Growth studies of marine and terrestrial lignicolous fungi with special reference to laccase and other lignin-modifying enzyme activities of xylariaceous fungi | 海洋和陸生真菌的生長, 尤其是炭角菌之漆酶和其它木質素修飾酶活性的研究 | Hai yang he lu sheng zhen jun de sheng zhang, you qi shi tan jiao jun zhi qi mei he qi ta mu zhi su xiu shi mei huo xing de yan jiu

Includes bibliographical references (leaves 216-256)

Wood degrading enzymes, especially the components of fungal ligninolytic systems, have attracted researchers’ interest increasingly not only because of their importance in controlling carbon recycling in the biosphere, but also due to their many potential biotechnological applications. In the past three decades, many investigations were focused only on terrestrial white-rot basidiomycetes. Little information is available on the enzymes from other fungal groups from different habitats. Using agar plate assays, seventy-four fungi collected from tropical and subtropical regions, including 29 marine isolates (mainly manglicolous) and 45 terrestrial isolates (mainly xylariaceous), were screened for the presence of lignocellulolytic activities. Most of these isolates were ascomycetes except for two basidiomycetes and one mitosporic fungus. Results showed that, among these fungi, laccase activity (as displayed by 2,2’-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) oxidation) was common in addition to cellulolytic and xylanolytic activities. However, ligninolytic peroxidase activities (as shown by Poly R-478 decolorization) were found only in the isolate Xylaria sp. CY829. The enzyme activity profiles of the test fungi indicate (1) these ascomycetes have potential to utilize the lignocellulosic components in wood, and (2) laccase appears to be the dominant enzyme for ligninolysis in ascomycetes, which is different from white-rot basidiomycetes that produce ligninolytic peroxidases predominately, i.e., manganese peroxidase (MnP) or lignin peroxidase (LiP) in addition to laccase. Detection of ligninolytic peroxidase activity in Xylaria sp. CY829 provides biochemical evidence suggesting that xylariaceous fungi can be involved in white-rot decay of wood. Physiological studies of five selected xylariaceous fungi, including two marine isolates (Halorosellinia oceanica CY325 and Hypoxylon sp. CY326) and three terrestrial isolates (Xylaria spp. CY829, CY1072, and CY1109) were conducted in liquid batch cultures. Laccase activity was detected in culture supernatants of all the selected fungi except for Xylaria sp. CY829, which was a producer of MnP with Poly R-decolorizing ability. All fungi preferred high nitrogen media (ca. 24 mM N) for better yields of biomass, concomitant with higher titers of laccase or MnP produced as compared with those in low nitrogen media (ca. 2.4 mM N). These enzymes were produced during the primary growth phase, and the titers of laccase and MnP usually declined after the cessation of fungal growth. Decolorization of Poly R-478 by Xylaria sp. CY829 in liquid cultures also occurred during the primary growth phase and was not suppressed by the high nitrogen condition. In addition, Xylaria sp. CY829 could also produce LiP activity as displayed by the Azure B assay. This enzyme activity could be detected occasionally in the culture supernatant, unlike MnP which was readily detectable almost throughout the entire period of incubation. Similar to MnP production, LiP could be detected in high nitrogen cultures. However, LiP of Xylaria sp. CY829 was not investigated in detail in this study. The selected xylariaceous fungi preferentially utilized simple nitrogen sources, such as ammonium tartrate, asparagine and urea for growth and enzyme production. Use of tryptone as the sole nitrogen source did not favor fungal growth. Of the three carbon sources tested, glucose and xylan supported more vigorous growth than did carboxymethyl cellulose (CMC), but higher titers of laccase and/or MnP were obtained in glucose media. The presence of Mn(II) in the media was required for production of MnP by Xylaria sp. CY829. For laccase producers, addition of 28 μM Cu(II) to the media stimulated the laccase production without any adverse effect on fungal growth. However, growth responses to a higher concentration of Cu(II) (280 μM) varied with the fungi tested. Fungal growth was examined at temperatures ranging from 15 to 37oC. For the selected terrestrial Xylaria spp., vegetative growth occurred between 15 and 30oC, with optimum recorded at 20-25oC for Xylaria sp. CY1072 and 30oC for Xylaria spp. CY1091 and CY829, respectively. The combined effects of temperature and salinity on the growth of marine isolates H. oceanica CY325 and Hypoxylon sp. CY326 were also investigated. A significant interactive effect between the two environmental factors was confirmed by two way ANOVA. Growth took place only between 20 and 30oC, with an optimum at 25oC for Hypoxylon sp. CY326 and 30oC for H. oceanica CY325, respectively. The Phoma-pattern was shown in these isolates: higher incubation temperature resulted in an increased salinity tolerance to some extent. Although the correlation between laccase titers and biomass yields was vague in such batch cultures, laccase production by these marine isolates under varying salinity conditions was demonstrated. However, presence of laccase activity in the artificial seawater (ASW) cultures was detectable only after the culture supernatant was dialyzed against distilled water, indicating a reversible inhibition of laccase activity by seawater. Halorosellinia laccases produced in the media with or without ASW exhibited similar pH-activity profiles: increasing pH from 2.5 to 6.0 led to a marked decrease in laccase activities toward ABTS, with zero activity at pH 7.5. Under nitrogen sufficient culture conditions, native polyacrylamide gel electrophoresis (PAGE) assay demonstrated two extracellular protein bands with laccase activity (L1 and L2) produced by Xylaria sp. CY1091 and one laccase-active band by H. oceanica CY325. These enzymes had acidic pIs and could be separated by anion exchange chromatography. The purified laccase of H. oceanica CY325 and L2 of Xylaria sp. CY1091 exhibited a blue color, a feature typical of laccases, while L1 of Xylaria sp. CY1091 was atypical with a light yellow color. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, L1 was found to be made up of two peptides with molecular weights of 25 and 54 kDa, respectively, whereas L2 had a single peptide of 80 kDa. For H. oceanica laccase, the molecular weight was estimated as 89 kDa. These molecular sizes lie within the range of laccases reported for other fungi. Similar reaction patterns were demonstrated by the laccases in response to pH changes, with optimum activity at pH2.5-3.0 toward ABTS. Laccase activities were significantly enhanced with increasing temperature, with optimum temperature for catalysis at 37oC for L1 and L2 of Xylaria sp. CY1091 and 37-45oC for H. oceanica laccase, respectively. However, all of the enzymes were inactivated after incubation at 60oC for 10 min. With respect to susceptibility of laccase to inhibitors, such as azide and halides, no significant differences were found between the laccases obtained from terrestrial Xylaria sp. CY1091 and marine isolate H. oceanica CY325. Moreover, of the halides present in seawater, the high level of chloride was identified as the predominant factor responsible for inhibition of H. oceanica laccase activity. The concentration of fluoride or bromide in seawater was too low to cause such inhibition. Multiple MnP isoenzymes can be shown in cultures of Xylaria sp. CY829 using anion exchange chromatography. Of these isoenzymes, only the predominant component MnP(H3) was purified. This enzyme had a molecular weight of 45 kDa as estimated on SDS-PAGE and showed the typical spectral feature of hemoprotein. Its optimum pH was 4.5 for oxidation of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB). The MnP activity was enhanced as the assay temperature increased from 22 to 37oC, but was inactivated after exposure at 60oC for 10 min. To my knowledge, this is the first MnP characterized from an ascomycete, which exhibits many characteristics similar to the MnPs reported from white-rot basidiomycetes. In general, the present study depicted the physiology of xylariaceous fungi with respect to the conditions required for vegetative growth and production of lignin modifying enzyme activities, as well as some biochemical characteristics of these enzymes. The information will no doubt provide a better understanding of wood degradation activities involving ascomycetes, especially in the marine environment such as mangroves.

Thesis (Ph.D.)--City University of Hong Kong, 2005