Selection Of High-yield Laccase Strains And Laccase Gene Cloning From Lentinula Edodes

Hign active laccase producing strains were selected from white rotfungus. Different substrates, different concentrations, different time and differentbasic mediums were used to select the best growth condition of laccase-producedfungus. The Lentinula edodes Cr8001,Cr241,As5.560 are the three best strainswith high laccase producing ability. A part of laccase gene from the Lentinulaedodes Cr241 was isolated and sequenced. The isolated gene consists of 112bp. The research methods and conclusions of this paper are as follows: First, the Bavendamm medium was used to make a first screen from 74 strains,there were Pleurotus ostreatus 36 strains; Lentinula edodes 25 strains, Volvariellavolvacea 3 strains, Pleurotus eryngii 3 strains and other kinds 7 strains. All thestrains were grown at 28℃ in PDA plate containing 0.4mM Bavendamm for 6d, 19strains with better laccase producing ability were selected through the color and sizeof the band produced by them on the plate. Second, the oxidative band was used to make a second study on the 19 strains.All the 19 strains are grown at 28℃ in PDA plate for 8d, then a piece of it with thediameters of 12mm were cultivated at 28℃ in the PDA plate containing 0.02 ％,0.04 ％,0.06 ％ O-methoxyphenol, observed the color and the size changes of theoxidative band .It proved that 0.04 ％ was the best concentration for observation. Igot 14 strains with better laccase producing ability. The 14 strains are grown at 28℃ in shaken flasks with PDA medium. Recordthe laccase activity of each strain. After 20d, the laccase activity produced byLentinula edodes Cr241,Cr8001 and As5.531 got the peak. They are the bestlaccase-producing strains. A part of laccase gene from the Lentinula edodes Cr241 was cloned by RT－PCR. For one step RT－PCR 2μl total RNA(include about 1～100ng mRNA) wasmixed with of 1.5μl buffer, 1μl of Mg2+(25mmol/L), 0.5μl of dNTP(2.5mmol/L), 2μl of each of the primers (2.5μmol/L), 2μl of Oligo dT, and 0.5μl of 47河南农业大学 2004 届硕士论文AMV-Optimized Taq polymerase, adjusted to 50μl with sterilized dH2O,andoverlaid with 25μl of sterile mineral oil. The reverse amplification experiments werecarried out at 37℃ for 30min .The amplification was then performed in a DNAthermal cycler under such a condition: ① 94℃ 10min 1 cycle；② 94℃ 1min,62.5℃ 2 min ,72℃ 3 min,30 cycles；③ 72℃ 10min 1 cycle；④ 4℃ 5min。The isolated gene consists of 112bp.