text:
Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79.
ABSTRACT Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)-1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55 degrees C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60 degrees C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99x10(6) and 3.07x10(6) M-1 s-1, respectively). Catalytic rate constants for typical N-OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s-1), violuric acid (8.4 s-1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s-1), were found to be higher than those reported for other high redox potential fungal laccases.
Article: Chlorobenzoic acid degradation by Lentinus (Panus) tigrinus: In vivo and in vitro mechanistic study-evidence for P-450 involvement in the transformation.[Show abstract] [Hide abstract]
ABSTRACT: Aim of this work was to investigate the ability of Lentinus (Panus) tigrinus to degrade and detoxify a chlorobenzoate (CBA) mixture composed of mono-, di- and tri-chlorinated isomers. The degradation process was investigated as a function of both the growing medium (i.e. low N Kirk's and malt extract-glucose medium) and cultivation conditions (i.e. stationary and shaken cultures). The majority of CBAs were quantitatively degraded within the early 15 d from spiking with the notable exception of the double ortho-chlorinated compounds, 2,6-di-, 2,3,6-tri- and 2,4,6-tri-CBA. Analysis of the degradation intermediates indicated the occurrence of side chain reduction, hydroxylation and methylation reactions. Although CBAs stimulated laccase production, in vitro experiments with a purified L. tigrinus laccase isoenzyme demonstrated its inability to participate in the initial attack on CBAs even in the presence of redox mediators; similar results were found with a Mn-peroxidase isoenzyme. Conversely, prompt degradation was observed upon 1h incubation of CBAs with a purified microsomal fraction containing cytochrome P-450 monooxygenase. The nature of some reaction products (i.e. hydroxylated derivatives), the dependency of the reaction on NADPH and its susceptibility to either CO or piperonyl butoxide inhibition confirmed the involvement of L. tigrinus cytochrome P-450 in the early steps of CBA degradation.Journal of hazardous materials 07/2013; 260C:975-983. · 4.14 Impact Factor
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ABSTRACT: Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anophelesgambiae larvae in a concentration of 180nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7μM, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.Bioorganic & medicinal chemistry 03/2012; 20(5):1679-89. · 2.82 Impact Factor
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ABSTRACT: This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations.Biomolecules. 01/2013; 3(3):597-611.
Page 1
tigrinus CBS 577.79 was produced in a bubble-
column reactor using glucose-containing medium
supplemented with 2,5-xylidine under conditions
of nitrogen sufficiency. The main laccase isoen-
zyme was purified to apparent homogeneity by
ultra-filtration, anion-exchange chromatography
and gel filtration that led to a purified enzyme
with a specific activity of 317 IU (mg protein)–1
and a final yield of 66%. Laccase was found to be
a monomeric protein with a molecular mass of
69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of
the high mannose type. Temperature and pH
optima were 55?C and 3.75 (2,6-dimethoxyphenol
as substrate). At 50 and 60?C, the enzyme half-
lives were 281 and 25 min, respectively. The P.
tigrinus laccase oxidized a wide range of both
compounds: the highest catalytic efficiencies were
for 2,2¢-azinobis-(3-ethylbenzthiazoline-6-sulfonic)
acid and 2,6-dimethoxyphenol (5.99 · 106and
3.07 · 106M–1s–1, respectively). Catalytic rate
constants for typical N–OH redox mediators, such
as 1-hydroxybenzotriazole (2.6 s–1), violuric acid
(8.4 s–1) and 2,2,6,6-tetramethylpiperidin-N-oxide
radical (7.8 s–1), were found to be higher than
those reported for other high redox potential
fungal laccases.
Production Æ Panus tigrinus Æ Purification Æ
Substrate specificity
Bioreactor Fermentation Æ Laccase
bubble column reactor
relative electrophoretic mobility
isoelectric focusing on
polyacrylamide gel
catalytic rate constant
Michaelis Menten constant
lignin-modifying enzyme
olive-mill wastewater
polyacrylamide gel electrophoresis
sodium dodecyl sulfate
oxide radical
violuric acid
D. Quaratino Æ F. Federici Æ M. Petruccioli Æ
M. Fenice Æ A. D'Annibale (&)
Dipartimento di Agrobiologia & Agrochimica,
Universita ` degli Studi della Tuscia, Via San Camillo
de Lellis snc, I-01100 Viterbo, Italy
e-mail: firstname.lastname@example.com
Antonie van Leeuwenhoek (2007) 91:57–69
DOI 10.1007/s10482-006-9096-4
Production, purification and partial characterisation of a
novel laccase from the white-rot fungus Panus tigrinus
Daniele Quaratino Æ Æ Federico Federici Æ Æ
Maurizio Petruccioli Æ Æ Massimiliano Fenice Æ Æ
Alessandro D'Annibale
Received: 11 April 2006/Accepted: 29 May 2006/Published online: 30 September 2006
? Springer Science+Business Media B.V. 2006
Page 2
Lignin is a complex aromatic biopolymer rather
recalcitrant to biological degradation. Fungi
belonging to the ecological group of white-rot
basidiomycetes are considered the most effective
lignin degraders in nature (Field et al. 1993).
Degradation efficiency of these fungi is associated
with the release of extracellular lignin-modifying
oxidases (Tuor et al. 1995). Among these oxi-
dases, laccase (E.C. 126.96.36.199 para-benzenedi-
ol:oxygen oxidoreductase) has been shown to be
unambiguously implicated in the breakdown of
lignin via mechanisms either involving natural
mediators, such as 3-hydroxyanthranilate (Eggert
et al. 1997) or reactive oxygen species (Guille `n
et al. 2000). Laccases are extracellular, glycosyl-
ated proteins with two disulphide bridges and
four copper atoms distributed in one mononu-
clear (T1) and one trinuclear (T2/T3) domain
(Thurston 1994). The T1 copper center acts as the
primary electron acceptor from the substrate.
From T1, electrons are then transferred to the
type-3 copper pair center that acts as the final
acceptor. The trinuclear T2/T3 center, that is in-
volved in O2binding, accepts these electrons with
subsequent reduction of molecular oxygen to two
water molecules. In the last decade, increasing
research effort has been aimed at developing
environmentally sustainable processes in the pa-
per industry based on the use of laccases for
mediator-assisted bleaching of cellulose pulps
(Call and Mucke 1997). Laccases have also found
industrial application in the manufacturing pro-
cess of Denim textiles. In addition, the capability
of laccases to polymerize ubiquitous pollutants,
such as substituted phenols and anilines (Dec and
Bollag 1994) and to oxidize industrial dyes
(Nyanhongo et al. 2002) has suggested possible
usage in wastewater treatment (Duran and
Esposito 2000). In this respect, an efficient use of
laccases and the development of tailor-made
applications require a thorough determination of
their kinetic and stability properties.
The white-rot fungus Panus tigrinus CBS 577.79
was found to be an efficient producer of both Mn-
dependent peroxidase and laccase (Fenice et al.
2003). In addition, it proved to efficiently degrade
and detoxify olive-mill wastewater (OMW), an
agro-industrial phenol-containing effluent, even in
the presence of high organic loads (D'Annibale
et al. 2004; D'Annibale et al. 2006). This promp-
ted us to purify a major laccase isoenzyme and to
characterize its main physicochemical and bio-
chemical properties in order to gain further
information on this promising strain for possible
future applications. These properties are com-
paratively discussed with those reported for the
reference strain P. tigrinus 8/18 (Golovleva et al.
1993; Leontievsky et al. 1994).
Materials and methods
Organism and inoculum preparation
Panus tigrinus (strain 577.79) was obtained from
the CBS culture collection (Baarn, The Nether-
lands). During the study, the strain was main-
tained on potato dextrose agar slants at 4?C and
sub-cultured every month. Inocula were prepared
by growing the fungus for 96 h at 28?C in 1 liter
shaken flasks (150 rpm) filled with 200 ml of a
medium containing 50 g l–1glucose and 2 g l–1
yeast extract. At the end of incubation, pre-cul-
tures were centrifuged (4,000 g, 10 min) and wa-
shed with deionized water. The mycelium was
homogenized by Ultra-Turrax (IKA Labortech-
nik, Staufen, Germany) (two subsequent steps of
30 s each, at ca. 7,000 rpm) and diluted with
deionized water to yield a biomass concentration
of approximately 10 g l–1.
Enzyme production
Fermentations were carried out in a 3-l jacketed
bubble-column reactor (BCR; diameter 12 cm,
height 27 cm) filled with 2 l of a medium con-
taining 70 mM glucose, 20 mM nitrogen (as
ammonium tartrate), 0.25 mM 2,5-xylidine and
0.01 g l–1
yeast extract (medium GATXY).
Experiments in BCR were performed under the
following conditions: inoculum size 0.35 g l–1;
aeration rate 0.3 vvm; temperature 28? C; incu-
bation time 13 days. Air was injected through a
fritted glass sparger (Diameter 11 cm). The fer-
mentation parameters were monitored in the
bioreactors by an adaptative/PID digital controller,
58Antonie van Leeuwenhoek (2007) 91:57–69
Page 3
ADI 1030 (Applikon Dependable Instruments,
Schiedam NL). Fungal growth was determined by
measuring biomass dry weight.
Enzyme purification
Ten-day-old BCR cultures were centrifuged
(12,000 · g, 20 min) and culture supernatants
concentrated on a Mini-Sart tangential flow
apparatus (Sartorius, Goettingen Germany) fitted
with a 10 kDa membrane cassette. Further con-
centration and buffer exchange against 10 mM
imidazole–HCl buffer pH 6.0 (buffer A) was per-
formed in stirred cells (Amicon, Lexington, Mass)
fitted with a 10-kDa flat membrane prior to its
application to a 30 ml Q-Sepharose Fast Flow
column pre-equilibrated with buffer A at a flow
rate of 1 ml min–1. After extensive washing with
175 ml equilibration buffer, laccase was eluted by
a linear NaCl gradient from 0 M to 0.25 M
(660 ml) in buffer A. The pooled active fractions
were concentrated as above and applied to a Su-
perdex 75 prep-grade column (100 cm · 1.5 cm)
equilibrated with buffer A added with 0.15 M
NaCl (flow rate 0.54 ml min–1). Active fractions,
filter-sterilized (0.22 lm) and stored at –20?C.
Laccase and analytical assays
Laccase activity was routinely assayed spectro-
photometrically at 35?C using 2,6-dimethoxyphe-
nol (DMP) as a substrate and monitoring the
formation of 3,3¢,5,5¢-tetramethoxy- p-dipheno-
quinone (cerulignone) at 477 nm (e = 14600 M–
1cm–1) (Slomczynsky et al. 1995). One unit of
enzyme activity (IU) is defined as the amount of
enzyme, which produces 1 lmol of product per
min under the assay conditions. Protein determi-
nation was performed according to the dye-
binding method (Bradford 1976), using bovine
serum albumin as the standard. Total sugars and
ammonium were determined as reported else-
where (Quaratino et al. 2006).
Physico-chemical characterization
The native enzyme molecular mass was deter-
mined by gel filtration chromatography on Super-
calibrated with tyroglobulin (670 kDa), c-globulin
(158 kDa), ovalbumine (44 kDa) and myoglobin
(17 kDa),vitaminB12(1.35 kDa).Polyacrylamide
(12%) sodium dodecyl sulfate gel electrophoresis
(SDS-PAGE) was performed at constant voltage
(200 V) on a Mini-Protean II apparatus (Biorad,
Richmond USA) according to the Laemmli
method (1970). Molecular mass determination
under denaturing conditions was performed by
comparison with low-molecular weight standards
(Roche, Manheim). Analytical isoelectric focusing
on polyacrylamide gel (IEF-PAGE) in the range
2.5–7.0 was performed on a Mini-IEF apparatus
(Biorad, Richmond USA). The pH gradient was
measured by using the following standards: human
carbonic anhydrase (pI = 6.55), bovine carbonic
anhydrase (pI = 5.88), b-glucosidase (pI = 5.20),
soybean trypsin inhibitor (pI = 4.50), glucose oxi-
dase(pI = 4.15),amyloglucosidase(pI = 3.50)and
pepsinogen (pI = 2.80). Proteins were visualized
on gels by the ultra-sensitive colloidal Coomassie
G-250 method (Neuhoff et al. 1988). Native poly-
acrylamide gel electrophoresis (PAGE) was per-
(1964). Gels were cast with a 4% stacking gel and
at 20 mA and separate at 30 mA. Prior to activity
staining, native gels were rinsed three times with
distilled water, equilibrated in 50 mM acetate
buffer pH 4.0 and finally stained with 2 mM 2,2¢-
(ABTS) in the presence of 50 IU of catalase. To
laccase, the enzyme (4 lg) was previously degly-
cosylated by incubation with 12 IU of N-glycosi-
dase F (Roche Mannheim) in 0.2 M phosphate
buffer pH 7.2 for 3 h. Quantification was per-
formed by comparing the relative electrophoretic
migrations of native and deglycosylated laccases
on SDS-PAGE gel. UV–vis absorbance spectra of
purified laccase (540 lg ml–110 mM imidazole–
HCl buffer at pH 6.0) were recorded in a Lambda
20 spectrophotometer (Perkin Elmer, Sweden).
Lectin assay
Laccase (1 lg) and standard glycoproteins (1 lg
each) supplied with the Dig Glycan Differentiation
Antonie van Leeuwenhoek (2007) 91:57–6959
Page 4
Kit (Roche, Manheim Germany) were spotted
onto a Protran BA85 nitrocellulose membrane
(Schleicher & Schuell, Dassel Germany) and de-
tected immunologically after binding to lectins
conjugated with digoxigenin according to the
manufacturer's instructions. The following lectins
were used: Galanthus nivalis agglutinin, specific
for terminal mannose; Sambucus nigra agglutinin
specific for sialic acid a(2–6)galactose; Maackia
amurensis agglutinin, specific for sialic acid a(2–
3)galactose; peanut agglutinin, specific for galac-
tose b(1–3)N-acetylgalactosamine and Datura
stramonium agglutinin specific for galactose b(1–
spectra of purified laccase (540 lg ml–110 mM
imidazole–HCl buffer at pH 6.0) were recorded
in a Lambda 20 spectrophotometer (Perkin El-
mer, Sweden).
UV–vis absorbance
Effect of pH and temperature on enzyme
activity and stability
Laccase activity as a function of pH was measured
at 30?C using DMP as a substrate and 0.1 M cit-
rate–phosphate buffer in the pH range 2.5–7.5.
The temperature–activity profile was determined
in 0.1 M citrate–phosphate buffer pH 3.75 in the
range 10–70?C. To test the pH stability, 10 IU of
laccase were incubated at 25?C for 325 h in 5 ml
0.1 M citrate–phosphate buffer over the pH range
3.0–5.0. Incubations in the same buffer at pH 5.5
and 6.0 were prolonged to 670 h. Aliquots (10–
30 ll) were assayed for residual laccase activity
using DMP as a substrate. Thermal stability
experiments were conducted by incubating 40 IU
of laccase in 4 ml 0.1 M citrate–phosphate buffer
pH 6.0 at 50 and 60 and 70?C for 360, 90 and
12 min, respectively. Aliquots (50 ll) were chilled
on ice and assayed for residual activity as de-
scribed above.
Steady-state kinetic measurements
Kinetic constants of laccase for different phenolic
and non-phenolic substrates were determined at
30?C in air-saturated 0.1 M citrate–phosphate
buffer pH 3.75 using a substrate concentration
range from 0.005 mM to 10 mM. For ABTS,
experiments were performed in the same buffer at
pH 3.5.
extinction coefficients used for the test substrates.
For kinetic studies on N–OH mediators, enzy-
matic activity was determined by measuring the
oxygen uptake rate with a SA 520 Clark oxygen
electrode (Orion Instruments, Boston MA) con-
nected with a LKB 481 single-channel potentio-
metric recorder. The reaction mixture (10 ml)
containing variableconcentrations
from 0.5 mM to 25 mM) of the tested mediator in
0.1 M acetate buffer pH 4.0 was equilibrated at
25?C in the electrode chamber, then the reaction
was initiated by adding appropriate amounts of
laccase. Activity towards N–OH compounds was
calculated from the oxygen uptake rate by
assuming a stoichiometric ratio of 4 moles of
oxidized mediator per mole of oxygen consumed
(Xu et al. 2000). All kinetic constants were cal-
culated by non-linear least squares regression,
fitting experimental data to the Michaelis Menten
equation. Data were then linearised by the
Eadie–Hofstee method.
Table 2 provides wavelengthsand
Laccase production
Figure 1
extracellular laccase production by P. tigrinus
CBS 577.79 grown in a bubble column reactor on
a glucose-containing medium supplemented with
2,5-xylidine (XYL), as an inducer. Addition of
2,5-xylidine to the medium led to a ca. twenty-
fold increase in laccase activity (data not shown).
However, althoughXYL
rather low (i.e. 0.25 mM), its presence led to
delayed growth with biomass formation increas-
ing very slightly within the first 7 days of fer-
mentation (Fig. 1B). The onset of laccase activity
occurred on day 4 and increased at almost linear
rate up to day 10 when laccase reached its maxi-
mal activity (1.71 IU ml–1). The activity peak was
observed when glucose and ammonium were
depleted by about 85 and 39.5% from the growth
medium, respectively (Fig. 1A). The time course
of extracellular protein production paralleled that
of, laccase activity reaching a concentration of
88 lg ml–1on day 10 (Fig. 1A): specific laccase
showsa typical fermentationfor
60Antonie van Leeuwenhoek (2007) 91:57–69
Page 5
activity and volumetric productivity were 19 IU
(mg protein)–1and 7.12 IU l–1h–1, respectively.
Native alkaline PAGE analyses followed by
activity staining with ABTS showed the presence
in 10-day-old cultures of a major laccase isoen-
zyme with Emof 0.44 and two minor bands with
Emvalues of 0.14 and 0.47, respectively (data not
shown). Under these culture conditions, no
manganese-peroxidase activity was detected. Ini-
tial additions of CuSO4to the growth medium at
concentrations ranging from 0.1 mM to 1.0 mM
did not result in any further stimulation of laccase
production leading, by contrast, to marked inhi-
bition of fungal growth (at concentrations higher
than 0.5 mM, data not shown).
Laccase purification
The purification of P. tigrinus CBS 577.79 laccase
from 10-day-oldcultures
Table 1. Culture supernatant was 100-fold con-
issummarized in
centrated with 93% recovery of the total activity
with an expected low purification of 1.18 fold. In
the first chromatographic step, that involved an-
ion-exchange chromatography on Q-Sepharose
Fast Flow, laccase eluted at a NaCl concentration
of 0.22 M with a yield of 75% and a purification
fold of 15.97. Laccase was eluted as a single and
symmetric peak (Kav= 0.230) in the second step
that involved gel permeation chromatography on
Superdex 75, resulting in a final purity-fold and
yield of 16.71 and 66%, respectively (Table 1).
The enzyme, which showed a bright blue color and
had a specific activity of 317 IU (mg protein)–1
(DMP as a substrate), turned out to be homoge-
neous by both SDS- (Fig. 2A) and IEF-PAGE
(Fig. 2B), as judged by ultra-sensitive colloidal
Coomassie G-250 staining.
Physico-chemical properties of P. tigrinus laccase
The molecular mass of the native enzyme, as
determined by gel permeation chromatography,
Laccase activity (IU ml-1)
Extracellular protein ( g ml-1)
Time (days)
02468 10 1214
Total sugars (g l-1)
(NH4)+ (mM)
Biomass dry weigh ( t g l-1)
Fig. 1 Time courses of
laccase activity (•),
extracellular protein (n),
biomass dry weight (,),
nutrient nitrogen (s) and
total sugars (m) in a 3 l
bubble-column reactor.
Data are the
mean ± standard
deviation of three
Antonie van Leeuwenhoek (2007) 91:57–6961
Page 6
slightly differed from the one determined under
denaturing conditions by SDS-PAGE (69.1 kDa
vs. 68.5 kDa, respectively) (Fig. 2A). The protein
was shown to be rather acidic exhibiting a pI value
of 3.15 (Fig. 2B). The carbohydrate content of the
laccase was found to amount to 6.9% of
the molecular mass. Analysis of the nature of the
protein-bound oligosaccharide showed that the
enzyme was specifically recognized by G. nivalis
agglutinin lectin, which is reported to bind termi-
nal mannose residues. Negative results obtained
with M. amurensis, D. stramonium and S. nigra
and peanut agglutinin lectins suggested absence of
salivated complex type glycans and O-linked
oligosaccharide chains containing b(1–3)-N-acet-
ylgalactosamine at their non-reducing ends. The
UV–vis spectrum showed a maximum of absor-
bance at 610 nm and a shoulder at ca. 330 nm,
which are indicative of the presence of type-1 site
and type-3 binuclear copper, respectively.
Effect of pH and temperature on activity and
stability of laccase
The bell-shaped pH-activity profile showed that
the optimal pH for laccase activity was at 3.75
(DMP as a substrate), while the enzyme exhibited
a relative activity of about 3% at 6.5 (Fig. 3A).
Table 1 Purification scheme of extracellular laccase from P. tigrinus CBS 577.79
Step Total activity
Total protein
Specific activity
UI (mg protein)–1
Culture supernatant
Ultra-filtration (cut-off 10 kDa)
Q-Sepharose chromatography
Superdex 75 chromatography
Table 2 Kinetic constants (Kmand Kcat) and catalytic efficiency (Kcat/Km) of extracellular laccase from P. tigrinus CBS
coefficient (M–1cm–1)
Violuric acide
457 ± 12
107 ± 8
132 ± 9
102 ± 4
77 ± 8
119 ± 5
1056 ± 86
422 ± 34
239 ± 12
9.1 ± 1.2
31 ± 4
3340 ± 198
609 ± 14
2730 ± 123
2548 ± 213
8270 ± 345
11450 ± 765
4.8 ± 0.4
34.4 ± 2.0
93.9 ± 5.6
22.8 ± 1.3
36.3 ± 2.1
365.6 ± 32
49.6 ± 5.0
64.7 ± 7.3
25.9 ± 0.8
24.5 ± 2.2
185.7 ± 21
327.4 ± 12
138.5 ± 6.7
7.8 ± 0.2
8.4 ± 0.3
2.6 ± 0.02
0.4 ± 0.05
1.06 · 104
3.21 · 105
7.11 · 105
2.23 · 105
4.71 · 105
3.07 · 106
4.69 · 104
1.53 · 105
1.08 · 105
2.69 · 106
5.99 · 106
9.80 · 104
2.27 · 105
1.44 · 103
1.66 · 103
1.52 · 102
0.34 · 102
n.d., not determined;a3,4-dihydroxyphenylacetic acid
c2,2¢-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid
d2,2,6,6-tetramethylpiperidin-N-oxide radical
eActivity determined by the Clark oxygen electrode
Data are the mean ± standard deviation of three experiments
62Antonie van Leeuwenhoek (2007) 91:57–69
Page 7
The pH optimum for ABTS oxidation was in the
range 2.7–3.2. Figure 3B shows that the optimal
temperature was at 55?C and that a relative
activity of 40% was observed at 15?C.
Laccase stability as a function of pH was
determined at values lower than or equal to 6.0,
due to the negligible activity exhibited by the
enzyme above that pH value. Figure 4A shows
that the enzyme was maximally stable at pH 6.0
and 5.5 with half-lives of 632 h and 607 h. How-
ever, in the pH range 3.0–5.0, laccase proved to be
significantly stable showing half-lives ranging
from 131 h to 372 h. Thermal stability profiles of
P. tigrinus laccase at 50, 60 and 70?C are shown in
Fig. 4B. Half-lives at these temperatures were
281, 25 and 4 min, respectively.
Substrate specificity
Table 2 reports the kinetic constants for the lac-
case-catalyzed oxidation of both naturally occur-
ring and synthetic aromatic compounds. Presence
of an additional methyl or methoxyl group re-
sulted in a notable increase in Kcat. This was
evident by comparing 4-methoxyphenol with
phenol (36.3 s–1vs. 4.8 s–1, respectively), 2,6-di-
methoxyphenol with 2-methoxyphenol (365.6 s–1
vs. 22.8 s–1, respectively) and 4-methyl catechol
with catechol (93.9 s–1vs. 34.4 s–1, respectively).
Comparison of the Kcatfor 2-methoxyphenol and
4-methoxyphenol oxidation (22.8 s–1vs. 36.3 s–1,
respectively) showed that the activity was signifi-
cantly affected by the position of methoxyl sub-
stitution on the aromatic ring. In addition, P.
tigrinus laccase also showed higher affinity for the
para- than the ortho-methoxyl-substituted sub-
strate (Km of 77 lM vs. 102 lM, respectively).
The enzyme was unable to oxidize tyrosine and
nitrophenols, whether they be ortho, meta- or
showed markedly lower affinity for synthetic
substrates than phenolic compounds with the
notable exceptions of o-dianisidine and ABTS,
the Km values of which were 9.1 and 31 lM,
respectively. Table 2 also reports kinetic con-
stants for four distinct redox mediators, namely 1-
hydroxybenzotriazole (HBT), N-hydroxyphthali-
mide (HPI) and violuric acid (VLA), that have
been reported to be very effective in laccase-
mediated reactions (Xu et al. 2000; Fabbrini et al.
2002). Catalytic rate constants for these mediators
were in thefollowing
TEMPO > HBT > HPI. Lowest affinities were
observedwith HBT
11450 lM, respectively). In general, the catalytic
efficiencies for these mediators were lower, by at
least two order of magnitude, than the majority
of substrates under study. For naturally occur-
ring and synthetic substrates, the highest val-
ues of catalytic efficiency were observed with
not shown). Laccase
order:VLA ‡
andHPI (8270and
1 2 3
1 2
Fig. 2 (A) SDS-PAGE of native (Lane 3, 4 lg, Lac) and
deglycosylated (Lane 2, 4 lg, d-Lac) laccase from P.
tigrinus CBS 577.79 and molecular weight standards (Lane
1). (B) IEF-PAGE of laccase (Lane 1, 4 lg) and pI
standards (Lane 2). NG-F stands for N-glycosidase F
Antonie van Leeuwenhoek (2007) 91:57–69 63
Page 8
ABTS (5.99 · 106M–1s–1), respectively.
(3.07 · 106M–1s–1) and
P. tigrinus is a white-rot basidiomycete able to
perform selective degradation of lignin in both
woody and non-woody plants (Costa et al. 2002).
This interesting capability has suggested the use
of this organism in biopulping techniques (Gon-
calves et al. 2002). The relevance of this fungus in
other potential biotechnological applications has
been inferred from its capability to decolorize
agro-industrial effluents (D'Annibale et al. 2006)
and to degrade both textile dyes (Nazareth &
Sampy 2003) and priority pollutants (Leontievsky
et al. 2002). Most data available on the lignin-
degrading system of P. tigrinus refer to strain 8/18
(Maltseva et al. 1991; Leontievsky et al. 1994;
Relative activity (%)
Temperature (˚C)
1020 3040 5060 70
Relative activity (%)
Fig. 3 (A) pH-activity
profiles of P. tigrinus CBS
577.79 laccase with 2,6-
(DMP, •) and 2,2¢-
sulfonic acid (s)
oxidation at 30?C. (B)
profile of P. tigrinus CBS
577.79 laccase in DMP
oxidation (n) in 0.1 M
citrate phosphate buffer
pH 3.75. Data are the
mean ± standard
deviation of three
64Antonie van Leeuwenhoek (2007) 91:57–69
Page 9
Lisov et al. 2003). In particular, extracellular
laccase was purified from both liquid (Leontiev-
sky et al. 1997) and solid-state cultures (Golovl-
eva et al. 1993) of strain 8/18. The enzyme
purified from liquid cultures exhibited a molecu-
lar mass of 64 kDa and typical UV–vis and EPR
spectroscopic features of copper oxidases (Leon-
tievsky et al. 1997). Recently, the enzyme has
been crystallized and a preliminary structure
analysis at 1.4 A˚ resolution has been reported
(Ferraroni et al. 2005). By contrast, laccase ob-
tained from solid-state cultures of the same strain,
albeit containing approx. four copper atoms per
molecule, exhibited atypical spectroscopic fea-
tures and was defined as a yellow laccase (Leon-
tievsky et al. 1997). The authors postulated that
the native enzyme did not maintain its copper
centers in the oxidized state and hypothesized
that this effect was due to the reaction of low
molecular mass lignin degradation intermediates
with ligands of the type-1 copper (Leontievsky
et al. 1997). However, manganese peroxidase was
found to be the main lignin-degrading enzyme in
P. tigrinus 8/18, since it was secreted at signifi-
cantly higher extents than laccase (Maltseva et al.
1991; Leontievsky et al. 1994; Podznyakova et al.
1999). In addition, the total ligninolytic capacity
of this strain was found to be highly correlated
Incubation time (hours)
0 50 100 150 200 250300350650700
Residual activity (%)
Incubation time (hours)
0 50 100150200250300350 400
Residual activity (%)
Fig. 4 (A) Residual
activity of laccase from P.
tigrinus CBS 577.79
throughout 325 h
incubation at 25?C in
0.1 M citrate phosphate
buffer at pH 3.0 (•), 3.5
(s), 4.0 (.), 4.5 (,), 5.0
(n), 5.5 (h) and 6.0 (rÞ.
The incubation of the
enzyme at pH 5.5 and 6.0
was prolonged to 670 h.
(B) Residual activity of P.
tigrinus CBS 577.79
laccase throughout 360, 90
and 12 min incubation in
0.1 M citrate phosphate
buffer pH 6.0 at 50 (•), 60
(s) and 70 (.) ?C,
respectively. Data are the
mean ± standard
deviation of three
Antonie van Leeuwenhoek (2007) 91:57–6965
Page 10
with manganese peroxidase production (Leon-
tievsky et al. 1994).
By contrast, laccase was the predominant lig-
nin-modifying enzyme (LME) produced by the
strain P. tigrinus CBS 577.79 in both solid (Fenice
et al. 2003) and liquid cultures in OMW-based
media (D'Annibale et al. 2004). The same strain,
grown on an agro-industrial effluent, was able to
both tolerate organic loads as high as 60,000 mg l–1
and to perform significant dephenolization and
decolorization of the waste (D'Annibale et al.
2004). The interesting characteristics of P. tigrinus
CBS 577.79 and the aforementioned differences
with the reference strain 8/18 prompted us to
produce, purify and to characterize the main
laccase isoenzyme produced in liquid cultures. To
this end, a pneumatically agitated reactor, i.e. a
BCR system, was used in the present study; in-
deed, the BCR system proved to be more ade-
quate than mechanically agitated reactors to
support laccase production by this strain (Fenice
et al. 2003) due to lower shear-stressing condi-
tions (D'Annibale et al. 2006). With regard to
nitrogen concentration, a condition of sufficiency
was adopted since it was previously observed that
laccase production levels by P. tigrinus CBS
577.79 were very low or absent under nitrogen
limitation (Quaratino 2005; Quaratino et al.
2006). In this respect, these findings are in
agreement with earlier studies reporting that
culture conditions in which nutrients, including
carbon, sulphur and, above all, nitrogen, were not
limiting resulted in markedly improved laccase
production (D'Souza et al 1999; Medeiros et al.
1999; Galhaup et al. 2002). However, when the
laccase activity peak was reached, the nitrogen
source was found to be only partially depleted
from the medium (present study). The addition of
2,5-xylidine to the liquid growth medium signifi-
cantly stimulated laccase production by P. tigrinus
CBS 577.79 in agreement with an earlier study
conducted with another strain of the same species
(Elisashvili et al. 2002) and with other studies
reporting 20-fold and 9-fold stimulation in Tra-
metes villosa (Yaver et al. 1996) and Pycnoporus
cinnabarinus (Eggert et al. 1996), respectively. By
contrast, both the failure of CuSO4addition to
enhance laccase production by strain CBS 577.79
and the evident toxic effects of this salt at con-
centrations higher than 0.5 mM were unexpected.
In contrast, a recent study (Chernykh et al. 2005)
showed that laccase production by the reference
strain P. tigrinus 8/18 was 10-fold stimulated by
the combined addition of 2 mM CuSO4 and
1 mM 2,4-dimethylphenol to a glucose/peptone
liquid medium. Although this is a further feature
that differentiates the strain employed in the
present study from P. tigrinus 8/18, the two strains
exhibited some similarities with regard to the
physiological regulation of LMEs production. In
particular, the enhancement of laccase production
was shown to require either nitrogen-sufficient or
nitrogen-rich conditions (Quaratino 2005; Cher-
nykh et al. 2005) while manganese peroxidase
was preferentially produced in nitrogen-limited
cultures in the presence of high concentrations of
Mn2+(Quaratino et al. 2006; Lisov et al. 2003).
The three-step purification scheme employed
in the present study proved to be highly efficient
resulting in a 66% final yield and a purity fold of
16.7. The purified enzyme was found to be N-
glycosylated with a glycosylation pattern of the
high-mannose type and differed slightly from P.
tigrinus 8/18 blue laccase regarding molecular
mass (69 kDa vs. 64 kDa, respectively), pI (3.15
vs. 3.0, respectively), pH and temperature optima
(3.75 vs. 4.8 with DMP as a substrate and 55?C
vs. 60?C, respectively) and apparent Km for
2,6-dimethoxyphenol (119 lM
respectively). Albeit being classified as para-
benzenediol:oxygen oxidoreductases, laccases are
rather non-specific as to their reducing substrates
(Thurston 1994; Xu 1996). The P. tigrinus CBS
577.79 laccase did not make any exception since it
was able to oxidize a wide range of aromatic
compounds albeit with different reaction rates
and affinities. The increase in Kcatobserved for
compounds bearing an additional methoxyl or
methyl group (2,6-dimethoxyphenol vs. 2-meth-
oxyphenol and 4-methylcatechol vs. catechol,
respectively) might be due to the electron-
donating effect of both substituents leading to
higher density at the phenoxy group. For the
opposite reason, the enzyme was unable to oxi-
dize nitro-substituted phenols. In fact, the nitro
substituent has a very strong electron-withdraw-
ing character thereby impairing the susceptibility
of the substrate to oxidation. With respect to the
vs.16.6 lM,
66 Antonie van Leeuwenhoek (2007) 91:57–69
Page 11
effect of substituents on laccase-catalyzed reac-
tion rates, Garzillo et al. (1998) emphasized the
polar effect of additional non-bulky substituents
with at least one lone pair electrons on the atom
adjacent to the aromatic ring such as the hydro-
xyl, the methoxyl or the amino-group. The
greater effect of these substituents than other
electron-donating groups such as –CH3 and –
CH=CH–COOH was explained in terms of their
higher impact on the electron density of the pri-
mary oxidizing group (Garzillo et al. 1998). Sev-
eral studies have emphasized the role played by
the difference between the redox potential at site
type 1 of laccase and that of reducing substrates in
governing reaction rates in laccase-catalyzed
oxidations (Xu 1996; Xu et al. 2000). In this re-
spect, the lower redox potentials of both DMP
and 4-methylcatechol than 2-methoxyphenol and
catechol can also account for their higher sus-
ceptibility to laccase-catalyzed oxidation. How-
ever, it has been shown that the catalytic
efficienciesof Trametes
lignosus and Pleurotus ostreatus laccases were
only partially due to their specific redox capabil-
ities (Garzillo et al. 2001). In another study, lac2
isoenzyme of basidiomycete C30 was more effi-
cient in phenol oxidation than lac1, although the
redox potential of the T1 copper of the former
was lower by 170 mV than that of lac1 (Klo-
nowska et al. 2002). The same investigators de-
duced that other factors, such as hydrogen
bonding and extent of protonation of ionisable
residues in the vicinity of the T1 copper, might
play considerable effects on the overall activity
(Klonowska et al. 2002). The applicative impli-
cations of laccase-mediated catalysis prompted us
to investigate the kinetic constants of P. tigrinus
laccase for four distinct redox mediators belong-
ing to the N–OH group including HBT, VLA,
TEMPO and HPI. The catalytic rate constants for
HBT and VLA were similar to those of T. villosa
I laccase or better than those reported for other
high redox potential laccases, such as those from
Pycnoporus cinnabarinus or Botrytis cinerea (Li
et al. 1999; Xu et al. 2000). In addition, P. tigrinus
laccase exhibited larger affinity for these com-
pounds than other fungal laccases with the sole
exception of Coriolus cinereus laccase-1, the Km
of which for HBT was 7000 lM (Xu et al. 2000).
trogii, Rigidosporus
In a recent study (Kulys and Vidzuinaite 2005)
aimed at investigating kinetic constants of TEM-
PO oxidation, three out of four recombinant
laccases were found to be scarcely competent on
that mediator. Among them, recombinant T.
villosa laccase exhibited better affinity and cata-
lytic efficiency on this mediator than P. tigrinus
laccase(391 lM and
2730 lM and 1.44 · 103M–1s–1, respectively).
Although a significant amount of information
is available on the lignin-degrading system of P.
tigrinus, this study reports on a laccase isoenzyme
with properties distinct from those previously
reported for this species and provides further,
important knowledge on the strain CBS 577.79,
the use of which has been shown to be very
promising in OMW treatment (D'Annibale et al.
2004; D'Annibale et al. 2006). The good stability
of the P. tigrinus CBS 577.79 laccase in the pH
range 5.0–5.5, which is generally that encountered
in OMW, and the high values of the specificity
constants (Kcat/Km) for several phenols that are
commonly found in that wastewater (i.e. catechol,
4-methylcatechol, 3,4-dihydroxyphenylacetic acid
and 4-methoxyphenol), clearly indicate the ade-
quacy of this enzyme in OMW treatment.
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Antonie van Leeuwenhoek (2007) 91:57–6969


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Food Revolutionaires
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Total Annihilation Walkthrough :
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Walkthrough - FAQ/Strategy Guide
_____ _____ _____ ___ _ |_ _| / _ \ |_ _| / \ | | | | | | | | | | / _ \ | | | | | | | | | | | /_\ | | | | | | |_| | | | | _ | | |__ ____ |_| \_____/ |_| |_| |_| |____| _ _ / \ | \ | | / \ _ _ _ _ _ _ _ _ _ ___ _____ _ ___ | \| | / \| \ | || \ | || || | | || || | / \|_ _|| |/ _ \| | | | /\ || \| || \| || || | | || || | / _ \ | | | ||| ||| | | /__\ || || || || |_| || || | | /_\ | | | | ||| ||| | | || || || || _ || || | | _ | | | | ||| ||| |\ | | /\ || |\ || |\ || || | | || || |__ | | | | | | | |||_||| | \ | |__| |__||_| \_||_| \_||_||_| |_||_||____||_| |_| |_| |_|\___/|_| |_| ------------------ Total Annihilation ------------------ FAQ/Strategy Guide For the PC By BrakZero +- [ Version 0.54 ] -+ This FAQ covers the following games: � Total Annihilation � Total Annihilation: The Core Contingency A R M C O R E _ /\ /\ ____/C\____ /// \\\ /////ORE\\\\\ /// /\ \\\ /////COREC\\\\\ /// /AR\ \\\ /////ORECORE\\\\\ /// /MARM\ \\\ /////COR/|\ECO\\\\\ ______/// /ARMARM\ \\\_______ (////REC/|||\ORE\\\\) \������/ /ARMARMAR\ \�������/ (///COR/|||||\ECO\\\) _______ \MARMARMA/ _______ V.S. \/RECO|||||||RECO\/ /�������\ \RMARMA/ /�������\ \RECO|||||||RECO/ \\\ \RMAR/ /// \REC|||||||ORE/ \\\ \MA/ /// \CO|||||||RE/ \\\ \/ /// \C|�����|O/ \\\ /// \| |/ \\\/// \\// \/ "What began as a conflict over the transfer of consciousness from flesh to machine, escalated into a war which has decimated a million worlds. The Core and the Arm have all but exhausted the resources of a galaxy in their struggle for domination. Both sides, now crippled beyond prepare, the remnant if their armies continue to battle on ravaged planets, their hatred fueled by over 4000 years of total war. This is a fight to the death. For each side, the only expectable outcome is the complete elimination of the other..." - Introduction Welcome to my Total Annihilation FAQ/Strategy Guide! It is not done yet, but do not fear! Updates are always coming! ~ BrakZero ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ =�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�= :: Revision History :: Version 0.54 [7/14/02] - Since I�m very very very lazy I stopped working on this guide after that last update. Now, more than seven months later, I�ve started to work on it again. Unfortunately, there are only a few new mission strategies added, but I just wanted to update this so that people will know I didn�t completely abandon this guide. (218K) Version 0.53 [12/9/01] - Very minor format changes and some more mission strategies scattered around. Mission strategies should be completely done with the next update. I hope. (214K) Version 0.5 [11/22/01 � Thanksgiving] - I�m done with the units� section... after working on it slowly for about 9 months. And I�ve even started the walkthrough for the Arm campaign. Man, I can�t believe this day has finally come!! *faints* (184K) Version 0.47 [10/25/01] - Finished everything up to the Sonar in the Structures section. Getting close to the end of the units; it should be done with the next update. ^___________^ (163K) Version 0.42 [9/26/01] - Finished Core Seaplanes. - Would have added more, but since I haven�t updated for quite a while, I thought I should make another update to let people know I�m still alive. (136K) Version 0.41 [8/3/01] - Finished Core Hovercraft. (133K) Version 0.4 [8/1/01] - Finished Core Ships. - Finished Core Advanced Ships. (128K) Version 0.3 [7/9/01] - Finished Core Aircraft. - Finished Core Advanced Aircraft. (121K) Version 0.2 [7/8/01] - Finished the Core Advanced Kbots. - Finished the Core Vehicles. (109K) Version 0.1 [7/7/01] � This guide is VERY VERY VERY incomplete. I only have the Arm units done, and the Core units are only done up to through the Kbots, which is the first part... -_- (98K) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ =�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�=�= ----------------------------------------------------------------------- ------------------------------- Credits ------------------------------- ----------------------------------------------------------------------- Triaxx2 - Thanks for the cost and statistics for the Core Leviathan! Cavedog (www.cavedog.com) - For making the best real-time strategy game I�ve ever played. I absolutely love this game. But why did you give up on the website??? CJayC (www.gamefaqs.com) - For all his amazingly hard work and determination he puts into his website, and for posting this FAQ. Myself (firstname.lastname@example.com) - Of course, this couldn�t be here without yours truly. ----------------------------------------------------------------------- ----------------------------------------------------------------------- ----------------------------------------------------------------------- ======================================================================= ============================= LEGAL STUFF ============================= ======================================================================= This document was meant solely for public use to the users of the World Wide Web. It is not, by any means, to be rewritten, reproduced, etc without permission of the author. It should not be used directly or indirectly to make any amount of profit. It�s against the law. Don�t be stupid. This document is copyright (C) 2001 by BrakZero (Alex Biedacha) ======================================================================= ----------------- Table of Contents ----------------- Battle Breakdown Campaign Walkthrough Strategies Units = Arm - Kbots - Advanced Kbots - Vehicles - Advanced Vehicles - Aircraft - Advanced Aircraft - Ships - Advanced Ships - Hovercraft - Seaplanes - Mines - Structures + Energy Production + Metal Production + Energy to Metal Converters + Energy Storage + Metal Storage + Unit Production Plants + Radar + Sonar + Defense + Targeting + Aircraft Repair + Attack Structures = Core - Kbots - Advanced Kbots - Vehicles - Advanced Vehicles - Aircraft - Advanced Aircraft - Ships - Advanced Ships - Hovercraft - Seaplanes - Mines - Structures + Energy Production + Metal Production + Energy to Metal Converters + Energy Storage + Metal Storage + Unit Production Plants + Radar + Sonar + Defense + Targeting + Aircraft Repair + Attack Structures - Krogoth Related + Krogoth Gantry + Krogoth = Others - Galactic Gate (Arm) - Galactic Gate (Core) - Hydration Plant - Sea Serpent - Truck Shortcut Keys Codes, Secrets, etc. ����������������� BATTLE BREAKDOWN If you�re confused with everything that�s going on in that screen of yours, and you have no idea what�s going on, you don�t need to look any further. ====================================== | | 2. | 3. | | 1. |------------------------- | | | | | | |-----------| | | | | | 4. | | | | 5. | | | | |-----------| | | | | | 6. | | | |------------------------| | | 7. | 8. | (9.) | ====================================== 1. Map and Radar/Sonar This displays the overall map as well as the radar and/or sonar on the map. However, depending on how you set it, this entire area can appear in different ways: - You can have it on Mapped status, in which all of the terrain is visible. This doesn�t mean that you can see everything, though. It means you can see the map in black and white, and that means only the terrain is visible. Enemy units can�t be seen in that way. But, if you combine Mapped with Permanent line of sight, all the terrain is visible AND colored, so you can go wherever you want and see whatever you want as well, including your enemy units. This eliminates your need for radar and sonar completely. - If you have the Unmapped status for your map, that means you start off the Skirmish with a black map, and it stays that way until you explore it. After you explore and leave the area, it stays visible, but not with a permanent line of sight. - Radar/sonar appears in the same color of your team color, and the same goes for the enemy. You can spot enemy units through radar/sonar only with Radar Towers and Sonar Stations, and their advanced versions. Also, some structures and units come with automatic radar devices that can be used. (Such as the Commander and the Advanced Aircraft Plant) - Just as a note, the settings for ALL Campaign missions are Unmapped, and the line of sight is on the True setting. Unmapped is the blacked- out-until-explored mapping, and the True setting for line of sight means that a unit�s view is affected by elevation. Also, the team colors appear as blue for you, and red for the enemy. Remember that the radar appears in the same color as the team the unit is on. 2. Metal Gauge This displays everything about your metal. The bar indicates how much metal you have left compared to your maximum amount. On the right of that, there is a green number with a plus sign and a red number with a negative sign. The plus sign number tells how much metal is being produced at the moment. The red negative number tells how much is being used up. So generally, you just the producing number to be higher and that way you won�t run into any metal shortage problems. 3. Energy Gauge This displays everything about your energy. The bar indicated how much energy you have left compared to your maximum amount. On the right of that, there is a green number with a plus sign and a red number with a negative sign. The plus sign number tells how much energy is being produced at the moment. The red negative number tells how much is being used up. So generally, you want the producing number to be higher and that way you won�t run into any energy shortage problems. 4. Unit Construction Choice/Commands When you click on a construction unit, a small screen should appear in this area with various pictures of units. These pictures can be clicked and placed somewhere, where the construction unit will nanolathe them. (Keep in mind that there can be more choices if you click on the arrow beneath the pictures.) This is the same for unit production plants such as a Kbot Lab. However, you can click "Commands" at the top of this section (Automatically "Commands" when clicking a non-construction unit) and this gives you a list of how you would like the unit to function. 5. Battle Screen Well, this is obviously where you see everything, battle everything, build everything, etc. I pity you for even reading this, if you don�t know what�s going on in this part of the screen. =P 6. Action Commands Whenever you click on any type of unit, these commands will appear. These give specific actions for the selected unit. These include basic commands such as Attack, Move, etc. A majority of the time, the unit will have at least one of these commands blacked out, and that simply means that that particular unit cannot use that command. This holds especially true with the D-Gun command... 7. Life Gauge/Unit Data When you hold your pointer over a unit, a gauge will appear in this portion of the screen. This is the amount of life they have left in them. Full life means a full green meter, and red is the killed off part. So the more green, the better! (Unless we�re speaking of an enemy unit.) Also, when you are using a construction unit and choosing your unit to nanolathe, look down here for the metal and energy cost. 8. Unit Action and Metal/Energy Production and Use First off, when you click a unit, there are those green and red numbers again. These show the units metal production, metal consumption, energy production, and energy consumption. The red and green numbers on the left display metal. (Green is production and red is consumption, in case you didn�t take a hint. =P) The two numbers on the right display energy. And above them displays what the selected unit is doing. For instance, if a unit is doing nothing, it is marked as "Ready". These actions include things like Moving, Attacking, Nanolathing, Ready with Orders, etc. 9. Unit Being Attacked and Secondary Building Gauge This doesn�t always appear when selecting a unit, so I�ve put it on my little map in parentheses. This DOES appear in two situations: 1) When a unit on your side is attacking or aiming for an enemy unit, the name of the enemy unit is displayed here, as well as the amount of life the enemy unit has. 2) When you are making nuclear missiles from nuke launchers or nuke protection systems, the building progress shows up here. This is unique because those missiles are not actual units, and cannot be seen while building. So, if you want to make a nuke from a CORE Silencer, then click the picture to make one, then hold your mouse pointer over the Silencer and look in this area. It should display how much of the missile is done. --- So now hopefully you know everything that�s going on in the battle screen, so good luck! ======================================================================= ======================================================================= CAMPAIGN WALKTHROUGH Stuck on a mission? Need an easier strategy for it? Just need some info about it? Well, here I�ll try to give some tips, info, and strategy for all of the missions for both the Arm and the Core. (And that includes the Core Contingency Campaign, but not the Battle Tactics missions) *** VERY IMPORTANT NOTE *** ��������������������������� These mission strategies are based on the EASY difficult setting. I did this because I find that many people naturally turn the difficulty level down to easy for their first time playing through, and many people referring to this section are generally playing the missions for their first time. If you are not playing on easy and are stuck, try checking out some of the other guides on the Internet, like the guide at www.cavedog.com. Besides, the basic strategies are pretty much exactly the same no matter what the difficulty is set on. You�ll only find yourself being attacked more constantly by a larger number of units as it goes up, that�s all... and you�ll start off with less units. +- [ Arm Campaign ] -+ ======================================================================= Mission #1 � A Hero Returns ----------------------------------------------------------------------- Starting Units � 5 Peewee, 3 Jeffy, 2 Hammer, 2 Stumpy, 2 Rocko, 1 Flash, (1 Galactic Gate) ======================================================================= As you might expect, the first mission is ridiculously easy. First, scroll upward towards the radar dot at the top of the screen to look at your Galactic Gate. That is your goal, and it is extremely simple. Just highlight all of your units, then click right next to the Galactic Gate within the Dragon�s Teeth, and just sweep upward. You�ll run into some very small patrols of Core A.K.�s and Weasels, but just ignore them and a Jeffy will quickly reach the Galactic Gate to finish the mission. --- {From here on, the Commander is always a starting unit in a mission along with the other units mentioned.} --- ======================================================================= Mission #2 � CORE KBot Base, Destroy It! ----------------------------------------------------------------------- Starting Units � 5 Peewee, 2 Rocko, 1 Hammer ======================================================================= First, take your Commander and throw up 3 Metal Extractors on the metal patches along with 3 Solar Collectors. During this time, you�ll probably be attacked by one measly unit, which should be disposed of quickly by your starting units. Once you�re done with that, make a Kbot Lab and make about 20 Hammers. Yes, it is boring to wait for them while doing absolutely nothing else, but hey, it�s worth it. Once you�re done with the Hammers, gather them all up (along with the starting units) and go southeast through the small canyon. (I recommend that you don�t take your Commander. He�ll be fine staying where he is; the enemy won�t attack there.) Plow through the resistance with your Hammers and cross the shallow part of the river right into the base. Find the Kbot Lab and blow it up to complete the mission. ======================================================================= Mission #3 � Spider Technology ----------------------------------------------------------------------- Starting Units � 4 Spider, 2 Peewee, 2 Rocko, 2 Hammer, 1 L.L.T. ======================================================================= Don�t listen to the part in the Briefing that talks about clearing a path for the spiders � you don�t need to. Just highlight the 4 Spiders at the top of the map and walk them back to your base. 99% of the time, at least one of the Spiders returns safely, if not all of them. Their paralyzing weapon stops the weak patrols in their tracks. Once one of the Spiders walks a little bit past the starting units, the mission is completed. ======================================================================= Mission #4 � CORE Contamination Spreads... ----------------------------------------------------------------------- Starting Units � 2 Hammer, 2 Rocko, 2 Weasel (captured Core units), 1 Zeus, 1 Light Laser Tower (captured Core unit) ======================================================================= Well, at least you�re actually starting to get SOME challenge. You�re going to be attacked right off the bat by a couple of units coming in one at a time, but they should be easily taken care of by your Light Laser Tower, Rocko units, and your Zeus. Still, don�t waste any time, and build 2 Metal Extractors on the nearby Metal patches, along with several Solar Collectors to get your energy going. Throw up a Radar Tower, and check out some of the radar picked up on the left. You should see three red dots lined up vertically, with another red dot a little bit above it. Take your Weasels and quickly drive over there to destroy the red dots, which are Radar Towers, and destroy the Metal Extractor a little bit above it. While they�re doing this, build a few L.L.T. units lining the left side of the base to get some early defense. The position in the corner of the map is perfect, as the Core does not attack from the north and so you only have one side to defend. Anyway, after you�re finished putting up some defenses, build a Kbot Lab and crank out a few Hammers, along with a couple of Rocko units if you wish. During this time, you�ll be attacked by groups of Core A.K.�s, Storms, Thuds, and Weasels. Hopefully you can hold everything up with some of those defenses while you build some Kbots to help them out. If you would like to, you can also build a Vehicle Plant to help the defenses with a few vehicles. In any case, make a Construction Kbot using the Kbot lab after making some basic units, and then make an Advanced Kbot Lab while you ward off a few attacks. After you�ve gotten an Adv. Kbot Lab, crank out lots of Fido bots. These are excellent for the mission, and are fantastic for defending your base once grouped over on the left. During all of this time, you�ll probably be very low on metal. Use the Commander or a Construction Kbot/Vehicle to reclaim any kind of wreckage that you find, and it will help you out. Be patient while the Fidos are being made and easily blast away any of the dying forces being sent your way. Once you�ve gotten about 20 Fidos and several other Kbots and Vehicles, you�re ready to sweep the map. Gather up your units, and start to explore the map. Leave the Commander and a couple of other units behind in case some troops attack while you�re away... it�s better not to risk anything with the Commander. You may want to start by going north, then slowly towards the center/west- center part of the map where the main base and resources are. Even so, you�ll still be required to check a majority of the blacked-out space, as the Core troops are scattered in small groups all over the entire map. Once you�ve destroyed every single Core unit, the mission is completed. ======================================================================= Mission #5 � The Gate to Thalassean ----------------------------------------------------------------------- Starting Units � 6 Fido, 4 Triton, 3 Bulldog, 3 Jeffy, 3 Zipper, 2 Lurker, 2 Skeeter, 2 Zeus, 1 Jammer, 2 L.L.T., 1 Metal Extractor ======================================================================= As unbelievably stupid as this sounds, I think that the best way to complete this mission is to gather up your units immediately and start sweeping the map. Seriously, this seems like an easier way than to build a base and ward off attacks until you�ve built up a big enough army. You�ve got to understand that the enemy is ALSO building up their troops while you do, and they are striking your base CONSTANTLY... and their strikes are hard. Since it is so difficult to keep them away, an immediate strike with your already quite powerful group would be nice. It is a little bit risky, and you might fail, but it should work... as long as you do one thing... REPAIR units whenever you can. While sweeping, you should stop between battles and repair every single unit to full health with you Commander. If the Commander is damaged, then stay in one spot for a while and wait for him to heal by himself. If you do this, you should successfully wipe out all the land units. If you don�t want to do this, you can always stay and build a base. Just do the regular things: build your resources and produce an army of vehicles. (You can now use some advanced vehicles) Just make sure you�ve got plenty of defenses for the Core onslaught, and then sweep the land part of the map once you are ready. Now, to capture the Galactic Gate. You must have your Triton tanks. If by some wild chance they didn�t survive, then you can either build a Vehicle Plant, Construction Vehicle, and Adv. Vehicle Plant to produce a couple, or just use the Commander for the next job. It would be better to use the Commander instead of waiting for it all to be done. In any case, first take your Lurkers and go right towards the southwest corner of the map and take out the scout ship patrolling along the coast of the tiny island. Then, use your Tritons (or Commander, but you should have Tritons =T) and go to that island. Take out the few light units there; they shouldn�t give you any problems. Just MAKE SURE YOU DO NOT DESTROY THE GALACTIC GATE. Take you Commander over there to capture the Galactic Gate. Once the gate is captured and every single Core unit on the map is destroyed, you have won. ======================================================================= Mission #6 � Beachhead on Thalassean ----------------------------------------------------------------------- Starting Units � None (Only Commander and Galactic Gate) ======================================================================= ======================================================================= Mission #7 � The Defense of Larab Harbor ----------------------------------------------------------------------- Starting Units � 8 Jethro, 5 Skeeter, 4 Hammer, 3 Samson, 2 Crusader, 1 Ranger, 1 Zeus, 3 Defender, 3 L.L.T, 2 Metal Storage, 2 Wind Generator, 1 Energy Storage, 1 Guardian, 1 Radar Tower, 1 Shipyard, 1 Tidal Generator, and 1 Moho Mine ======================================================================= ======================================================================= Mission #8 � The Gate to Tergiverse IV ----------------------------------------------------------------------- Starting Units � 6 Jethro, 6 Skeeter, 3 Lurker, 1 Crusader, 1 Ranger, 1 Zeus, 5 L.L.T., 2 Wind Generator, 1 Shipyard ======================================================================= ======================================================================= Mission #9 � The Hydration Plant ----------------------------------------------------------------------- Starting Units � None (Only Commander and Galactic Gate) ======================================================================= Very easy mission. Start by D-gunning the Pyro, as you have no need for land units in this mission. Capture the Radar Tower and the Solar Collector, then throw up another Solar Collector and two Metal Extractors on the metal patches, then build an aircraft plant like the briefing says. You�ll notice that the radar dots are extremely close to you, but don�t worry about them. The Kbots will swarm around you, but they can�t get up the steep hill you start out on, nor can any of your units besides the Commander go down. (You are put here purposely to make sure you use aircraft instead of land units) Build a Peeper and scout out the northern-central part of the map until you find the Hydration Plant. Nothing will shoot you down. Once you find it, build an Atlas, load the Commander onto it, and drop the Commander off at the Hydration Plant and capture it. (D-Gun anything if necessary) Alternatively, just don�t bother doing anything, and just march your Commander up to the Hydration Plant and D-Gun anything in your path. :) ======================================================================= Mission #10 � The Bromid Maze ----------------------------------------------------------------------- Starting Units � 2 Hammer, 2 Spider, 1 Zeus (and 9 Zipper cut off from you at the north-west corner of the map) ======================================================================= Start off by highlighting your Zippers that are cut off and run them back to your normal units. They will run into some very light resistance, but most, if not all, should make it back. Repair them, then you can start. This is not a very hard mission, mainly due to the fact that the Core will not attack you, and they do not build their base any bigger. Not to mention that their base is weak, too. So start by laying down a couple of Metal Extractors and several Solar Collectors, then build a Kbot Lab. You�re going by low on metal just with those two Metal Extractors, so go a little bit to the right and you should find two more metal patches. Put up a L.L.T. close by just in case they happen to get attacked with light forces, and a Defender once you have built a Construction Kbot with the Kbot Lab. Build a couple more Defenders around the base, too. Once you have done this, build an Advanced Kbot Lab and make about 10 or so Fido; this is plenty to take care of the weak forces at the Core base. You are pretty much ready to take care of everything, but I recommend that you quickly make an Aircraft Plant and pop out a couple of Thunders to take care of the Punisher on a small hill near the Core base. Build a Peewee to scout it out and find it quickly without being destroyed by the Crashers, then go and bomb the Punisher until destroyed, then bomb anything you can until the Thunders are finally shot out of the sky. Once the Punisher has been taken care of, gather up your Fidos, Zippers, and other starting units and sweep the map. It should be quite simple. If by any chance you happen to come across a unit that shows up on radar but cannot be reached to its placement on a steep hill, just walk the Commander over and D-Gun it. (Commanders can walk up nearly anything) Destroy every last unit to win this mission. ======================================================================= Mission #11 � The Gate to Barathrum ----------------------------------------------------------------------- Starting Units � 3 Jethro, 2 Brawler, 2 Fido, 2 Zipper, 1 Zeus ======================================================================= The one huge problem with this level is metal. In the area that you begin in, there is only ONE metal patch that is really close, others may be a bit too far away. Make a Metal Extractor on it, then make some Solar Collectors. You�ll need to make a lot of Solar Collectors to provide enough energy for Metal Makers, but make only a few, then make a Kbot Lab. Once you�ve done that, continue making them. After every 4 or so that you make, make a Metal Maker, then continue until you have about 20 Solar Collectors and 5 Metal Makers. You will have to use this for everything. While you are doing that, make a Construction Kbot and some Hammers if you can. You will be attacked early on in the mission, so some of these defenses will help. Making a L.L.T. or two wouldn�t hurt, but make sure to build several Defenders in case the Core uses air strikes. A Radar Tower would also help, or an Advanced Radar Tower once you get the chance to build one. After a while, you probably will not be attacked at all, so take your time and build your usual land attack units (Fido bots are recommended). If you have surplus energy and metal to be used, make an Aircraft Plant and try to use a few Peepers to scout out some of the landscape in the northwest, as that is where the Galactic Gate is, but do not expect them to last very long with all those anti-air towers scattered around. Build a few bombers, as well as some of those strategic bombers if you have an advanced aircraft plant. Use these bombers to destroy a couple of things up in the enemy territory, particularly the Punisher positioned right in front of the Galactic Gate at the end of the dried up river. Keep trying to destroy as much as you can until they get destroyed. Once you have made a nice army of 30 or so Fidos as well as several Jethro to get rid of aircraft, start to sweep the map. Destroy all the units; just make sure you do not blow up the Galactic Gate. Once everything is cleared out, walk your Commander over from your base and capture the Gate to go on to Barathrum. ======================================================================= Mission #12 � Barathrum! ----------------------------------------------------------------------- Starting Units � None (only Commander and Galactic Gate) ======================================================================= ======================================================================= Mission #13 � Landown�s Interface ----------------------------------------------------------------------- Starting Units � 6 Zipper, 4 Jethro, 3 Fido, 1 Atlas, 1 Construction Kbot, 1 Zeus ======================================================================= ======================================================================= Mission #14 � The Heat Increases ----------------------------------------------------------------------- Starting Units � 4 Zipper, 4 Fido, 2 Jethro, 1 Construction Vehicle, 1 Solar Collector, 2 L.L.T. ======================================================================= ======================================================================= Mission #15 � Rougpelt ----------------------------------------------------------------------- Starting Units � 1 half-dead Zipper ======================================================================= A pretty easy mission. Almost immediately a half-dead Zipper, the only survivor of the scouting team sent ahead (the remains of the rest are right in front of you), and a Core Pyro will head your way. Quickly capture the Pyro before your Zipper kills it, and then destroy the A.K.s that come just a few seconds later. Repair both the Pyro and the Zipper, and start searching for the Advanced Radar Tower. (Don�t bother to capture the Solar Collectors, you do not need them) It is located at the southern point of the map, sort of between the south-central part and the southwest corner. You�ll meet some resistance, but you�ll have to take care of them with your D-Gun; just watch out for Roaches and don�t blow them up when you�re too close. Once you find the Advanced Radar Tower, capture it to finish this simple mission. ======================================================================= Mission #16 � Stockpile at the Abutment. ----------------------------------------------------------------------- Starting Units � 3 Zipper, 2 Fido, 1 Construction Kbot, 1 Zeus ======================================================================= Okay. There is a lot to do right away here. First, lay down a Metal Extractor on the nearby metal patch and two solar collectors. Put up a Kbot Lab immediately afterwards, and crank out a few Jethros. During this time, have your Construction Kbot put up several Defenders and some L.L.T.s. You might have some trouble putting them up because of the little resources you have, but you�ll manage. Also put up a radar tower. Metal is a big problem here. There�s only like 2 metal patches near you, so you�ll have to resort to using Metal Makers. Fortunately, there are a bunch of geothermal cracks just a little to the west, so bring a Construction Kbot over there and build a couple, also putting up Defenders around it. However, don�t do this until you�ve got a few L.L.T.s because a couple squads of Raiders and A.K.s will attack you, so you�ll have to be able to destroy them easily. They also send down some planes early on, so make sure you have those Defenders put up. Anyway, once you�ve got all your resource problems settled (you should try having 2 Geothermal Powerplants and 12 Solar Collectors to support about 9 Metal Makers), start assembling an army. You�ll need a Vehicle Plant and a Kbot Lab, as well as their advanced versions. Start making about 15 Fidos, 5 Bulldogs, 10 Hammers, and some random Zeuses, Zippers, and Diplomats thrown in. You also should build an Aircraft Plant and make a few Peepers to scout the area and locate the passage through the cliff mentioned in the briefing, the Abutment. This cliff is impenetrable, so you�ll have to find that opening (should be somewhere near the middle) if you want to get your land units through. By this time, the enemy will probably have stopped sending the little groups of land units, but they�ll constantly send down planes to harass your base. They shouldn�t pose any kind of threat at all as long as you have Defenders. Once you�ve located the passage through the cliff and gotten all your land units assembled make an Advanced Aircraft Plant and pop out 10-15 Brawlers. Send them up to the enemy base and take out as many Pulverizers and Gaat Guns as you can before they get shot down. Once you�ve done that, send your land units up and rip through the base, destroying everything. If you made what I told you to, they should have absolutely no problem at all. ======================================================================= Mission #17 � Fortress at Brooban�s Isle ----------------------------------------------------------------------- Starting Units � 3 Jethro, 2 Skeeter, 1 Conqueror, 1 Construction Kbot, 1 Construction Ship, 1 Crusader, 1 Zeus, 1 Shipyard ======================================================================= ======================================================================= Mission #18 � Vengeance! ----------------------------------------------------------------------- Starting Units � 10 Hammer, 9


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Originally published October 19, 2009 at 12:20 AM | Page modified October 19, 2009 at 9:32 AM
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Seattle mayoral candidates trade sharp exchanges
In a race for Seattle mayor in which candidates Joe Mallahan and Mike McGinn agree on much, they traded criticisms Sunday about the few issues they differ on, including the Alaskan Way Viaduct, management experience, and — in a new line of attack — a gun ban in city parks.
Seattle Times staff reporter
In a race for Seattle mayor in which the two candidates agree on much, they traded criticisms Sunday about the few issues they differ on, including the Alaskan Way Viaduct, management experience, and — in a new line of attack — a gun ban in city parks.
The sharpest exchanges in the KIRO-TV debate occurred when Joe Mallahan, clearly angry, accused foe Mike McGinn of making "baldfaced lies" in recorded phone calls in which the McGinn campaign accused Mallahan of "siding with the NRA" in backing a "repeal" of the ban.
"Mike and his campaign chose to conduct a lie campaign," said Mallahan, adding that his wife received one of the calls. "Mike, the Seattle voters deserve an apology. I deserve an apology."
When asked if he would defend in court the ban enacted by Mayor Greg Nickels, Mallahan said he may not, because it is "not a pragmatic way of addressing public-safety concerns."
"I don't owe an apology, because we told the public that Mr. Mallahan did not support the gun ban, and you heard that right now, here, he doesn't," McGinn said.
The two candidates — both first-time candidates for office — have been a puzzle for Seattle voters since Nickels' surprise defeat in the August primary. After two months of Seattle-style civility, the tone of the race now has an edge.
The candidates expressed similar views on protecting the environment, encouraging outreach to at-risk youth and their support for Referendum 71, which upholds the state's "everything but marriage" law. Both said they would support some public spending to return the NBA to Seattle, with conditions.
Mallahan, a vice president at T-Mobile, emphasized his business experience, while McGinn, a lawyer, campaigned on his environmental and neighborhood work.
McGinn said among the past Seattle mayors, he most admired Norm Rice because Rice convened an education summit that led to the popular Families and Education Levy. Mallahan trumpeted his endorsements by past Mayors Charles Royer and Wes Uhlman.
But as in past debates, the issue of replacing the Alaskan Way Viaduct with a deep-bore tunnel was a line of attack. After eight years of contentious debate, the tunnel was picked this winter by the governor, Legislature and city.
McGinn's opposition — and support for a surface and transit option — is the signature of his campaign. Mallahan supports the tunnel, calling it a "stimulus project" that is overdue.
In the debate, Mallahan said by opposing the tunnel, "my opponent, on the other hand, wants his first act of leadership to be conducting war with the governor, the Legislature and the City Council." State lawmakers have earmarked $2.4 billion for the project.
McGinn said he opposed the tunnel because the city is responsible for cost overruns. "It's not a solution for the future. It exposes the city to too much risk. The problem is the elected officials did not listen to the people," said McGinn.
But he conceded if other issues are resolved, such as the risk being shared by or shifted to the state, "it would be my job to implement it."
When given the chance to ask each other questions, both men focused on management experience, a critical quality for mayor of a city with 11,000 employees.
McGinn attacked Mallahan's central campaign theme — that private business experience enabled him to make the city more efficient. McGinn noted that Mallahan's payroll of campaign staff was larger than "my entire budget."
Mallahan asked McGinn about something he recently said in a TV interview, in which McGinn conceded "detail is not my strength." "Why should we not make the conclusion that you have zero management experience?" Mallahan asked.
The two candidates will appear together for at least five more debates, including one on KING 5 on Wednesday.
The Nov. 3 election is being conducted entirely by mail-in ballots, which began hitting mailboxes last week.
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O'Neill Evolution 2011 - Slopestyle Session
A Snowboarding video by quattro_media - 3,540 views
The 6 star Swatch TTR World Snowboard Tour event is once again pushing new boundaries. This year's event now holds a 6 star TTR rating for both the women's Slopestyle as well as the Halfpipe. And this year also sees the 6 star rating for men extended to the 2-hit Slopestyle course for the first time.
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After 4 days of shaping the course offers a new level of riding to the O'Neill Evolution. They went for a fun set-up that riders can enjoy riding and trying new tricks. It's safe so they can push their riding. At the bottom they went for as much variation as possible to bring a good show to everyone involved.