Isolation, Identification, Liquid Fermentation And Enzymological Characterization Of A New Strain With High Laccase Production From Agricultural Waste Composting

Composting is a technology which has been widely used in the disposing of agricultural waste. Since lignin is the major organic compound that limiting composting speed, its degradation is essential for the operation of composting. Laccase, capable of oxidizing a wide range of aromatic compounds, is a type of multieopper polyphenol oxidases. The enzyme plays an important role in the biodegradating of lignin and formatting of humus in composting.In this paper, aerobic composting was processed with agricultural waste such as straw. A strain with high laccase production was isolated and identified from the agricultural waste composting (Termed as C2). The study was focus on the enzyme isolation and purification, the enzymatic properties and the gene of laccase. The results were as follows:According to analytical mehod established in our laboratories taking guaiacol as substrates to be oxidized by laccase and showing color change, one bacterial strain with high enzyme production was obtained. Based on its cultural characteristic, morphologic culture, ITS sequence analysis, the strain was identified as Hypocrea lixii and termed as Hypocrea lixii sp. C2. The GenBank accession numbers of the ITS gene sequence was HQ699075.The liquid state fermentation of Hypocrea lixii sp. C2was optimized. The obtained optimal parameters were glucose50g·L-1, carbamide2.21g/L, KH2PO43g/L, MgSO4·7H2O2g/L, CuSO40.3g/L, guaiacol0.05%, initial medium pH4.0,30℃of the fermentation temperature and4day of the cultivation time. After the optimization, the highest laccase yield hit200U·mL-1which was3times of previous conditions, for the substrates ABTS.The laccase produced by the strain Hypocrea lixii sp. C2in liquid-state fermentation was isolated and purified by using TCA-acetone precipitation, membrane filtration, ion-exchange chromatography. After purification, we obtained a electrophoresis pure laccase, its purity and yield were13.2folds and7.4%of the crude extract respectively. The molecular mass of the laccase was about60kDa by SDS gel electrophoresis. The optimum temperature and pH of the purified laccase was35℃and4.0separately. The remaining enzymic activity is80%respectively at50℃in1h. The half-time of the enzyme was more than1h at60℃. Laccase activity was inhibited by several metal ions, such as Na+, Fe2+, Fe3+, Pb2+and Zn2+Laccase could be activated by Cu2+. And at the low concentration, the enzyme activity was promoted by K+and Mg2+. Under standard assay conditions, Km values was1.00mmol·L-1towards ABTS.The partial laccase gene of the new strain was successfully amplified by using primer pair CulAF and Cu2R. Then the PCR product was purified and cloned, gaining a148bp fragment of laccase gene. Compared with other laccase genes in NCBI, there are the highest homology in the laccase genes’ amino acid sequence from strain Trichoderma sp. C1LL-2011.