High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes / 生物工程学报

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).

Inducing synthesis of LacA from Trametes sp. AH28-2 and cloning & analysis of 5'-terminal sequence of transcription control of the gene / 生物工程学报

Copper ion was necessary for the transcription of all laccase isozyme genes from Trametes sp. AH28-2, with higher concentrations of Cu2+ (1-2 mmol/L) being more favorable to the synthesis of laccase. In the glucose media containing 0.5 mmol/L Cu2+, the laccase activity of the supernate was rather low (44.3 u/L) and had an increase of 60.3% (71.0 u/L) when 4.0 mmol/L o-toluidine was added. Moreover, the activity reached up to 2584 u/L as the glucose was replaced by cellobiose. And Native-PAGE showed that LacA was the main laccase component if fungus was induced by o-toluidine or copper ions. It had been demonstrated by quantitative RT-PCR that the regulation of lacA expression, induced by o-toluidine, occurred at the transcriptional level, with the accumulation of mRNA transcripts being accompanied by the increase of laccase activity of the culture fluid. In addition, the structural gene of lacA interrupted by 10 introns was 2110 bp in length and the corresponding cDNA sequence was 1560 bp encoding a 520 aa protein, which had high similarities with other laccases from basidiomycetes. Furthermore, a length of 1881 bp of 5'-terminal sequence of transcription control of lacA, amplified by the improved inverse PCR, contained a TATA box, seven CAAT boxes as well as a number of putative cis-acting elements important for its expression, including five MREs, nine CreA-binding sites, four XREs, two STREs and seven nitrogen factor binding sites. The existence of these elements was well in agreement with the data obtained from Trametes sp. AH28-2 shaken-flask cultures.

Studies on production, purification and partial characteristics of the extracellular laccase from Armilliria mellea / 生物工程学报

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).

Immobilization of Fungal Laccase on Nylon Net and Application of the Immobilized Enzyme / 微生物学通报

Condition optimization for immobilization of Fungal laccase with Nylon Net and glutaraldehyde and the nature of the immobilized enzyme were studied. The optimum conditions of the immobilization are: Nylon Net is crosslinked with 5% glutaraldehyde 15mL for 6 hours; the 30U laccase is added for immobilization for 8 hours. On this case, the recovery of enzyme activity was 50.3%. Compared with free enzyme, the thermal stability of immobilized enzyme was improved evidently but the optimal pH decreased slightly. 52% enzyme activity of immobilized laccase was hold after 8 cycles treatment with low concentration pulp wastewater.

STUDIES ON THE EXPRESSION CONDITION OF SEGMENT OF GABA_A RECEPTOR a1 SUBUNTT IN E. COLI / 微生物学通报

In order to improve the expression level of segment of GABAA receptor a1 subunit in E. coli, growth conditions of the recombatant, which influence the final yield of protein expression, including growth medium, inoculation ratio, temperature, pH, rotation speed, inducing time and concentration of IPTG and so on, were studied in shaking flasks. The results indicated that, with 3% inoculation ratio, cultured 3.5 hours at 37℃, and then induced 5 hours by IPTG at 32℃, the yield of GABAA receptor protein was 95mg/L and the biomass was 3.25 g/ L. In contrast, using a 16 L stirred fermentor instead of shaking flasks, the highest level of the protein expression, 136mg/L with 4.95g/L of biomass, was achieved after fermenting 5.5 hours.

PRELIMINARY STUDIES ON LACCASE PRODUCTION BY A WHITE-ROT FUNGUS AH28-2 / 微生物学通报

A novel white-rot fungus AH28-2,which was isolated from 224 fungi samples,ability to produce effectively laccase by induction.Several factors influencing laccase production were investigated.The optimum conditions were as follows:the 300mL Erlenmeyer flask containing 150mL of liquid medium was inoculated with 7.5mL of mycelial fragments and the medium was supplied with lignin at a concentration of 0.1%(initial pH8.5).The cultures was incubated at 28℃ on rotary shaker(150r/min) for 4～5 days.The maximum enzyme activity was 20184IU/L.