions that yielded a cell mass with a high level of lovastatin hydroxylation activity were determined. Enzyme activity was measured by assaying monacolin J production in vegetative-phase mycelia. To further maximize the enzyme 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.02.018