Elsevier

Bioresource Technology

Volume 101, Issue 19, October 2010, Pages 7592-7597
Bioresource Technology

Improving the simultaneous production of laccase and lignin peroxidase from Streptomyces lavendulae by medium optimization

https://doi.org/10.1016/j.biortech.2010.04.087Get rights and content

Abstract

To improve laccase (Lac) and lignin peroxidase (LiP) production from Streptomyces lavendulae, the Lac and LiP activity models were constructed and the C–N sources formulation in the medium optimal for the simultaneous production of Lac–LiP complex was asparagine 16.44%, urea 23.48%, fructose 18.01%, malt extract 21.21%, (NH4)2SO4 20.86%. By nutritional analysis, the optimal initial mole ratio of C/N for laccase and LiP production were 1.48, 1.43 with C, N levels of 1.298–1.308 mol L−1 and 0.875–0.912 mol L−1, respectively. Verification experiment revealed that the initial C/N ratio in medium was the key factor affecting laccase activity, and the combined C, N sources could improve ligninolytic enzymes activity. Thus, enzyme production cost could be cut down effectively, not only by the simultaneous fermentation of Lac–LiP complex, but also by maintaining the initial C/N mole ratio at 1.43–1.48 in the medium with low levels, and different kinds of C and N sources.

Introduction

Lignocellulose degradation is the central step for carbon recycling in land ecosystems (Martínez et al., 2009), because around 20% of the total carbon fixed by photosynthesis is incorporated into lignin (Francisco et al., 2009), which accounting for up to 25% of the dry weight of woody plants (Breen and Singleton, 1999). Being part of a complex matrix in which cellulose microfibrils are embedded, not only lignin itself is highly resistant towards chemical and biological degradation (Martínez et al., 2009), but also the degradation of cellulose is strongly inhibited by the presence of lignin (Martínez et al., 2009). Therefore, lignin degradation, removal or modification is not only the key step in lignocellulose decay (Martínez et al., 2009), but also the rate-limiting step of carbon recycling, even a central issue for the industrial utilization of plant biomass (such as bio-fuel production) (Francisco et al., 2009).

Up to now, the key enzymes directly involved in the depolymerization of lignin in natural lignocellulosic substrates include laccase (Lac, E.C. 1.10.3.2) and peroxidases, mainly manganese peroxidase (MnP, E.C. 1.11.1.13) and lignin peroxidase (LiP, E.C. 1.11.1.14) (Breen and Singleton, 1999). Up to the present, laccase is mainly produced by fungi such as white rot, brown rot, soft rot and stain fungi (Martínez et al., 2009), but shows low activity and stability in alkaline pH range or at high temperature (Singh and Chen, 2008). In contrast, laccase of bacterial origin is highly active and stable under the condition of high temperature or high pH value (Jordaan, 2005, Singh and Chen, 2008). As one of the most studied aerobic cellulolytic bacteria (Béguin and Aubert, 1994), Streptomyces is good at producing ligninolytic enzymes having great application potential under extreme conditions. Some strain like Streptomyces psammoticus has the ability to produce all the three major ligninolytic enzymes of LiP, MnP, Lac (Niladevi and Prema, 2005), and exhibits laccase activity even in alkaline environment (pH 7.5–10.0) (Niladevi et al., 2007, Tuncer et al., 2009). When the laccase from Trametes versicolor is completely inhibited by 1 mM NaN3, the laccase from Streptomyces coelicolor is still fully active under the same condition (Dubé et al., 2008). Especially, laccase from Streptomyces lavendulae can be stable at 70 °C (Suzuki et al., 2003), even withstand a pH scope from 3.0 to 9.0 (Dubé et al., 2008).

Although laccase is generally regarded as the most preferred enzyme, laccase alone has a limited effect on lignin bioremediation (Mazumder et al., 2009). In fact, laccase has so low redox potential that it can directly oxidize phenolic subunits only (Breen and Singleton, 1999, Martínez et al., 2009), which usually comprises less than 10% of the total polymer in natural lignin (Martínez et al., 2009). On the other hand, LiP is regarded as the most effective oxidizer capable of catalyzing the oxidation of phenolic and non-phenolic compounds and aromatic amines, aromatic ethers and polycyclic aromatic hydrocarbons (Breen and Singleton, 1999, Martínez et al., 2009). Therefore, Lac–LiP complex system is expected to have a much higher efficiency on lignin degradation due to their potential synergism.

Medium optimization has become one of the main ways to realize the production of Lac–LiP complex by simultaneous fermentation because ligninolytic enzyme production is highly regulated by the media composition or ingredients (Singh and Chen, 2008), or essentially by the nutrient levels in fermentation medium (Ben Hamman et al., 1999). As two most critical components of nutritional medium for any fermentation process, carbon and nitrogen sources are the most useful tools to improve or to stimulate the production of ligninolytic enzymes (Jordaan, 2005). Moreover, medium optimization is an important and realizable option to reduce production cost (Singh and Chen, 2008). The most efficient method for large scale fermentation is submerged fermentation (SmF) or agitated liquid culture (Jordaan, 2005). To produce Lac–LiP complex from S. lavendulae simultaneously, the medium optimization was studied by submerged fermentation here.

Section snippets

Strains

The adopted strain S. lavendulae was preserved in BOKU – University of Natural Resources and Applied Life Sciences, Vienna, Austria.

Inoculum preparation

To prepare inoculum, the autoclaved medium composed of malt extract (15 g L−1, w/v), yeast extract (15 g L−1), (NH4)2SO4 (0.5 g L−1), CaCO3 (0.1 g L−1), MgSO4 (0.5 g L−1) and trace element solution 0.5% (v/v) in baffled flask was inoculated with S. lavendulae slant and then cultured with shaking at 130 rpm under 25 °C for 3 days until its cell density reached 1 × 107 CFU (mL)−1.

Principal Components Analysis

After PCA, four principal components (F1, F2, F3, F4) were figured out as Eqs. (2), (3), (4), (5) showed. According to the total variance of malt extract, asparagine, urea, fructose and (NH4)2SO4, the explained variance of F1, F2, F3, F4 reached 28.778%, 25.631%, 24.856%, 20.735%, respectively, and their cumulative percentage of explained variance was 100%.F1=0359+1.025×10-2×(asparagine)+4.063×10-2×(urea)+0.235×(fructose)-0.457×(malt extract)(1.000,0.000)F2=-0.641+0.528×(urea)-0.166×(fructose)-

Conclusions

Inferring from the optimized C–N sources formulation to produce laccase (asparagine 19.26%, urea 19.60%, fructose 19.03%, malt extract 18.75%, (NH4)2SO4 23.36%) and LiP (asparagine 14.95%, urea 24.37%, fructose 20.93%, malt extract 18.87%, (NH4)2SO4 20.88%) from S. lavendulae, the optimal initial C/N mole ratios were 1.48, 1.43. Verification revealed C/N ratio was the key factor affecting laccase activity and the combined C, N sources improved enzyme activity. Thus, production cost would

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant No. 30700102), Branch of the Water Pollution Control and Management Project (2008ZX07209-010), Beijing Nova Program (Grant No. 2008A074).

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