Development of a reporter gene-based assay for the bioactivity determination of rhLH pharmaceutical products.

To establish an in vitro biological activity detection method for luteinizing hormone (LH), the hLHCGR-CREB-HEK293 cell line was constructed to stably express human luteinizing hormone/chorionic gonadotropin receptor (hLHCGR). After optimization, the rhLH starting working concentration was 800 mIU/mL with 4-fold serial dilutions, 10 concentrations and an incubation time of 5 h. The method was confirmed to be highly specific, with good accuracy, precision and linearity, meeting the needs of process research and release testing, and can be used as a routine detection method for LH biological activity. With the increasing demand for research and development of rhLH biologically similar drugs, establishing a stable and simple activity assay method to evaluate the biological activity of rhLH can provide technical support for quality control of rhLH products and powerful tools for comparability research of similar products.

Rational design of styrene monooxygenase mutants with altered substrate preference.

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an α- or ß-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of α-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by site-directed mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene.

Mutations at the putative active cavity of styrene monooxygenase: enhanced activity and reversed enantioselectivity.

Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-ß-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene.