Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

Abstract

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ϳ13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl ␤-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an ␣␤␥ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

“…Washed membrane fractions from Methylococcus capsulatus Bath were used, as procedures for the isolation of membrane fractions with high pMMO activity have only been developed in this species (Basu et al, 2003;Choi et al, 2003;Yu et al, 2003). mb from Methylosinus trichosporium OB3b T was used in these studies for a variety of reasons.…”

Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer

“…Furthermore, the specific activity in the presence of duroquinone, NADH, and NDH-2 was Ϸ40% greater than that with duroquinol alone (11). However, Chan and coworkers recently reported pMMO activity in detergent-solubilized membranes with either NADH or duroquinol as the reductant but 10-fold greater specific activity with the quinol (49). Their purified pMMO samples had similar specific activity with either NADH or duroquinol as the reductant (49), but 4-to 10-fold lower than reported by others (4,11).…”

“…AMO has not been purified to homogeneity with activity, and although several laboratories have reported purification of active pMMO (4,31,34,49,51), the specific activity of the purified enzyme is a small fraction of the activity in vivo. Disruption of the physiological electron transfer pathways to AMO and pMMO may be in part responsible for the deterioration of activity in vitro (40,41).…”

“…Although there are other enzymes that are able to oxidize small alkanes (32,46,47), they are distinct in structure and properties from AMO and pMMO. These two enzymes thus represent a novel mechanism for alkane oxidation, one that has yet to be elucidated due to the difficulty in isolating pure samples of either enzyme with high activity (4,17,28,31,49,51).AMO and pMMO initiate oxidative metabolism of NH 3 and CH 4 in nitrifying and methanotrophic bacteria, respectively (3,19). Both types of bacteria obtain energy solely from the subsequent oxidation of the products of the monooxygenase reaction.…”

A Physical Chemist's Expedition to Explore the World of Membrane Proteins

“…The conversion of methane to methanol by pMMO has been one of the research targets of the Chan laboratory in Taiwan during the past decade. During this period, Steve Yu, Kelvin Chen, Charlie Chen, and others developed methods to obtain large quantities of high-quality pMMO that were judged to be homogenous according to subunit composition, complement of metal cofactors, and molecular mass (28,111). Progress in our work on pMMO was also facilitated by major technological advances in the study of proteins since our work on CcO some 30 years earlier, including high-throughput genome sequencing, bioinformatics, MALDI-TOF MS and ESI MS (electrospray ionization mass spectrometry) for proteomics and peptide mass fingerprinting, and ATR FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) for secondary structure determination of proteins in membranes.…”