대학원 공지

— 세미나 시리즈 –

건국대학교 생명과학특성학과/대학원 생명과학과

4단계 BK21 미래통합형 생명과학 인재양성 사업단

Biocatalytic Production of a Potent Inhibitor of Adipocyte Differentiation from Phloretin Using Engineered CYP102A1 and Roles of Human CYPs for Phloretin Oxidation in Human Liver Microsome

In this study, we investigated an efficient enzymatic strategy for producing potentially valuable phloretin metabolites from phlorizin, a glucoside of phloretin that is rich in apple pomace. Almond β-glucosidase efficiently removed phlorizin’s glucose moiety to produce phloretin. CYP102A1 engineered by site-directed mutagenesis, domain swapping, and random mutagenesis catalyzed the highly regioselective C-hydroxylation of phloretin into 3-OH phloretin with high conversion yields. Under the optimal hydroxylation conditions of 15 g cells L^–1 and a 20 mM substrate for whole-cell biocatalysis, phloretin was regioselectively hydroxylated into 3.1 mM 3-OH phloretin each hour. Furthermore, differentiation of 3T3-L1 preadipocytes into adipocytes and lipid accumulation were dramatically inhibited by 3-OH phloretin but promoted by phloretin. Consistent with these inhibitory effects, the expression of adipogenic regulator genes was downregulated by 3-OH phloretin. We propose a platform for the sustainable production and value creation of phloretin metabolites from apple pomace capable of inhibiting adipogenesis. Additionally, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed k_cat values of 3.1 and 5.8 min⁻¹, respectively. However, CYP3A4 has a 3.3-fold lower K_m value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h⁻¹ productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6β-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

연사: 윤철호 교수 (생명과학기술학부, 전남대학교)

일시: 2021년 05월 25일 화요일. 오후 5시~6시

https://us02web.zoom.us/j/87162544958?pwd=UGVTTlphRmFheW9TNlhRMHFzNWptZz09

회의 ID: 871 6254 4958

암호: 299187