Selection of High Laccase-Producing Coriolopsis Gallica Strain T906
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J. Microbiol. Biotechnol. (2016), 26(9), 1570–1578 http://dx.doi.org/10.4014/jmb.1604.04011 Research Article Review jmb Selection of High Laccase-Producing Coriolopsis gallica Strain T906: Mutation Breeding, Strain Characterization, and Features of the Extracellular Laccases Xiaoli Xu1,2, Lei Feng1, Zhenya Han2, Sishi Luo1, Ai’min Wu3, and Jun Xie1,2* 1Institute of New Energy and New Materials, South China Agriculture University, Guangzhou 510642, P.R. China 2Key Laboratory of Energy Plants Resource and Utilization, Ministry of Agriculture, Guangzhou 510642, P.R. China 3Key Laboratory of Biomass Energy of Guangdong Regular Higher Education Institutions, Guangzhou 510642, P.R. China Received: April 5, 2016 Revised: May 26, 2016 Commercial application of laccase is often hampered by insufficient enzyme stocks, with very Accepted: June 1, 2016 low yields obtained from natural sources. This study aimed to improve laccase production by mutation of a Coriolopsis gallica strain and to determine the biological properties of the mutant. The high-yield laccase strain C. gallica TCK was treated with N-methyl-N-nitro-N- First published online nitrosoguanidine and ultraviolet light. Among the mutants isolated, T906 was found to be a June 10, 2016 high-production strain of laccases. The mutant strain T906 was stabilized via dozens of *Corresponding author passages, and the selected ones were further processed for optimization of metallic ion, Phone: +86-13825131965; inducers, and nutritional requirements, which resulted in the optimized liquid fermentation Fax: +86-020-85288355, medium MF9. The incubation temperature and pH were optimized to be 30°C and 4.5, +86-020-85282633; E-mail: [email protected] respectively. The mutant strain T906 showed 3-times higher laccase activity than the original strain TCK under optimized conditions, and the maximum laccase production (303 U/ml) was accomplished after 13 days. The extracellular laccase isoenzyme 1 was purified and characterized from the two strains, respectively, and their cDNA sequence was determined. Of note, the laccase isoenzyme 1 transcription levels were overtly increased in T906 mycelia compared with values obtained for strain TCK. These findings provide a basis for C. gallica pISSN 1017-7825, eISSN 1738-8872 modification for the production of high laccase amounts. Copyright© 2016 by The Korean Society for Microbiology Keywords: Coriolopsis gallica, mutagenesis, laccase and Biotechnology Introduction of laccases is often hampered by insufficient enzyme stocks, with very low yields obtained from natural sources Laccase (p-diphenol oxidase, E.C. 1.10.3.2) is part of a [11]. Laccases are widely spread among fungi, bacteria, and group of copper-containing polyphenol oxidases of the plants. Fungal laccases usually show higher redox blue oxidase family [27]. Laccases can modify organic and potential and yield than their bacterial counterparts [15], inorganic substrates via four-electron reduction of O2 to and their secretion patterns are affected by the fungal H2O with the concomitant oxidation of phenolic compounds species, substrates, and eco-physiological factors [4]. The [20]. Therefore, these enzymes have broad application most important fungal laccase sources are white-rot fungi prospects and potential value, such as in pulp bio- (WRF), which are involved in lignin degradation [16]. The bleaching, dye decolorization, wastewater treatment, food potential applications of lignocellulolytic enzymes in processing, medical care, and biomass energy. In previous industrial and environmental technologies require large reports, the immobilization of laccase on several supports amounts of biodegradation enzymes at low cost. Therefore, has proved to be promising for its biotechnological it is urgent to select new organisms with high synthesis of applications [10, 23, 24]. However, commercial application enzymes such as laccase, and develop strategies for their J. Microbiol. Biotechnol. C. gallica Strain T906: A High Laccase-Producing Mutant 1571 overproduction. There are many reports about using morphology observations. The strain was maintained on potato conventional mutants, such as UV irradiation or chemical medium (PDA), and used as the parent for mutation breeding of a mutagenesis, in order to improve enzyme production, higher laccase-producing mutant in this study. including laccase, cellulase, and hemicellulase [1]. Recently, an attempt was made to develop a UV mutant for enhanced Laccase Enzyme Assays Culture of the WRF C. gallica strains, and laccase production production of cellulase with reduced sensitivity to catabolite were carried out as described previously [19]. Protein concentration repression: the mutant strain Trichoderma asperellum SR1-7 was determined using a Bradford Protein Assay Kit (Tiangen, was shown to produce filter paper enzyme, carboxymethyl China) with bovine serum albumin as the standard. Laccase cellulose, and β-glucosidase under optimized conditions, activity was assayed by measuring oxidation of ABTS at 30°C in reaching 1.4-, 1.3-, and 1.5-fold increases compared with 0.1 M NaAc-HAc (pH 4.0). The reaction solution consisted of the wild type [26]. 1.95 ml of 0.1 M NaAc-HAc, 2 ml of 0.5 mM ABTS, and 50 μl of Although constitutive blue oxidases, such as fungal laccase, enzyme solutions. After 3 min, substrate oxidation by laccase was are usually produced in small amounts, the yield can be monitored at 420 nm. One unit of activity was defined as the significantly enhanced by a wide variety of substances, amount of enzyme needed to oxidize 1 μM of ABTS per minute including phenolic compounds (e.g., 2,5-xylidine) and [18]. All samples were measured in triplicates. metal ions (e.g., copper or cadmium). Therefore, the effects of different inducers, such as aromatic compounds and Mutant Screening The conidia of C. gallica TCK were collected after culture on lignin-containing plant extracts, on laccase production PDA medium for 21 days at 30°C, dispersed with sterilization were assessed. Indeed, copper was shown to increase glass beads by vortexing, and cultured in PDA liquid medium at laccase gene expression levels in Podospora anserina [12], 30°C for 10 h. Ten milliliters of log-phase culture was transferred Trametes versicolor [14], and Pleurotus ostreatus [22]. Hence, onto PDA-remazol brilliant blue R (PDA-RB) medium plates. TCK in the regulatory sequence upstream of the laccase genes, spore suspensions were treated with 20 μg/ml NTG for 30 min, metal regulatory element consensus sequences were found followed by UV irradiation under magnetic stirring as following: in the promoter regions in Podospora anserina [12] and a 15 W or 30 W UV lamp was used at a distance of 30 cm with Phanerochaete chrysosporium [5]. irradiation times of 10, 20, 30, 40, 50, 60, 80, 100, and 150 sec, In this study, using the high laccase-producing Coriolopsis respectively. The PDA-RB plates were incubated at 30°C. Colonies gallica strain TCK as parental strain, a mutant strain named with large color rings were inoculated into MF medium and T906 was obtained by a combination of chemical and UV cultured for 13 days at 30°C. Laccase activities of the isolated treatments. Based on the optimization of temperature, pH, strain were determined as described above. carbon source, nitrogen source, and metallic ion, the Optimization of Submerged Fermentation Conditions for Laccase optimized liquid fermentation medium MF9 was developed. Production Comparing with wild-type C. gallica strain TCK, the laccase Culture of the WRF C. gallica strains were carried out as activity in mutant strain T906 was 303 U/ml under described previously. To enhance the laccase production, the optimized conditions, which reached about 7.5-times to the fermentation conditions were optimized. First, the fermentation initiate MF liquid culture, and showed 3-times higher temperature and initial pH values were screened ranging from laccase activity than the parental TCK wild type. These 25°C to 40°C and pH values 3 to 7, respectively. Then, based on findings demonstrated that C. gallica can be modified to the liquid fermentation culture MF (millfeed 25 g/l, glucose 10 g/l, improve its laccase production. KH2PO4 0.2% (w/v), MgSO4·7H2O 2 g/l, CaCl2·2H2O 0.5 g/l, ammonium tartrate 0.1 g/l, VB1 1.84 g/l, Tween-80 10 mg/l, MnSO ·H O 0.05% (w/v), NaCl 7 mg/l, FeSO ·7H O 7 mg/l, CaCl Materials and Methods 4 2 4 2 2 7 mg/l, ZnSO4·7H2O 1.0 mg/l, CuSO4·7H2O 0.5 mg/l, and H3BO3 0.5 mg/l (pH 4.0)), the effects of carbon source (maltose, sucrose, Chemicals and Microorganisms glycerol, glucose, carboxymethylcellulose sodium, starch, or All chemicals were of analytical grade and purchased from millfeed), nitrogen source (casein, yeast powder, peptone, Invitrogen (USA), except 2,2’-azino-bis (3-ethylbenzthiazoline-6- ammonium nitrate, ammonium sulfate, or ammonium dihydrogen sulfonate) (ABTS), syringaldazine, and vanillic acid, which were phosphate), metal elements (Cu2+, Fe2+, Mn2+, Zn2+, Ca2+, Co2+, and from Sigma-Aldrich (Germany). C. gallica strain TCK was isolated Mg2+), the initial reaction pH, and optimal temperature of the from poplar in the north of China (CCTCC number: M2011317). It fermentation cultures were assessed. Meanwhile, several laccase was identified as C. gallica by comparison of internal transcribed inducers (veratryl alcohol, ABTS, gallic acid, tannic acid, and spacer region sequences of the 18S rRNA and 5.8S rRNA genes guaiacol) were added at 0.5 mM after 96 h of incubation. in GenBank (GenBank Accession No. EF458487) as well as September 2016 ⎪ Vol. 26⎪ No. 9 1572 Xu et al. Biochemical Characterization of the Purified Laccase from TCK PCR was carried out on an Eppendorf Mastercycler Ep Realplex, and T906 Strains with SYBR Premix Ex Taq (Takara Bio, China). The PCR conditions Culture supernatants of TCK and T906 at 11 days were collected were as follows: 95°C for 30 sec; 40 cycles at 95°C for 15 sec and by centrifugation at 12,000 rpm for 15 min at 4°C.