ABBS 2005,38(02): Novel Cold-adaptive Penicillium Strain FS010 Secreting Thermo-labile Xylanase Isolated from Yellow Sea

Abstract        A novel cold-adaptive xylanolytic Penicillium
strain FS010 was isolated from

Key words        marine fungus; Penicillium
chrysogenum; cold-active xylanase; gene expression

Since the pioneering
work of Barghoorn and Linder [1], much work has been carried out on the
taxonomy, distribution, occurrence, bioactive compounds and significance of
marine fungi [2–4]. To
colonize in habitats with low temperatures, enzymes are produced by marine
fungi, which are adapted to low temperatures. Compared with their mesophilic
counterparts, these cold-active enzymes display a higher catalytic efficiency
over a temperature range of roughly 0–30 ºC and a
higher thermosensitivity [5]. Using protein modeling and X-ray crystallography,
the molecular adaptation of cold-active enzymes resides in a weakening of the
intramolecular interactions, and in an increase of the interaction with the
solvent in some cases, leading to more flexible molecular edifices capable of
performing catalysis at a lower energy cost [6]. These properties can be
extremely useful for various applications, such as cold washing, food
industries, leather softening, environmental bioremediation and molecular
biology applications [7].

Xylan, the main
hemicellulose in plant cell walls, is the most abundant renewable
polysaccharide in nature behind cellulose. It comprises a backbone of b-1,4-linked xylopyranosyl residues highly branched
with acetyl groups and various sugars [8]. The xylan backbone is hydrolyzed by
xylanase (XYL; 1,4-b–D-xylan
xylanohydrolase; EC In this paper, we first
report the isolation and characterization of a novel psychrotrophic marine
fungus FS010, as well as the cloning and expression of the cold-active xylanase
in Escherichia coli.

Materials and Methods

Strains, vectors and
media

Penicillium chrysogenum FS010 was
isolated from deep-sea sediments collected in the area of the

Characterization of marine
fungi

The optimal temperature
for fungal isolation was determined by measuring the colony diameters after
incubation of MM agar plates for 96 h at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45
and 50 ºC. The optimal temperature for XYL production was determined by
measuring the diameters of hydrolytic halo on the selective medium containing
0.5% (W/V) xylan at different temperatures. FS010 was grown in
liquid culture in one-liter Erlenmeyer flasks on a
rotary shaker (300 rpm) at 15 ºC in 300 ml of minimum medium, using 108 conidia per
liter (final concentration) as the inoculum. The growth curve under the optimal
temperature was performed by the measurement of the wet weight of filtered
mycelia. To induce xylanase formation by birchwood xylan, the replacement technique
described by Sternberg and Mandels [16] was used.

Xylanase assay

Xylanase activity was
assayed by measuring the production of reducing-sugar ends from birchwood xylan
with 3,5-dinitrosalicylic acid (DNS) reagent. Samples of 1 ml of crude xylanases
in sodium acetate buffer (pH 5.0) were incubated with 2 ml of 0.5% (W/V)
birchwood xylan in the same buffer at 25 ºC. After 30 min, 2 ml of DNS reagent
was added and the samples were boiled for 10 min. Reducing sugar production was
followed by measuring the absorbance at 540 nm. Using a standard curve
generated with D-xylose, absorbance was converted into moles of reducing
sugars produced. One unit of enzyme activity was defined as 1 mmol/min of xylose released at 25 ºC.

Cloning and sequencing of
the 18S rDNA gene

Polymerase chain
reaction (PCR) amplification of the 18S rDNA gene fragment was carried out with
the universal primers EF3 (5‘-CCTCTAAATGACCAAGTTTG-3‘) and EF4 [5‘-GGAAGGG(G/A)TGTATTTATTAG-3‘]
[17], using high-fidelity pfu DNA polymerase (Sangon,

Cloning of the xyl
from P. chrysogenum FS010

Based on the xylanase
gene sequence of mesophilic P. chrysogenum [20], two oligonucleotide
primers were synthesized to amplify fragments of the FS010 cold-active xylanase
cDNA: upstream primer 5‘-CGAAGAACCAACATGATTCC-3‘ and downstream
primer 5‘-CCACCATCCTTCTTCTCTCA-3‘. P. chrysogenum FS010
induced by xylan was grown as described above and mycelia were harvested by
filtration. The recovered mycelia were frozen in liquid nitrogen and total RNA
was isolated from powdered mycelia with Trizol reagent (Sangon) according to
the supplier’s manuals. The total RNA was treated with Dnase I (TaKaRa,

Nucleotide sequence
accession number

The nucleotide sequence
of 18S rDNA was deposited in the GenBank with the accession No. AY593254. The
xylanase cDNA sequences from P. chrysogenum FS010 and A3969.2 were
deposited in the GenBank nucleotide sequence database with accession No.
AY583585 and DQ304546, respectively.

Construction of an
expression vector

A plasmid encoding a
glutathione S-transferase (GST) fusion protein containing the coding
sequence of xyl, corresponding to amino acids 1–353 of the cold-active XYL from FS010, was constructed by
insertion of the corresponding xyl cDNA sequence into the EcoRI/XhoI
sites of the pGEX-4T-1. The inserted DNA fragment encoding XYL was prepared by
high-fidelity PCR using pGEM-T-xyl as the template. The forward primer 5‘-GCGCGAATTCATGATTCCCAATATCACTCAACTC-3‘
and the reverse primer 5‘-GCGCCTCGAGTCAATCTGAATGTAACCTGCTTAG-3‘
contained the underlined EcoRI and XhoI restriction site. The PCR
was carried out as follows: an initial denaturation at 94 ºC for 5 min followed
by 30 cycles of amplification (94 ºC for 30 s, 52 ºC for 30 s, and 72 ºC for 1
min) and an additional extension at 72 ºC for 10 min. The amplified PCR product
was digested with these two enzymes and then purified by the PCR clean-up
system (Promega). The purified PCR product carrying the cohesive ends was
cloned into the multi cloning sites of EcoRI/XhoI-treated
pGEX-4T-1. The constructed plasmid was designated as pGEX-4T-xyl. Using the same
procedure, the xyn cDNA isolated from P. chrysogenum A3969.2 was
inserted into pGEX-4T-1, generating recombinant plasmid
pGEX-4T-xyn.

Expression and
purification of the recombinant XYL

E. coli BL21
transformed with pGEX-4T-xyl was used to inoculate into 400 ml of Luria-Bertani
medium containing ampicillin (100 mg/ml). After
incubation for 3–4 h at 37 ºC
with shaking (230–250 rpm)
until the cells were in mid-log phase, isopropyl-b–D-thiogalactopyranoside (IPTG) (Dingguo, Beijing,
China) was added at a final concentration of

Protein analysis and
enzymatic properties

SDS-PAGE (10%) was
performed as described by Laemmli [21]. Protein concentrations were determined
by the method of The substrate
specificity assays were performed by incubating the purified recombinant XYL
with 0.5% (W/V) substrates such as oat spelts xylan, birchwood
xylan, wheat arabinoxylan, b-xyloside,
sodium carboxymethylcellulose (CMCNa), p-nitrophenylcellobioside (pNPC) and
Avicel (Sigma-Aldrich) [23] under standard conditions. The optimal­ pH and
temperature were determined in the range of pH ­3.­0­­ to 9.5 (

Results and Discussion

Morphological and
molecular identification of the FS010 strain

Fifty-one marine fungi
strains were isolated at 4 ºC from deep-sea sediments collected from the The kinetics of mycelia
growth in MM broth at 15 ºC is shown in Fig. 2. FS010 rapidly entered
the stationary phase after 48 h, indicating that the strain had a great
advantage in shortening the fermentation cycle. Compared to A3969.2, FS010 grew
more luxuriantly at 15 ºC, indicating FS010 is a true cold-adaptive marine fungus.
FS010 colonies on agar medium are fast-growing, in shades of green, and mostly
consist of a dense felt of conidiophores. The vegetative mycelium in agar was
white, whereas the aerial mycelium was dark green. The medium was yellow
because the fungi could produce yellow pigments. The brush-like conidial head
was developed from the aerial medium. Branching is an important feature for
identifying Penicillium species. The branching pattern of FS010 is
two-stage branched. The conidia are globose and greenish. Based on
light-microscopy observations (Fig. 3), the strain FS010 was
preliminarily identified as Penicillium spp.. To further identify the
strain, the 18S rDNA gene of strain FS010 was cloned and sequenced. The
amplified product was 1538 bp in length. Sequence analysis indicated that the
18S rDNA sequence of FS010 showed 99.9% identity to that of mesophilic P.
chrysogenum. Based on conventional morphological and molecular
identification, the novel isolate belongs to P. chrysogenum, deposited
in the State Key Laboratory of Microbial Technology Center for typical
collection.

Cloning and sequencing
of the cold-active xyl cDNA

Using RT-PCR, a specific
band of 1128 bp was amplified from the total RNA of P. chrysogenum FS010
(Fig. 4). The nucleotide sequence of the 1128-bp DNA fragment was
determined for both strands. Open reading frame (ORF) was located between
nucleotides 1 and 1062, and its G+C content
ratio was 53.58%. Sequence analysis confirmed that the RT-PCR product had high homology
to those of the xylanase gene family. In comparison with xyl of the
mesophilic P. chrysogenum, there are five different sites in FS010 xyl:
two mutations causing silent substitutions in coding region (G®A at nt 528; C®T at nt 1053) and three
causing amino acid substitutions in XYL catalytic domains (C®G at nt 457, leucine
replaced with valine at position 153; C®G, G®C at nt 821 and 822,
threonine replaced with serine at position 274). The serine substitution may be
important in protein efficient catalytic efficiency and thermolability by
influencing noncovalent interactions such as hydrogen bond, van der Waals force
and hydrophobic interaction, which was in accordance with the results reported
by Methe et al. [25]. The ORF encoded a 353-amino acid protein, with a
deduced molecular mass of approximately 38 kDa. The potential pI was 4.97. At
the N-terminus of the deduced sequence, a putative signal sequence was
identified by the SignalP 3.0 serve system (http://www.cbs.dtu.dk/services/),
with cleavage predicted to occur after amino acid 23 of the pre-protein. Two
potential N-glycosylation sites were found at Asn4 and Asn325.

Comparison of the
deduced xyl amino acid sequence from P. chrysogenum FS010 with
those available on the databases revealed identity values of 80%, 79%, 59%, 45%
and 44% of the xyl from Penicillium canescens (GenBank accession
No. AY756109), Aspergillus aculeatus (GenBank accession No. AB013110), Fusarium
oxysporum (GenBank accession No. AF052583), Phanerochaete chrysosporium (GenBank
accession No. AF301903) and Neurospora crassa (GenBank accession No.
XM_324353.1), respectively. All of the homologous sequences belonged to
glycosyl hydrolase family 10. It indicated that the FS010 XYL was also a member
of this family. A prosite pattern search [26] performed on the deduced FS010
protein sequence indicated an XYL active site signature pattern [GTA]-xX-[LIVN]-x-[IVMF]-[ST]-E-[LIY]-[DN]-[LIVMF] (E is the active site
residue and X is random amino acid residue) at amino acid 259. An alignment by
the Conserved Domain Architecture Retrieval Tool [27] shows that the deduced
polypeptide sequence belongs to a single domain family.

Expression of the FS010 xyl
gene in E. coli

Considering expression level
and solubility of fusion proteins, the GST fusion expression system was chosen
to express the xyl cDNA in E. coli [28]. The expression plasmid
was constructed, as described above, and designated as pGEX-4T-xyl,
which was introduced into E. coli BL21 for high-level expression as a
fusion protein with GST under the control of the tac promoter.

A 64-kDa XYL fusion
protein (26-kDa GST+38-kDa XYL)
band from the cell-free extracts of BL21 (pGEX-4T-xyl) induced by IPTG was shown
on SDS-PAGE, whereas no 64-kDa protein band from cell-free extracts of
non-induced BL21 (pGEX-4T-xyl) was detected (Fig. 5). It was therefore
concluded that xyl cDNA was highly expressed as a fusion protein with
GST in E. coli BL21. The XYL fusion protein content was 16.7% of the
total proteins, determined by the scanning of the PAGE gel using the GeneTool
software. The recombinant XYL activity of E. coli cell extracts was
measured against the birchwood xylan under standard conditions. The specific activity
of XYL was 1868 U/mg, whereas no xylanolytic activity was detected in the cell
extracts from non-induced BL21 (pGEX-4T-xyl), indicating that the xyl cDNA
from strain FS010 was successfully overexpressed in the E.
coli. According to the cold-active xyl cDNA expression protocol, the
xyn cDNA from strain A3969.2 was also successfully
overexpressed and purified in the E. coli (Fig. 5). Generally,
most of fungal genes encoding glycosyl hydrolases could not be expressed in the
prokaryotic system as a catalytically active enzyme because of the shortage of
intact protein modifications. However, Xue et al. [29] had iso­­­lated
cellulase genes using enzyme assay plates from a cDNA library in E. coli
from the rumen fungus Neocallimastix patriciarum. It was assumed that
the post-translational modification of FS010 XYL was not necessary for XYL
activity. The purified XYL treated with thrombin displayed a single protein
band on SDS-PAGE gel stained with Coomassie brilliant blue R250 (Fig. 5).
The molecular weight of XYL was approximately 38 kDa, which is similar to the
deduced molecular mass, indicating that the purified protein was
the expression product of the xyl gene. The specific activity of the
purified XYL was 10,210 U/mg, while the specific activity of the purified XYN
was 9863 U/mg, indicating that there was no significant difference between XYL
and XYN.

Properties of the
recombinant XYL

The XYL was observed to
specifically hydrolyze xylan, the highest activity on soluble birchwood xylan (Table
1). It had no cellulase, CMCase and b-xylosidase activity but acted on pNPC.

As shown in Fig. 6,
the optimal temperature for the reaction of recombinant XYL was 25 ºC, whereas
the optimal temperature of recombinant XYN was 40 ºC. XYL was sensitive when
heating up to 55 ºC for 30 min. The recombinant XYL was
found to be thermolabile, as nearly 80% of xylanase activity was lost after a
30-min treatment at 55 ºC (Fig. 7), which was consistent with the
typical characteristic of cold-active enzymes. At 4 ºC, the recombinant XYL
displayed nearly 80% of the maximal activity, whereas the recombinant XYN
showed nearly 5.9% of the maximal activity. The specific activity of XYL at 4
ºC was 11-fold as high as that of XYN. The optimal temperature and
thermolability indicate that XYL is a cold-active enzyme (Fig. 6). In
spite of the significant difference (15 ºC) for optimal temperature between XYL
and XYN, there are two amino acid substitutions in XYL. Further work would be
needed to clarify the effect of two amino acids on the three-dimensional
structure of the recombinant XYL.

The optimal pH of XYL
was 5.5 (Fig. 8), which is similar to the optimal pH of XYN (data not
shown). The recombinant XYL showed activities at pH range of 3.0 to 9.5 and was
stable from pH 4.5–8.0 (Fig.
8).

Here, we have cloned and
analyzed the complete sequence of the xyl cDNA encoding cold-active XYL
from marine P. chrysogenum FS010. The FS010 XYL was expressed and
purified as a catalytically active enzyme in E. coli, and some
properties of the enzyme were characterized. This is the first demonstration of
a marine P. chrysogenum producing cold-active XYL. In comparison with
mesophilic and thermophilic XYL [30], the cold-active XYL had a great advantage
in that it could efficiently hydrolyze xylan into oligosaccharides at room
temperature, which means saving energy. The biochemical properties of
cold-active XYL would make it attractive for exploitation in many biochemical,
bioremediation and industrial processes. The adaptations to protein
architecture essential to cold-active enzymes are still not well understood,
and inquiries to unlock these adaptations continue to be an active area of
investigation.

Acknowledgement

We thank our colleague
Jin-Dong HU (

References

 1   Barghoorn ES, Linder DH. Marine fungi: Their
taxonomy and biology. Farlowia 1944, 1: 395–467

 2   Petrescu I, Lamotte-Brasseur J, Chessa JP,
Ntarima P, Claeyssens M, Devreese B, Marino G et al. Xylanase from the
psychrophilic yeast Cryptococcus adeliae. Extremophiles 2000, 4: 137–144

 3   Tsuda M, Kasai Y, Komatsu K, Sone T, Tanaka
M, Mikami Y, Kobayashi J. Citrinadin A, a novel pentacyclic alkaloid from
marine-derived fungus Penicillium citrinum. Org Lett 2004, 6: 3087–3089

 4   Dalsgaard PW, Blunt JW, Munro MH, Larsen TO,
Christophersen C. Psychrophilin B and C: Cyclic nitropeptides from the
psychrotolerant fungus Penicillium rivulum. J Nat Prod 2004, 67: 1950–1952

 5   Marx JC, Blaise V, Collins T, D’Amico S,
Delille D, Gratia E, Hoyoux A et al. A perspective on cold enzymes:
Current knowledge and frequently asked questions. Cell Mol Biol
(Noisy-le-grand) 2004, 50: 643–655

 6   Feller G. Molecular adaptations to cold in
psychrophilic enzymes. Cell Mol Life Sci 2003, 60: 648–662

 7   Chessa JP, Feller G, Gerday C. Purification
and characterization of the heat-labile a-amylase secreted by the psychrophilic bacterium TAC
240B. Can J Microbiol 1999, 45: 452–457

 8   Biely P. Microbial xylanolytic systems.
Trends Biotechnol 1985, 3: 286–289

 9   Beg QK, Kapoor M, Mahajan L, Hoondal GS.
Microbial xylanases and their industrial applications: A review. Appl Microbiol
Biotechnol 2001, 56: 326-338

10  Coughlan MP, Hazlewood GP. b-1, 4-D-xylan-degrading enzyme systems:
Biochemistry, molecular biology and applications. Biotechnol Appl Biochem 1993,
17: 259–289

11  Polizeli ML, Rizzatti AC, Monti R, Terenzi HF,
Jorge JA, Amorim DS. Xylanases from fungi: properties
and industrial applications. Appl Microbiol Biotechnol 2005, 67: 577–591

12  Ray RR. Production of alpha-amylase and
xylanase by an alkalophilic strain of Penicillium griseoroseum RR-99. Acta Microbiol Pol 2001, 50: 305–309

13  Viikari L, Kantelinen A, Sundquist J, Linko M.
Xylanases in bleaching: From an idea to the industry. FEMS Microbiol Rev 1994,
13: 335–350

14  Raghukumar C, Muraleedharan U, Gaud VR, Mishra
R. Xylanases of marine fungi of potential use for biobleaching of paper pulp. J
15  Inglis GD, Popp AP, Selinger LB, Kawchuk LM,
Gaudet DA, McAllister TA. Production of cellulases and xylanases by
low-temperature basidiomycetes. Can J Microbiol 2000, 46: 860–865

16  Sternberg D, Mandels GR. Induction of
cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol
1979, 139:761–769

17  Smit E, Leeflang P, Glandorf B, van Elsas JD,
Wernars K. Analysis of fungal diversity in the wheat rhizosphere by sequencing
of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel
electrophoresis. Appl Environ Microbiol 1999, 65: 2614–2621

18  Al-Samarrai TH, Schmid J. A simple method for
extraction of fungal genomic DNA. Lett Appl Microbiol 2000, 30: 53–56

19  Sambrook J, Russell DW. Molecular Cloning: A
Laboratory Manual. 3rd ed. 20  Haas H, Friedlin E, Stoffler G, Redl B.
Cloning and structural organization of a xylanase-encoding gene from Penicillium
chrysogenum. Gene 1993, 126: 237–242

21  Laemmli UK. Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 1970, 227:
680–685

22  23  Bok JD, Yernool DA, Eveleigh DE. Purification,
characterization, and molecular analysis of thermostable cellulases CelA and
CelB from Thermotoga neapolitana. Appl Environ Microbiol 1998, 64: 4774–4781

24  Morita RY. Psychrophilic bacteria. Bacteriol
Rev 1975,39: 144–167

25  Methe BA, Nelson KE, Deming JW, Momen B,
Melamud E, Zhang X, Moult J et al. The psychrophilic lifestyle as
revealed by the genome sequence of Colwellia psychrerythraea 34H through
genomic and proteomic analyses. Proc Natl Acad Sci USA 2005, 102: 10913–10918

26  Gilkes NR, Henrissat B, Kilburn DG, Miller RC
Jr, Warren RA. Domains in microbial b-1,4-glycanases:
Sequence conservation, function, and enzyme families. Microbiol Rev 1991, 55:
303–315

27  Marchler-Bauer A, Anderson JB, Cherukuri PF,
DeWeese-Scott C, Geer LY, Gwadz M, He S et al. CDD: A Conserved Domain
Database for protein classification. Nucleic Acids Res 2005, 33: D192–196

28  Fang J, Ewald D. Expression cloned cDNA for
10-deacetylbaccatin III-10-O-acetyltransferase in Escherichia coli: A
comparative study of three fusion systems. Protein Expr Purif 2004, 35: 17–24

29  Xue GP, Orpin CG, Gobius KS, Aylward JH,
Simpson GD. Cloning and expression of multiple cellulase cDNAs from the
anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli.
J Gen Microbiol 1992, 138: 1413–1420

30  Haas H, Herfurth E, Stoffler G, Redl B.
Purification, characterization and partial amino acid sequences of a xylanase
produced by Penicillium chrysogenum. Biochim Biophys Acta 1992, 1117:
279–286