Newly-Isolated Laccase High Productivity Streptomyces Sp. Grown In Cedar Powder as the Sole Carbon Source

Microorganisms with greater potential to degrade lignin than well-known white-rot fungi were sought and identified, but the fungi were less frequently employed for slow growth and little enzyme productivity. They were subjected to enriched cultures in order to explore the bacteria instead from 300 soil samples with cedar powder as the sole carbon source were prepared. From these, a culture with actinomycetes which showed the most oxidation activity of 2,6-Dimetoxyphenol (2,6-DMP) known as laccase substrate was selected and labeled as KS1025A strain. Characteristics of the bacteria and behavior of the secreted enzymes were examined. As a result, it was identified as a strain of Streptomyces sp. from the 16S rDNA gene sequence homology. The optimum temperature and pH for laccase activity of the secreted enzyme of this strain are 50°C and 4.5, respectively. Since Mn2+ was not directly oxidized, it was assumed that it did not contain manganese peroxidase. However, when MnSO4 was added during 2,6-DMP oxidation reaction, activity increased. After 120 hours of culture, 14 U/mL of laccase activity could be achieved by this strain, greatly exceeding known values by white rot fungus, namely 1.8U/mL after approximately 20 days. Furthermore, since reaction could continue without the addition of H2O2 during 2,6-DMP oxidation reaction, the culture solution is thought to contain free oxidizing agents. In addition, approximately 50% of 0.05% lignin sulfonic acid was decolorized by this strain in 5 days. The strain or the enzyme produced by it may be utilized for rapid biodegradation of lignin when adding hard (or soft) biomass containing lignin to produce bioethanol.


Introduction
In recent years, effective use of wood-based (or grass-based) plant waste material (wood, bagasse, etc.) for biomass has been studied. For example, cellulose/hemicellulose within the plant material comprises approximately 50% of its dry weight, and by hydrolyzing, sugars such as glucose, etc. can be extracted [1][2][3]. The obtained sugars can be used to produce bioethanol, etc [4] through fermentation. However, plants generally contain very strong polymer fibers which are formed by bonding cellulose fibers or hemicellulose fibers with lignin to maintain their shape. Although, lignification by lignin is advantageous when these plants are used as construction material; when used as biomass, this is an obstacle. Since various problems arise from the standpoint of the environment and processing costs, such as the need for alkali or acidic degradation or mechanical grinding, the use of plant material is limited [5]. Therefore, reduction of processing costs is an important issue [6,7].
In addition, if lignin degradation is carried out chemically, phenol derivatives are generated, inhibiting the hydrolyzation of cellulose by enzymes [8] and subsequent fermentation of sugars gained by hydrolyzation [9].
On the other hand, plants also serve as important storage sites for organic matter in the biosphere. Some natural microorganisms can degrade and utilize such organic matter as a source of carbon and energy. In particular, white-rot fungi degrades lignin by extracellularly secreting enzymes [10,11] and are considered to play an important role in carbon circulation in the ecosystem. However, enzyme production requires approximately 20 days in culture [12,13] and is difficult to use. Hence, focusing on lignin degradation by extracellular enzymes from fast-growing microorganisms, microorganisms with a high ability to degrade lignin, excluding filamentous fungi, were sought.

Enriched culture
Two hundred milligrams of soil sample was placed in a microtube and suspended in 300 μL of added sterile saline solution. After standing for one-minute, 50 μL of the supernatant was added to 500 μL of screening medium (composition as below) with cedar powder as the sole carbon source. The medium was covered with a gas-permeable plate seal and cultured for 2 weeks at 150 spm. After 2 weeks, 50 μL of the cultured medium was transferred to a new medium of 500 μL and was further cultured. This process was repeated four times and the medium was subcultured four times.
After subculturing for the fourth time for 1 week, the medium was centrifuged at 4725 g for 10 minutes at room temperature. The supernatant was extracted as an extracellular crude enzyme liquid. Approximately 300 soil samples were used as enriched cultures.
In addition to the above composition, 15 g/L of agar was added to the screening plate medium.

Lignosulfonate (a water soluble lignin derivative [20]) decolorization
For the lignosulfonate medium, 0.5 or 1 g/L of lignosulfonate was added instead of cedar powder. One loop was taken from the PD medium with the preserved strain, inoculated into 5 mL of ISP2 medium (composition g/L: 4 yeast extract, 10 malt extract, 4 glucose), and precultured at 28°C, 150 spm, for 48 hours.
One hundred microliter of preculture liquid was inoculated into 10 mL of lignosulfonate and cultured at 28°C, 150 spm, for 120 hours. Next, the culture was centrifuged for 10 minutes at 12000g, and A480nm was measured for the supernatant, which is the maximum absorption amount in the visible range of lignosulfonate.

Strain separation and preservation
The culture fluid was appropriately diluted, spread on the screening plate medium, and cultured statically at 28°C for up to 2 weeks. A single colony was transferred to the PDA medium as the preserved strain. PDA medium: potato dextrose agar medium (hereinafter referred to as "PDA medium").

Identification of the strain by 16S rDNA gene sequencing
A platinum loop was taken from the colony grown on the PDA medium and extracted by a DNA extraction kit, ultra clean microbial DNA Isolation kit (12224-50, MO BIO Laboratories, Carlsbad, CA, USA), as a template. As primers, 27f (5'-AGAGTTTGATCMTGGCTCAG-3') [21] and 519r (5'-CWATTACCGCGGCKGCTG-3') [22] were used. Sequencing was implemented from the template DNA, and the gene sequence was identified. The homology of gene sequence was searched in DNA Data Bank of Japan (DDBJ) [23]. In addition, the collected sequence data were multiple aligned by ClustalW in DDBJ.
The neighbor joining method was used to reconstruct the phylogenetic tree [24].

Enzyme production
The preserved strain was inoculated into 500 μL of screening medium, and cultured at 30°C, 110 spm for 120 hours, and the medium was centrifuged for 10 minutes at 4,725 g. The supernatant was extracted as the extracellular enzyme.
Enzyme activity is shown in Units (U). One Unit is the enzyme level at which 1 μmol of substrate oxidizes in 1 minute.

Measurement method of lignin peroxidase activity
An assay was carried out at pH 3.0 using 0.1 M sodium tartrate buffer and 0.01 mM veratryl alcohol as the substrate. Reaction was initiated by adding 4 mM H 2 O 2 to 100 mM sodium lactate buffer pH 4.5, 0.1 mM H 2 O 2 , and 0.5 ml culture supernatant and increased absorbance at 310nm could be observed [27].

Measurement method of Manganese (II) peroxidase activity
Manganese (II) peroxidase activity was identified by observing the formation of a Mn 3+ -malonate complex at 275 nm [28]. The reaction mixture contained 0.5 ml of 10 mM Mn 2+ in 100 mM sodium maloate buffer pH 4.5, 0.1 mM H 2 O 2 and 0.5 ml culture supernatant.

Enzyme protein concentration measurement
Measurement of enzyme protein concentration was carried out by the Bradford method [29], using the Bradford Protein Assay kit (Biorad Laboratories, Atlanta, GA, USA) with BSA as the control.

Enzyme concentration
The enzyme was concentrated to 10 times using a centrifugal ultrafiltration filter, Vivaspin 20-3K (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

SDS-PAGE and Zymogram method
SDS-PAGE and Zymogram analysis was conducted with 8% acrylamide gel using a modified method of Díaz et al. [13]. The protein of the gel was embedded and denatured by SDS for 10 minutes at 45°C in the presence of a reducing agent.
After electrophoresis at 20 mA, the gel was placed in a cleaning solution (0.1% Triton X and 100 mM sodium acetate buffer, pH5.0). The cleaning solution was replaced after 15 minutes. Cleaning was

Screening
Three hundred types of soil samples were individually added to a screening medium to form an enriched culture. As a result, a high level of 2,6-DMP oxidation activity could be observed by enzymes in the enriched cultured with a soil sample collected from Aichi, Japan. Laccase activity of the sample could progress without the addition of H 2 O 2 .

Isolation of the KS1025A strain
The active culture fluid was appropriately diluted and spread on a screening plate medium, and an actinomycete-like colony could be subsequently obtained. This strain was designated as KS1025A. After it was preserved, the enzyme was measured, and a high level of laccase activity as illustrated in Figure 1 could be observed. The enzyme of this strain was active even without the addition of MnSO 4 and H 2 O 2 , but activity increased with such addition. The assay of the Mn 3+ -malonic acid complex indicated oxidation activity from Mn 2+ to Mn 3+ (as manganase peroxidase activity). However, although veratryl alcohol oxidation (as lignin peroxidase activity) was examined, no activity was observed (data not shown). Hence, Laccase is thought to be the main enzyme group. After culturing the KS1025A strain in a PDA medium at 28°C for 7 days, a wrinkled, white to grey colony formed in 7 days. Mycelium was verified by microscope observation, but sporulation was not observed after a month of culture.
By identifying the 16S rDNA gene sequence of this strain, homology with Streptomyces atratus was determined to be 97.39% and a phylogenetic relationship could be shown (Figure 2).

Enzymological properties of enzymes produced by KS1025A
The optimum temperature and pH for laccase activity are 50°C and pH 4.5 respectively, as shown in Figure 3. The zymogram image is shown in Figure 4A, where 2,6-DMP oxide turned yellow. From the culture supernatant of KS1025, two large bands could be verified approximately between MW>175 kDa and MW 90 kDa. The CBB stained image is shown in Figure 4B. A large band could be seen at approximately 42 kDa for the KS1025A enzyme, but a band could barely be seen in the area where a strong reaction could be verified by Zymogram. From these observations, KS1025A derived Laccase is thought to be an enzyme with highly specific activity.

Discussion
In this study, actinomycete KS1025A with strong lignindegradation was newly isolated and identified as Streptomyces sp., using a universal primer for PCR amplification and DNA-sequencing. The universal primer mentioned above contained V1-4 regions which could be used to determine the specific sequences of the microorganism [30]. In the phylogenetic tree, the isolated actinomycete was slightly distant to its most homologenic microorganism, Streptomyces atratus (97.39%). Thereby we named the identified microbe as Streptomyces sp. KS1025A.
Crude enzymes derived from KS1025A do not show manganese peroxidase or lignin peroxidase activity. Therefore, laccase activity is considered to be the main cause of the lignin degradation ability of KS1025A. There are no reports concerning laccase or peroxidase activity in Streptomyces atratus (97.39%), Streptomyces pulveraceus (97.38%) or Streptomyces sanglieri (97.38%).
Optimal pH and temperature of the crude enzyme(s) derived from KS1025A are 4.5 and 50°C, respectively. Acidic pH (4)(5) has been reported to be optimal for most laccases, and pH 7-8 for others [31]. In the zymogram analysis, two types of laccases derived from KS1025A could be observed; low molecular weight laccases approximately 90 kDa and others greater than 175 kDa. However, most fungal laccases reported thus far are monomeric proteins with molecular weights between 50 and 80 kDa [17,32,33]. With the exception of the following, laccases from Agaricus bisporus [34] and Trametes villosa [35] are comprised of two subunits, and those derived from Podospora anserine [36] are comprised of four subunits. In addition, the molecular weight of laccases derived from Streptomyces coelicolor [31] and Streptomyces ipomoea [37] are approximately 32-33 kDa in monomeric form, and exist as dimers in their natural form with molecular weights of 67-69 kDa. The molecular weight of laccase derived from KS1025A is heavier than the above mentioned Streptomyceses laccase. Therefore, laccases from KS1025A may be comprised of more than two subunits, which may have been caused by the mild denaturing conditions for SDS-PAGE and zymogram analysis. This analysis was carried out at 45°C for 10 minutes to preserve enzyme activity, although SDS and reducing agents were added. Hence, subunits of laccases may not have completely separated.
Laccase activity and enzyme productivity of this strain was compared with white-rot fungus. Pleurotus ostreatus [13,[38][39][40][41] is known as a powerful lignin-degrading enzyme producer. When laccase activity of Pleurotus ostreatus is compared, a maximum of 1.8 U/mL of enzyme is produced in 21 days of culture, provided that copper is added as a Laccase catalyst [41]. Meanwhile, KS1025A strain produced a maximum of 14 U/ml of 2,6-DMP oxidative enzyme in 5 days of culture. From this, culture time can be reduced by 1/4 if KS1025A is used for enzyme production, and enzyme yield per day would increase 33 times. Possible applications of this strain for lignin degradation include wood-based biomass process, rapid decomposition of lignin colored material, and the industrial production of laccase. To the best of our knowledge, this is the first report on isolating laccase high-productive Streptomyces sp. Application of this strain is potential for de-lignin in wood-based biomass and rapid decomposition of lignin colored substance. Moreover, laccase has wider applications for degrading environmental pollutants such as bisphenol A; endocrine-disrupting chemical.

Conclusions
In this study we have screened high de-lignin enzyme producing microorgasms, and Streptomyces sp. KS1025A strain was isolated. The strain produces laccase in a short period and at high activity than previously reported white rot fungi. Remarkably, the laccase from the KS1025 strain was not-needed in addition of oxidation agent such as H 2 O 2 . By adding lignosulfate to the medium of the strain, incubation of the A480 nm was decreased about 50%.