Laccases have been paid much attention both from researchers and from industries in recent decades due to their possible involvement in the transformation of a wide phenolic and non-phenolic compounds and lignin, as well as bioremediation of highly recalcitrant aromatic environmental pollulants.

In nature, we can observe various occurrences of symbioses, synergies or competitions between or among diverse microorganisms, and interestingly, the secretion of fungal laccase is also involved in the biocontrol phenomenon. It is found that some fungi can promote the production of laccase during interactions with other organisms. This leads us to a possible way to upgrade laccase production to solve environmental problems, and to make or improve pretreatment processes of lignocellulosics by interspecies interactions.

Therefore, the aim of this study is to screen out the active lignolytic fungi and laccase-promoting interspecies interactions among white-rot fungi and its application to the pretreatment process of lignocellulosic biomass.

Chapter I. Laccase expression induced by co-culture and inductive materials

Firstly, this section was performed to screen out the most highly efficient cellulolytic fungi and the most active lignolytic fungi from these 10 species of fungi shown in Table 1:

It was found that T.reesei (No.5) and A.nidulans (No.6) were the most efficient to degrade avicel and xylan (as culture media), while D. squalens (No.8) and A. mellea (No.10) exhibited the most predominant ability to delignify rice straw by the indicators of laccase and Manganese peroxidase (MnP) activity.

It is postulated that the two cellulolytic fungi and two lignolytic fungi would cooperate each other due to the synergy of cellulases and lignolytic enzymes in degradation of lignocellulosics. To construct the synergic combination between fungi, four sets of two-species groups, D.squalens and T. reesei, D.squalens and A.nidulans, A.mellea and T. reesei or A.mellea and A.nidulans have been incubated for 40d in rice straw by simultaneous inoculation or successive inoculation (the latter was inoculated after the former had been cultivated for 20d). Unexpectedly, hardly laccase activites were detected in all simultaneous inoculated co-cultures during the whole incubation, but in successive inoculated co-cultures, the laccase activities in pure culture phase (for 20 days) can be amplified by inoculating the latter fungus in three two-species groups, D.squalens and T.reesei, D. squalens and A.nidulans, or A. mellea and T. reesei, significantly, laccase activity was upgraded 20 folds in D.squalens and T.reesei, and 7 folds in D. squalens and A.nidulans.

This study took the group of D.squalens (Ds) and T.reesei (Tr) as an example to verify the phenomenon of laccase increase. Obviously, it was shown that laccase from D.squalens was induced by inoculation of T.reesei. It was not because cell growth by some synergies between them brought laccase increase as T.reesei showed little growth in successive inoculated co-culture after vaccination in 20d and since 20d, D.squalens has exhibited no obvious changes in its quantities, too (Fig.2), and moreover, no laccase activity was detected in T.reesei's pure culture (Fig.1).

What are the inductive materials? No laccase induction phenomenon appeared in non-lignin media(PDB) by the same way of successive inoculated co-culture, but the addition of cell-free supernatant from pure culture T.reesei's 5-day cultivation in rice straw (Supernatant Tr) to PDB media cultivating D.squalens could strongly induce laccase, and the autoclaved supernatant can also bring laccase induction. So the inducers proved to be heat-stable, and may relate to lignin or its derivatives. Then three basic lignin component Units, p-hydroxyphenyl (H) acid, Guaiacyl (G) acid and syringyl (S) acid were added to D.squalens non-lignin PDB media, respectively and S unit was found to be able to induce laccase at the same extent as rice straw.

Additionally, all the ultrafiltrate fractions from Supernatant Tr by 10K-cut-off or 5K-cut-off ultrafilter can actively induce laccase as Supernatant Tr, which shows that not only a sole component but a series of materials with continuous molecular sizes from 0 to over 10KD containing S unit can be the inductive materials, therefore it is concluded that the partially degraded lignin with 0 to over 10KD molecular mass degraded by T.reesei have induced laccase expression from D. squalans.

To verify this postulation, soluble lignin in Supernatant Tr was completely depolymerized to monomolecular lignin component units by alkaline nitrobenzene oxidation and about a 3 folds of S component unit concentration was detected than in D.squalens culture in rice straw. Besides it, few laccases were found in D. squalens culture cultivated in submerged powder media of red pine, a softwood species, in which lignin contains no S unit. Undoubtedly, the conclusion above has been proved.

Chapter II. Purification and characterization of pure culture's laccase and induced laccase

In this chapter, the laccases from D.squalens pure culture and induced by co-culture were purified and characterized. Two kinds of laccases, Lac1 and Lac 2 have been purified, and Lac 1 was the minor laccase while Lac 2 represented the major laccase in D.squalens laccase systems. And the peak of Lac 2 was apparent to be amplified by induction, but it is unclear whether Lac 1 was induced or not.

Based on the characteristics between two Lac 2 (from pure culture and co-culture) shown in Table 2 and elution profiles, it was concluded that no novel laccase was induced and the two laccases are of the same laccase.

Chapter III. Lignolytic pretreatment of rice straw by crude induced laccase

As yet there are no reports that lignocellusic biomass was pretreated efficiently by laccase. This work has had a trial to delignify rice straw by crude laccase as pretreatment process using the alkaline pretreatment (NaOH) and no treatment as control experiments and assessed its effect from various angles.

From Fig.3, crude laccases from Ds pure culture and co-culture can depolymerize lignin of rice straws by 44% and 59% in 5d, respectively. Moreover, by further analysis of dioxane extraction from pretreated solid residues, it can be known that 18.6% and 24.7% rice straw have been partially depolymerized by laccase from Ds pure culture and co-culture, respectively, and what is more favorable is that, in the laccase-pretreated liquid residues, concentrations of aromatic compound were even lower than no-treated samples. This showed that the depolymerized lignin in liquids had completely oxidized. Although alkaline pretreatment (NaOH) delignified 95% of lignin (Fig.3) and the 5% lignin residue can also be extracted by dioxane as partially degraded lignin, the residual aromatic compounds in pretreated liquids showed a very high concentration (104.4 g/L).

Moreover, results of saccharifying the pretreated rice straw showed that there was no change in detected glucose conc. compared to not-treated straw. By further analysis of adsorption technique using two dyes having high affinity to cellulose, results showed that the increase of exposed cellulose was little, and from Fig.3, negligible silica (ash) removal was observed, showing that cellulose was still encrusted by silica.

Conclusions

D.squalens is a more efficient lignin-degrading fungus in rice straw media than many famous wood-rotting fungi and its laccase production can be further induced by co-culture with T.reesei. The lignin partially degraded by T.reesei strongly induced the D.squalens laccase, Lac 2. The crude induced laccase delignified 59% of rice straw lignin, and further partially depolymerized 24.7% of lignin and moreover, thoroughly decomposed these fall-off lignin to non-aromatic materials.

Though laccase cannot remove silica affecting saccharification from rice straw, still its use is promising for pretreatment of herbaceous plants with low silica content as well as woody materials.

Table1 White-rot basidiomycete strains used in this study.

Fig.1 Laccase activities of D.squalens (NO.8) and T.reesei(No.5)

Fig.2 The microbial quantities during incubation

Table 2. Comparison on the characteristics between laccase from pure culture or co-culture

Fig.3 Constituents of rice straw after pretreatment

自然界では、様々な微生物間に共同作用やシナジー効果、競争関係などの相互作用がみられる。本研究では、種間相互作用によるラッカーゼ産生の促進効果を検証し、さらにはそのようにして生産されたラッカーゼをリグノセルロース系バイオマス分解の前処理プロセスへ利用することを目的とした。

1.ラッカーゼ発現を誘導する種間相互作用

まず、木材腐朽菌10種から高効率リグニン分解菌Dichomitus squalens(Ds)およびArmillaria melleaと、高効率セルロース分解菌Trichoderma reesei(Tr)およびAspergillus nidulansを選抜した。稲わらをリグノセルロース源とした培地で、これらリグニン分解菌とセルロース分解菌を様々な組み合わせで共培養し(同時接種および逐次接種)、リグニン分解活性に対する菌株間の相乗効果を評価した。意外なことに、すべての組み合わせで、同時接種した系ではラッカーゼ活性が全く検出されなかったのに対して、逐次接種(リグニン分解菌単独で20日間培養後、セルロース分解菌を接種)した系では、リグニン分解菌単独に比べてラッカーゼ活性が顕著に増加した。特に、DsをTrと共培養した際には、Ds単独の20倍および10倍以上の大幅な活性上昇がみられた。そこで、DsとTrの組み合わせについてより詳細に解析した。

本実験条件ではTr純粋培養ではラッカーゼ活性が検出されず、また、逐次接種した共培養系ではラッカーゼ活性の上昇時にも総菌体量は増加していなかったことから、Trの存在がDsのラッカーゼ発現を誘導していることが示された。

リグニン非存在培地(PDB)で同様に共培養をおこなった際にはラッカーゼの誘導はみられなかったが、稲わらを基質とした培地でのTr純粋培養液を遠心分離した上清(5日間)をPDB培地に添加したところ、Dsのラッカーゼは誘導された。オートクレーブした上清にも同様の誘導効果がみられたことから、なんらかの熱安定性の誘導物質が、Trによって稲わらから生産されたと考えられた。また、分子量による文革の結果から、誘導物質は単一物質ではないことが示唆された。誘導物質としてリグニンの部分分解物が考えられたため、リグニンの基本構造ユニットであるp-ヒドロキシフェニル酸(H)、グアイアシル酸(G)、シリンガ酸(S)の3種について、誘導効果を検証した結果、PDB培地にSを添加することで、Ds純粋培養系でも稲わらと同程度のラッカーゼ誘導現象がみられた。Tr培養上清中の各基本構造ユニットの濃度を測定したところ、S成分の濃度がDs培養上清のおよそ3倍程度高かった。したがって、Trが稲わらを分解する過程で、S成分を含む可溶性のリグニン部分分解物が生じ、Dsのラッカーゼ産生を誘導したと考えられた。実際、S成分含量のほとんどないリグニンを有する基質(red pine)を用いて同様に共培養実験をおこなった際には、Dsラッカーゼの誘導は全くみられなかった。

2.純粋培養時および誘導時のラッカーゼの精製とその特性

Ds純粋培養時およびTrとの共培養時の培養上清から、ラッカーゼ活性を指標に精製をおこなったところ、異なるピークを示す2種のラッカーゼが得られ(Lac1とLac2)、どちらの上清でもLac2が主要活性画分であった。それぞれのLac2を詳細に性状解析したところ、分子量、等電点、至適温度、至適pH、N末端アミノ酸配列がすべて一致した。したがって、ラッカーゼLac2が共培養によって誘導されていることが示された。

3.粗精製ラッカーゼによるバイオマス前処理

粗精製ラッカーゼに活性を向上させるメディエータを添加して稲わら処理を行なった。その結果、処理前後でリグニン含量が59%減少し、24.7%のリグニンが部分分解された。これに対して通常用いられるアルカリ処理では95%のリグニンが分解された。処理後の液体画分における可溶性リグニンを調べると、アルカリ処理にくらべてラッカーゼ処理したものでは完全酸化されたものの割合が非常に大きかった。

しかし、ラッカーゼ処理した稲わらの糖化効率は、未処理の稲わらと同程度であった。ラッカーゼ処理後の稲わらの表面構造を解析したところ、セルロースの露出程度は未処理のものと同様であった。成分分析の結果とあわせると、ラッカーゼ前処理によってリグニン含有量は減少するものの、セルロースをおおうシリカの割合は変化しなかったため、糖化効率に影響を与えなかったと考えられた。

以上、本研究は、T. reeseiとの共培養によってD. squalensのラッカーゼの生産性が著しく高まる現象を発見し、さらに生産されたラッカーゼによって稲わらのリグニンが効率よく分解することを実証したものであり学術上・応用上貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。