Ecological function of myroilysin, a novel bacterial M12 metalloprotease with elastinolytic activity and a synergistic role in collagen hydrolysis, in biodegradation of deep-sea high-molecular-weight organic nitrogen

Abstract

Nearly all high-molecular-weight (HMW) dissolved organic nitrogen and part of the particulate organic nitrogen in the deep sea are present in hydrolysis-resistant amides, and so far the mechanisms of biodegradation of these types of nitrogen have not been resolved. The M12 family is the second largest family in subclan MA(M) of Zn-containing metalloproteases and includes most enzymes from animals and only one enzyme (flavastacin) from a human-pathogenic bacterium (Flavobacterium meningosepticum). Here, we characterized the novel M12 protease myroilysin with elastinolytic activity and collagen-swelling ability from the newly described deep-sea bacterium Myroides profundi D25. Myroilysin is a monomer enzyme with 205 amino acid residues and a molecular mass of 22,936 Da. It has the same conserved residues at the four zinc ligands as astacin and very low levels of identity (<or=40%) to other metalloproteases, indicating that it is a novel metalloprotease belonging to subfamily M12A. Myroilysin had broad specificity and much higher elastinolytic activity than the bacterial elastinase pseudolysin. To our knowledge, it is the first reported elastase in the M12 family. Although it displayed very low activity with collagen, myroilysin had strong collagen-swelling ability and played a synergistic role with collagenase in collagen hydrolysis. It can be speculated that myroilysin synergistically interacts with other enzymes in its in situ biotic assemblage and that it may play an important role in the degradation of deep-sea HMW organic nitrogen.

FIG. 1.

Growth of (A) and protease production by (B) M. profundi D25 in different media at 15°C. Seawater medium was artificial seawater (pH 8.0); casein medium was artificial seawater containing 0.4% casein (pH 8.0); Casamino Acids medium was artificial seawater containing 0.4% Casamino Acids; and fermentation medium was prepared as described in Materials and Methods. OD600nm, optical density at 600 nm.

FIG. 2.

Cleavage of oxidized insulin B chain by myroilysin. The cleavage pattern of myroilysin with the oxidized insulin B chain was determined as described in Materials and Methods.

FIG. 3.

Hydrolysis of fibrous elastin by myroilysin observed using an inverted microscope. Ten milligrams of fibrous elastin in 1 ml 50 mM Tris-HCl (pH 9.0) containing 100 μg/ml myroilysin was incubated at 37°C with continuous stirring. Fibrous elastin in 50 mM Tris-HCl (pH 9.0) without myroilysin was used as a control. After treatment for 1, 2, and 3 h, the samples were photographed with an inverted microscope (Olympus IX71) at room temperature. Magnification, ×960.

FIG. 4.

(A) Collagen swelling with myroilysin and urea. Ten milligrams of collagen in 2 ml 50 mM Tris-HCl buffer (pH 9.0) was incubated for 1 h at 37°C with continuous stirring. Tubes 1 to 5 contained 0, 25, 50, 75, and 100 μg/ml myroilysin, respectively. Tube 6 contained 6 M urea. Tube 7 contained 100 μg/ml myroilysin and 4 mM 1,10-phenanthroline. Tube 8 containing 10 mg collagen in artificial seawater and 100 μg/ml myroilysin was incubated for 5 h at 4°C with continuous stirring. (B) Collagen swollen by myroilysin as observed by SEM (Hitachi S-570). The control was 10 mg of collagen in 2 ml 50 mM Tris-HCl buffer that was incubated at 37°C for 1 h. For treatment, 10 mg of collagen in 2 ml 50 mM Tris-HCl buffer containing 100 μg/ml myroilysin was incubated at 37°C for 1 h. The samples were observed by SEM (Hitachi S-570) by using the method of Usha and Ramasami (22).

FIG. 5.

Synergistic action of collagenase and myroilysin for collagen hydrolysis. Five milligrams of type I collagen fiber in 1 ml 50 mM Tris-HCl buffer was incubated at 37°C with continuous stirring. Bar M, sample containing 100 μg/ml myroilysin that was incubated for 6 h; bar C, sample that was incubated for 1 h, after which 20 μg/ml collagenase was added and the preparation was incubated for 5 h; bar M+C, sample containing 100 μg/ml myroilysin that was incubated for 1 h, after which 20 μg/ml collagenase was added and the preparation was incubated for 5 h.