Controlling the simultaneous production of laccase and lignin peroxidase from Streptomyces cinnamomensisby medium formulation
© Jing and Wang; licensee BioMed Central Ltd. 2012
Received: 17 December 2011
Accepted: 20 March 2012
Published: 20 March 2012
Use of crude ligninase of bacterial origin is one of the most promising ways to improve the practical biodegradation of lignocellulosic biomass. However, lignin is composed of diverse monolignols with different abundance levels in different plant biomass and requires different proportions of ligninase to realize efficient degradation. To improve activity and reduce cost, the simultaneous submerged fermentation of laccase and lignin peroxidase (LiP) from a new bacterial strain, Streptomyces cinnamomensis, was studied by adopting formulation design, principal component analysis, regression analysis and unconstrained mathematical programming.
The activities of laccase and LiP from S. cinnamomensis cultured with the optimal medium formulations were improved to be five to eight folders of their initial activities, and the measured laccase:LiP activity ratios reached 0.1, 0.4 and 1.7 when cultured on medium with formulations designed to produce laccase:LiP complexes with theoretical laccase:LiP activity ratios of 0.05 to 0.1, 0.5 to 1 and 1.1 to 2.
Both the laccase and LiP activities and also the activity ratio of laccase to LiP could be controlled by the medium formulation as designed. Using a crude laccase-LiP complex with a specially designed laccase:LiP activity ratio has the potential to improve the degradation of various plant lignins composed of diverse monolignols with different abundance levels.
Lignocellulose degradation is the central process for carbon recycling in land ecosystems . As the key step in lignocellulose decay, lignin degradation, removal or modification is the rate-limiting step of carbon recycling [1, 2], and also the central issue for industrial utilization of plant biomass (for example, biofuel production from abundant and renewable lignocellulosic material) [3, 4]. Compared with the lignocellulosic biomass degradation by fungi, in vitro treatment of such biomass by lignin-degrading enzymes has a number of advantages, such as shorter incubation period without bacterial growth, reduced possibility of infection during large-scale microbe culture, lack of inhibitory effect of toxic byproducts (such as furfural) on bacterial or fungal mycelial growth, and improved reaction efficiency of bacterial enzymes at higher temperatures [1, 5]. However, lack of commercially available, robust, and inexpensive enzymes is a major barrier for the widespread application of ligninolytic enzymes in various industrial sectors [5, 6]. To avoid the high costs associated with enzyme purification procedures, one of the most promising ways to promote lignin biodegradation is to use crude enzymes [5–8].
Lignin is composed of chemically distinct subunits or monolignols (such as p-hydroxyphenyl, guaiacyl, and syringyl) whose abundance or proportions vary among plant species, and lignins of different plant origins require different proportions of laccase (E.C. 22.214.171.124), lignin peroxidase (LiP; E.C. 126.96.36.199) or manganese peroxidase, (MnP; E.C. 188.8.131.52) to promote the efficiency of degradation [2, 3, 9, 10]. Laccase is the most preferred ligninase enzyme , but can directly oxidize only phenolic lignin units [12–14], which usually comprise less than 10% of the total polymer content of natural lignin [1, 12]. By contrast, LiP is the most effective oxidizer ligninase known to date, and is capable of catalyzing the oxidation of phenolic or non-phenolic compounds, aromatic amines, aromatic ethers, and polycyclic aromatic hydrocarbons [13, 14]. Thus, a laccase-LiP complex can be expected to be more efficient at lignin degradation than either of the two ligninases alone, owing to their potential synergism [5, 6]. Further, laccases of fungal origin have low activity and stability at alkaline pH range or at high temperature, whereas laccases of bacterial origin have higher activity and stability under the same conditions [10, 15]. One of the most studied aerobic cellulolytic bacteria species, Streptomyces, can produce ligninase with promising application potential under extreme conditions of high temperatures or high pH ranges [5, 10, 15].
Generally, strain selection or improvement by genetic modification, optimization of medium formulation, and fermentation conditions are the main options to improve enzyme activity and reduce production cost [5, 6, 10, 15]. Compared with the high cost and uncertain results of genetic engineering, optimization of the medium formulation (including medium ingredients or composition, nutrient limitation, removal of potential toxic inducers) and culture conditions is a more dependable method in regulating the production of ligninolytic enzyme [5, 16]. Carbon and nitrogen are the two most important components of the nutritional medium for any fermentation process [17, 18], thus modifying or altering either or both in the medium should provide a marked improvement in the production of extracellular ligninolytic enzymes [10, 18].
Currently, the most efficient method for large-scale fermentation is submerged fermentation (SmF; that is, an agitated liquid culture) . Traditionally, production of ligninolytic enzyme by SmF usually has a higher production cost because the liquid media usually contains some relatively expensive constituents such as glucose, ammonium tartrate, nitriloacetic acid, MgSO4, NaCl, FeSO4, CoCl2, and ZnSO4 . To decrease production cost, a potential solution is to adopt less expensive natural substrates as opposed to a chemically defined medium . Coffee pulp, wheat bran, and yeast extract have been shown to be excellent substrates (nitrogen sources) for Streptomyces psammoticus to produce laccase by either SmF  or solid-state fermentation (SSF) . Urea and sawdust have been used as nitrogen and carbon sources for laccase production from Pycnoporus sanguineus by SSF  and Lentinula edodes by SmF , respectively.
In this study, we aimed to produce a high activity laccase-LiP complex from a new strain of Streptomyces cinnamomensis by SmF using low cost liquid medium composed of yeast extract, coffee pulp, wheat bran, sawdust and urea.
Results and discussion
Principal component analysis
Simultaneous production of laccase and lignin peroxidase (LiP) from Streptomyces cinnamomensis by submerged fermentation
Medium ingredients, ga
Mean ± SD
The explained variances in F1, F2, F3, F4 were 28.649%, 25.610%, 24.721% and 20.661% respectively, and their cumulative percentage of variance reached 99.641%.
Regression analysis and mathematical programming
Solutions to the expected maxima of laccase and lignin peroxidase (LiP) activities
Exp. max. activity
-4.500 × 10-8
-2.700 × 10-8
Design of verification experiment
For ligninolytic enzyme production, the types and concentrations of the carbon and nitrogen sources and the carbon:nitrogen ratio were shown to be key factors influencing extracellular laccase or peroxidase production from Penicillium chrysogenum , Streptomyces sp. F2621 and F6616 , Streptomyces viridosporus , Streptomyces albus , Botryosphaeria rhodina , Pleurotus ostreatus and Pleurotus sajorcaju , and Streptomyces lavendulae .
Theoretical solutions to medium formulations designed to produce laccase-lignin peroxidase (LiP) complexes with different laccase:LiP activity ratios
Designed laccase:LiP ratio
0.1 laccase: 1 LiPa
1 laccase: 1 LiPb
2 laccase: 1 LiPc
-7.800 × 10-8
-5.500 × 10-8
-5.400 × 10-8
Result of verification
Designed medium formulation for the verification experimenta by submerged fermentation of Streptomyces cinnamomensis
Expected maximum activity
Designed laccase:LiP ratio
0.1 laccase: 1 LiP
1 laccase: 1 LiP
2 laccase: 1 LiP
After 7 days of SmF culture using the optimal liquid medium (w/v) of yeast extract 1.48%, coffee pulp 1.94%, wheat bran 0.38%, sawdust 1.22% and urea 4.98% (Eqn 6; Table 2; Table 4), LiP from S. cinnamomensis reached a peak of 0.1450 U/mL, which was approximately 8.2 times activity of the initial average value of 0.0176 U/mL (Table 1) and higher than the expected peak of 0.1339 U/mL (Table 2). This confirmed that the LiP activity could also be controlled by medium nutrition, and the optimized medium formulation was reliable for LiP production.
Measured activity ratios of laccase-LiP complexes produced from Streptomyces cinnamomensis by submerged fermentation with optimized medium formulation
0.1 laccase/1 LiP
1 laccase/1 LiP
2 laccase/1 LiP
0.0537:1e to 0.1:1
0.537:1f to 1:1
1.074:1g to 2:1
Compared with strain improvement by genetic engineering , optimization of medium formulation is a low-cost way to control enzyme activities and their ratio. By adjusting medium formulation, we produced a crude laccase-LiP complex with designed laccase:LiP activity ratio specially for the degradation of species-specific plant lignins with fixed proportion of phenolic units to non-phenolic compounds. Using such specially designed laccase-LiP complexes, the degradation of lignin and lignocellulose biomass should be improved. By improving the biodegradation of abundant and renewable lignocellulosic biomass, laccase-LiP complex with specially designed laccase:LiP activity ratio will be beneficial to industrial processes.
The S. cinnamomensis strain used was obtained from BOKU University of Natural Resources and Applied Life Sciences (Vienna, Austria). Wheat bran and sawdust were purchased, and coffee pulp was obtained from a common coffee machine in the Division of Food Biotechnolgy, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Applied Life Sciences , Austria.
To prepare the inoculum, a growth medium composed of 15 g/L malt extract, 15 g/L yeast extract, 0.5 g/L (NH4)2SO4, 0.1 g/L CaCO3, 0.5 g/L MgSO4, and 0.5% (v/v) trace element solution (1% FeSO4, 0.09% ZnSO4, 0.02% MnSO4 (w/v) and distilled water) in 250 mL baffled flasks was autoclaved at 120°C for 20 minutes, then inoculated with S. cinnamomensis slant. The flasks were cultured at 25°C with shaking at 130 rpm for 3 days until spore concentration reached 1.5 × 107 colony-forming units (CFU)/cm3.
Experimental design and fermentation
The medium for laccase and LiP production from S. cinnamomensis by SmF (Table 1) was prepared in Erlenmeyer flasks with veratryl alcohol as inducer [5, 29]. After inoculation with 10% (7.5 mL v/v) of bacterial cell suspension containing 1.5 × 107 CFU/mL, 75 mL medium from each run was cultured at were cultured at 25°C with shaking at 120 to 130 rpm.
Laccase activity assay
The laccase activity was determined by oxidation of 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS, Sigma Chemical Co., St Louis, MO, USA) , with 1 U defined as the amount of laccase oxidizing 1 μmol of ABTS per minute .
Lignin peroxidase activity assay
Spore concentration assay
Spore concentration was determined by measuring the absorbance at 650 nm . The control had the same medium, but without inoculation.
where y represents laccase or LiP activity; F1, F2, F3 and F4 were the principal components corresponding to the four ingredients of yeast extract, coffee pulp, wheat bran and sawdust in culture medium; βi was the partial regression coefficient with β0, β1, β2, β3 being the linear terms and β11, β22, β33, β44 being the quadratic terms. R2 indicated to what extent of variability in the response could be explained by statistical model, and significance indicated the aptness of statistical model.
List of abbreviations
principal component analysis
submerged(/liquid) state fermentation
We are grateful to all the colleagues in Division of Food Biotechnology, Department of Food Sciences and Technology, BOKU University of Natural Resources and Applied Life Sciences for their general support or technical suggestions. This research was supported by the Eusia-Pacific Uninet Scholarship Award, National Natural Science Foundation of China (grant number 30700102), Branch of the Water Pollution Control and Management Project (2008ZX07209-010), Beijing Nova Program (grant number 2008A074) and Training Fund for Talents of Beijing (grant number 2010A005016000001).
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