In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1.34 × 104 M−1 s−1 (≈25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 106 M−1 s−1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 × 10−6 M−1 s−1 vs. 22.3 × 10−6 M−1 s−1; koff, 24.3 s−1 vs. 24.2 s−1; KO2, 0.91 × 10−6 M−1 vs. 0.92 × 10−6 M−1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 × 10−6 M−1s−1 vs. 0.51 × 10−6 M−1s−1; koff, 5.08 s−1 vs. 3.51 s−1; KCO, 1.77 × 10−7 M−1 vs. 1.45 × 10−7 M−1). All four substitutions are in the heme pocket and within 5 Å of the heme group.

The growth of demand will limit output growth for food over the next quarter century

The rate of growth of world food demand will be much slower for 1990–2010 than it was for the prior three decades. The major factor determining the increase in food demand is population growth. Income growth has a much smaller effect. From 1960 to 1990, population growth accounted for approximately three fourths of the growth in demand or use of grain. For 1990–2010, it is anticipated that population growth will account for nearly all of the increase in world demand for grain. The rate of population growth from 1990 to 2020 is projected to be at an annual rate of 1.3% compared with 1.9% for 1960 to 1990—a decline of more than 30%. World per capita use of grain will increase very little—perhaps by 4%. The increase in grain use is projected to be 40% less than in 1960–1990. It is anticipated that real grain prices will decline during the period, although not nearly as much as the 40% decline in the previous three decades. Concern has been expressed concerning the deterioration of the quality and productivity of the world’s farmland. A study for China and Indonesia indicates that there has been no significant change in the productive capacity of the land over the past 50 years. Contrary to numerous claims, the depth of the topsoil has not changed, indicating that erosion has had little or no impact.