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Breeding of Inductive-Mutant Strain with High Yield of Laccase and the Characterization of Laccase

Author: WangShiRan
Tutor: ZhaoShiGuang
School: Anhui University of Engineering
Course: Microbiology
Keywords: Laccase Low energy ion beam implantation Breeding Optimization Enzymatic Properties
CLC: Q93
Type: Master's thesis
Year: 2010
Downloads: 162
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Abstract

In recent years, the study of the laccase has attracted extensive attention. The laccase (Laccase. EC.1.10.3.2) polyphenol oxidase, a copper ion is widely distributed in fungi, especially white rot fungi. Laccase with polyphenols and aromatic diamine substrate effect, it is widely used in various fields of industrial production. In this paper laccase Ganoderma (Ganoderma lucidum) U60 strain was low energy N beam injection Mutation Breeding UIM-281 of laccase producing strain and strain conditions for enzyme production, laccase purification, enzymology The properties were studied. The main findings are as follows: 1, the use of low energy the N beam injection technology laccase U60 mycelia of the fungus Ganoderma (Ganoderma lucidum) irradiation mutagenesis. Biological effect relationship of different injection dose Ganoderma survival and mutation rate, determined by studying the energy of 15 keV under high proportion are available in 2.6 × 1015 ~ 3.9 × 1015 ions/cm2 implantation dose range mutants. 3:12 × 1015 ions/cm2 implantation dose parameters selected after several rounds of injection mutagenesis to obtain a stable hereditary laccase yield the sudden strain UIM-281; ??Fermentation experiments show that the UIM-281 laccase production vitality peak of 1.7 times and 2.28 times the original strain U60, and enzyme production fermentation cycle is relatively short 24 h, more cost-effective industrial fermentation Ganoderma lucidum laccase fermentation strains. By single factor test and Plackett-Burman design method laccase of Ganoderma lucidum UIM-281 fermentation medium major factor screening, corn flour, wheat bran and ABTS mass concentration of three significant factors. Steepest ascent experiments approaching the optimal level of the above three factors, the application of central composite design and response surface analysis, the establishment of a laccase in response to the value of the quadratic regression equation model, the optimal fermentation conditions: corn flour 17.56 g / L, of the bran 17.91 g / L ABTS 0.0274 g / L glucose, 10 g / L, and soy flour 10 g / L, and KH 2 PO 4 4.5g / L, proven in this condition, laccase 6325.3 U / L, a chemical synthetic medium laccase production increased nearly 1.27 times, the study provides a theoretical basis for the industrial fermentation laccase. 3, first using Plackett-Burman (PB) design method to filter out the main effect of factors that affect the fermentation of Ganoderma G. lucidumUIM-281 laccase medium volume, shaking speed and incubation time. Steepest ascent experiments approaching the optimal level of the above three factors, the application of central composite design and response surface analysis, established in response to the value of the quadratic regression equation model laccase, the optimal fermentation conditions: medium volume 52 mL, shaking speed 158 r / min, incubation time 172 h, temperature 28 ℃, 10% of the inoculum size, initial pH 5.5, seed age 96h. The the theoretical predictions 8342.6U above conditions / L, verify that the measured activity 8387.7U / L, and the predictive value of close, high reliability prediction model can be applied to the optimization of fermentation conditions laccase. 4, UIM-281 produced by a crude enzyme solution, a non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the the activity staining results showed that the laccase isozyme. Was purified by 35% -65% acetone in the secondary sedimentation and the filter membrane, the apparent molecular weight of about 46 kDa measured by SDS-PAGE of laccase. Enzymatic properties of laccase study results show that: the laccase optimal reaction temperature is 50 ℃, the optimum pH 6. Stored at 10 ℃ and pH 4.8 has the best stability; metal ions of Mn2, Ba2, Mg2, Ca2, Fe2 and inhibitors mercapto-ethanol, sodium lauryl sulfate and sodium azide on the enzyme have different degrees of inhibition of the Fe2, sodium lauryl sulfate and sodium azide showed strong inhibition of the enzyme, Co2 and Cu2 apparent activation Zn2 ??and Na have little effect on the enzyme activity of; Michaelis The constant Km to 1.552 mmol / L.

Breeding of Inductive-Mutant Strain with High Yield of Laccase and the Characterization of Laccase

Abstract

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