Genome mining for glycoside hydrolases from the psychrophilic yeast glaciozyma antarctica PI12

Abstract

Glycoside hydrolases are enzymes that hydrolyse glycosidic bonds in carbohydrate chains to produce simple molecules. In fungi, glycoside hydrolases are important enzymes that hydrolyse complex carbohydrates into simple sugars that can subsequently be consumed for energy metabolism. Glaciozyma antarctica is a psychrophilic yeast isolated from sea ice in Antarctica. The G. antarctica genome has been completely sequenced, and a total of 7,857 genes have been predicted. The objective of the present study was to determine different classes of glycoside hydrolases from the G. antarctica genome and predict the localisation of these enzymes. Using genome mining, a total of 97 G. antarctica genes were predicted to encode glycoside hydrolases. The majority of the enzymes, including endoglucanases, xylanases, and chitinases, were identified from GH family 5 (12 genes), followed by GH family 45 (11 genes), GH family 10 (11 genes) and GH family 18 (9 genes). The secreted glycoside hydrolase enzymes were primarily endoglucanases from GH family 45, and these enzymes degrade celluloses in the cell walls of plants and algae. Extracellular glycoside hydrolases have been implicated as important in nutrient scavenging and organic decomposition in Antarctic sea ice. © 2015, Malaysian Society of Applied Biology. All rights reserved.

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GLYCOSIDE HYDROLASES FROM THE PSYCHROPHILIC YEAST Glaciozyma antarctica PI12 167
Malays. Appl. Biol. (2015) 44(1): 167–172
* To whom correspondence should be addressed.
GENOME MINING FOR GLYCOSIDE HYDROLASES FROM THE
PSYCHROPHILIC YEAST Glaciozyma antarctica PI12
NOORAISYAH, M.N.1, SITI NUR HASANAH, M.Y.1, MAHADI, N.M.2, ABU BAKAR, F.D.1
and MURAD, A.M.A.1*
1School of Biosciences and Biotechnology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Malaysia
2Malaysia Genome Institute, Jalan Bangi, 43000 Kajang, Selangor, Malaysia
*Email: munir@ukm.edu.my
ABSTRACT
Glycoside hydrolases are enzymes that hydrolyse glycosidic bonds in carbohydrate chains to produce simple molecules. In
fungi, glycoside hydrolases are important enzymes that hydrolyse complex carbohydrates into simple sugars that can
subsequently be consumed for energy metabolism. Glaciozyma antarctica is a psychrophilic yeast isolated from sea ice in
Antarctica. The G. antarctica genome has been completely sequenced, and a total of 7,857 genes have been predicted. The
objective of the present study was to determine different classes of glycoside hydrolases from the G. antarctica genome and
predict the localisation of these enzymes. Using genome mining, a total of 97 G. antarctica genes were predicted to encode
glycoside hydrolases. The majority of the enzymes, including endoglucanases, xylanases, and chitinases, were identified
from GH family 5 (12 genes), followed by GH family 45 (11 genes), GH family 10 (11 genes) and GH family 18 (9 genes).
The secreted glycoside hydrolase enzymes were primarily endoglucanases from GH family 45, and these enzymes degrade
celluloses in the cell walls of plants and algae. Extracellular glycoside hydrolases have been implicated as important in nutrient
scavenging and organic decomposition in Antarctic sea ice.
Key words: Glycoside hydrolases, psychrophiles, Glaciozyma antarctica, genome mining
INTRODUCTION
Glaciozyma antarctica PI12 is a psychrophilic
yeast isolated from Antarctic sea ice (Hashim et al.,
2013). Psychrophiles have evolved a complex
range of adaptation strategies to survive in cold
environments. Several mechanisms have been
suggested for cold adaptation, such as changes in
membrane fluidity, the secretion of exopoly-
saccharides and antifreeze proteins and the
production of enzymes that optimally function at
subfreezing temperatures (Kawahara, 2002). The
Malaysian Genome Institute has fully sequenced
the G. antarctica genome, comprising 7,857
predicted genes (http://www.genomemalaysia.gov.
my/glaciozyma_antarctica/). Glycoside hydrolases
are enzymes that hydrolyse or cleave the glycosidic
bonds between carbohydrate-carbohydrate or
carbohydrate-non carbohydrate moieties, such as
proteins and lipids. Based on the carbohydrate
active enzymes (CAZy) database (http://www.cazy.
org/), 133 glycoside hydrolase families have been
identified to date. The families are grouped based
on amino acid sequence similarities. In fungi,
glycoside hydrolases are required to facilitate
infection, gain nutrition and degrade organic matter
in the surrounding environment (Zhao et al., 2013).
Based on a comparative analysis of the glycoside
hydrolases in fungi, these enzymes are extra-
cellularly secreted. The characterisation of these
secreted proteins would facilitate the prediction of
the function of this enzyme in terms of the
hydrolysis of polysaccharide materials. A detailed
study of these enzymes will increase the current
understanding of how glycoside hydrolases
facilitate G. antarctica survival in the extreme
Antarctic sea ice habitat and provide information on
the nutrition patterns of this yeast. In addition,
glycoside hydrolases are a group of enzymes with
industrial importance, and cold active glycoside
hydrolases have great potential in food, pharma-
ceutical and detergent industries.

168 GLYCOSIDE HYDROLASES FROM THE PSYCHROPHILIC YEAST Glaciozyma antarctica PI12
MATERIALS AND METHODS
All known glycoside hydrolase protein sequences
were downloaded from NCBI non-redundant
database and the UniprotKB/Swissprot and
UniprotKB/TrEMBL databases (http://www.uniprot.
org/). These protein sequences were blasted against
the G. antarctica genome database available at the
Malaysian Genome Institute (http://27.126.156.144/
glaciozyma_antarctica/). All matching sequences
based on a set parameter were filtered into a list of
potential glycoside hydrolase enzymes for G.
antarctica. The set parameters were 1) sequence
identity equal or higher than 30% and 2) an e-value
equal or lesser than 1x10-5. Once the list of potential
enzymes was obtained, a functional analysis of the
sequences was performed to identify potential
glycoside hydrolase domains using the InterProScan
software available at EMBL/EBI (http://www.ebi.
ac.uk/Tools/pfa/iprscan/) (Quevillon et al., 2005).
Alternatively, the putative function for each
sequence was also confirmed through a blast
analysis against the Protein Data Bank (PDB
database) (http://www.rcsb.org/pdb/home/home.do)
available at NCBI Blast (http://blast.ncbi.nlm.
nih.gov/Blast.cgi). The enzymes were classified into
different glycoside hydrolase families according to
the CAZY database (http://www.cazy.org) (Lombard
et al., 2014). The signal peptide prediction was
performed using three software programs: SignalP
4.1 (http://www.cbs.dtu.dk/services/SignalP/)
(Petersen et al., 2011), Signal 3L (http://www.csbio.
sjtu.edu.cn/bioinf/Signal-3L/) and Phobius (http://
phobius.sbc.su.se/).
RESULTS
Among the 7857 genes in the G. antarctica genome,
a total of 97 genes (1.24%) were predicted to encode
glycoside hydrolases (Table 1). The prediction was
based on the presence of a glycoside hydrolase
domain or a glycoside hydrolase binding subgroup
in the sequence. Subsequently, these proteins were
grouped into a respective glycoside hydrolase
family, according to a CAZy database (Fig. 1). The
number of hydrolase-encoding genes seems low in
this fungus, as a comparative analysis of fungi
genomes revealed a large number of CAZy enzymes,
ranging from 171-285 genes per genome (Damasio
et al., 2014). For instance, Trichoderma reesei
contains 200 glycoside hydrolase enzymes, while
Aspergilus niger contains 250 glycoside hydrolase
enzymes. The low number of glycoside hydrolases
in G. antarctica might suggest that in psychrophiles,
these enzymes might contain different coding
sequences for glycoside hydrolase domains. A
recent comparative analysis of glycoside hydrolases
in fungi revealed the distribution of common GH
families in fungi. The most prevalent families were
GH5, GH13, GH31, and GH61 (Zhao et al., 2013).
However, in the G. antarctica genome, the
distribution of the most prevalent GH family is GH5
(12 genes), followed by GH45 (11 genes), GH10 (11
genes) and GH18 (9 genes). These families belong
to mannanases, endoglucanases, cellulases, and
chitinases, respectively. Notably, two genes fall into
unclassified families due to non-similarity with any
sequence in the database, although these sequences
contain glycoside hydrolase domains. These two
domains are named GH-type carbohydrate-binding
subgroup domain (LAN_11_346) and galactose
binding domain (LAN_14_284).
We also screened the genes listed in Table 1 for
the presence of signal peptides in each of the
sequences. A total of 46% of the glycoside
hydrolases in the list had a signal peptide,
suggesting that these enzymes could be secreted
(Fig. 2). The majority of the secreted enzymes were
endoglucanases, followed by xylanases (Table 2).
Other secreted enzymes identified were mannanases,
α-amylases, lichenases, chitinases and mannosidases.
DISCUSSION
The extreme cold environment in which G.
antarctica inhabits requires special modifications,
including scavenging for scarce nutrients in the
environment, for the growth and survival of these
microorganisms. Glycoside hydrolase is an enzyme
that degrades carbohydrate linkages to generate
carbon sources. The release of various extracellular
enzymes in the cold environment might reflect
the metabolic adaptations of psychrophilic
microorganisms for significant functions, such as the
decomposition of organic materials and nutrient
cycling. This idea is supported by the presence of
organic carbon and nitrogen sources originating
from melting glacier ice (Brizzio et al., 2007), the
abundance of organic matter from the death and
lysis of the sea-ice organisms, and the secretion of
organic polymers by algae and bacteria (Yu et al.,
2009), suggesting that psychrophilic yeasts, such as
G. antarctica induce the secretion of extracellular
enzymes into the environment. The majority of
glycoside hydrolases present in G. antarctica
include GH 5, GH 45, GH10, and GH 18 families.
For GH families 5 and 45, most of the enzymes
comprise endo-β-glucanases, which hydrolyse -1,3
or -1,4 linkages in beta glucans. GH 45 is also the
largest group of signal peptide-containing enzymes,
suggesting that the members of this group could be
extracellularly secreted from G. antarctica. The
substrates for endo-β-glucanases are beta glucans,
namely cellulose, laminarin and lichenin. Cellulose

GLYCOSIDE HYDROLASES FROM THE PSYCHROPHILIC YEAST Glaciozyma antarctica PI12 169
Table 1. List of proteins with glycoside hydrolase domains identified in the
G. antarctica
genome
G. antarctica
gene Annotation E-value GH classification
identification
number*
1 LAN_01_115 Glucosidase I (
Pseudozyma antarctica
) 0 GH63
2 LAN_01_126 Barwin-related endoglucanase (
Hordeum vulgare
) 8e-18 GH45
3 LAN_01_290 Barwin-related endoglucanase (
Laccaria bicolor
) 5e-36 GH45
4 LAN_01_370 Endoglucanase-like protein (
Cryptosporangium arvum
) 7e-05 GH45
5 LAN_02_055 Endo-glycoceramidase (
Rhodococcus sp.)
8e-05 GH5
6 LAN_02_121 Exo-beta-1,3-glucanase (
Candida albicans
) 2e-22 GH5
7 LAN_02_122 1,3-beta-glucosidase (
C. albicans
) 8e-07 GH5
8 LAN_02_123 1,3-beta-glucosidase (
C. albicans
) 1e-07 GH5
9 LAN_03_017 Lichenase (
Clostridium thermocellum
) 7e-06 GH26
10 LAN_03_046 Endo-1,3(4)-beta-glucanase, GH 16 (
Bacillus licheniformis
) 5e-12 GH16
11 LAN_03_240 Glycosyl hydrolase 5 (cellulase A) (
Triticum aestivum
) 0 GH5
12 LAN_03_255 Transglycosylase SLT domain protein (
Neosartorya fischeri)
1e-25 GH23
13 LAN_03_593 Alpha-glucosidase (
Ruminococcis obeum
) 3e-76 GH31
14 LAN_03_646 Mannosyl oligosaccharide glucosidase (
Saccharomyces cerevisiae
) 0 GH63
15 LAN_03_748 Endo-β-1,3-glucanase (
Thermotoga petrophila
) 4e-08 GH16
16 LAN_03_749 Endo-β-1,3-glucanase (
Rhodosporidium toruloides
) 9e-10 GH16
17 LAN_04_028 Alpha-galactosidase (
Ajellomyces capsulatus
) 3e-35 GH23
18 LAN_04_169 Barwin-related endoglucanase (
Dichomitus squalens
) . 61 GH45
19 LAN_04_221 Chitin deacetylase (
Aspergillus nidulans
) 5e-13 GH10
20 LAN_04_222 Chitin deacetylase (
A. nidulans
) 2e-17 GH10
21 LAN_04_430 Beta-galactosidase (
Escherichia coli
) 1e-52 GH72
22 LAN_05_070 Beta-hexosaminidase (
Streotomyces coelicolor
) 1e-93 GH20
23 LAN_05_143 Beta-D-glucan exohydrolase (
Aspergillus aculeatus
) 6e-26 GH3
24 LAN_05_160 Endo-1,3(4)-beta-glucanase (
A. nidulans
) 4e-08 GH16
25 LAN_05_489 Chitinase (
Bacillus circulans
) 2e-17 GH18
25 LAN_05_492 Polysaccharide deacetylase (
R. toruloides
) 6e-08 GH16
27 LAN_05_524 Barwin-related endoglucanase (
Stereum hirsutum
) 1e-09 GH45
28 LAN_05_528 Barwin-related endoglucanase (
Gloeophyllum trabeum
) 3e-05 GH45
29 LAN_06_001 Glycosidase (
Auricularia delicata
) 3e-06 GH88
30 LAN_06_080 Polysaccharide deacetylase (
Roseomonas cervicalis
) 1e-33 GH10
31 LAN_06_221 Polysaccharide deacetylase (
Verticillium dahliae
) 1e-09 GH10
32 LAN_06_222 Polysaccharide deacetylase (
Moniliophthora roreri
) 4e-36 GH10
33 LAN_07_007 B-mannosidase (
R. toruloides
) 1e-39 GH2
34 LAN_07_048 Barwin-like endoglucanase (
Neurospora tetrasperma)
5e-06 GH45
35 LAN_07_165 Endo-beta-mannanase (
R. toruloides
) 8e-05 GH5
36 LAN_07_220 Exo-beta-1,3-glucanase (
Rhizoctonia solani
) 2e-08 GH55
37 LAN_07_233 Exo-beta1,3-glucanase (
Laccaria bicolor
) 8e-10 GH55
38 LAN_08_020 Endo-1,4-beta-D-mannanase (
R. toruloides
) 6e-52 GH5
39 LAN_08_021 Endo-1,4-beta-D-mannanase (
R. toruloides
) 1e-45 GH5
40 LAN_08_171 Alpha-1,2 mannosidase (
Rhodotorula glutinis
) 7e-42 GH47
41 LAN_08_180 Exo-oligoxylanase (
Macrophomina phaseolina
) 4e-65 GH10
42 LAN_08_216 Alpha-galactosidase (
Trametes versicolor
) 8e-55 GH31
43 LAN_08_305 Mannosidase (
Fomitiporia mediterranea
) 2e-90 GH92
44 LAN_09_198 Glucan beta-glucosidase (
Pseudozyma aphidis
) 2e-108 GH17
45 LAN_09_234 Mannan endo-1,6-alpha-mannosidase (
Verticillium dahliae
) 4e-26 GH76
46 LAN_09_291 Alpha-1,2 mannosidase (
R. toruloides
) 5e-68 GH47
47 LAN_10_095 Alpha-glucosidase (
R. solani
) 4e-21 GH31
48 LAN_10_097 Glucoamylase (
R. toruloides
) 3e-60 GH15
49 LAN_10_172 Chitinase (
R. toruloides
) 2e-43 GH18
50 LAN_10_180 Beta-lactamase (
R. toruloides
) 1e-06 GH3
51 LAN_10_181 Beta-lactamase (
R. toruloides
) 5e-14 GH3
52 LAN_10_189 Glycosidase (
Melampsora larici-populina
) 5e-05 GH16
53 LAN_10_210 Barwin-like endoglucanase (
R. toruloides
) 9e-07 GH45
54 LAN_10_275 Beta-lactamase (
R. toruloides
) 1e-120 GH3
55 LAN_11_034 Mannan endo-1,6-alpha-mannosidase (
Schizosaccharomyces octosporus
) 5e-27 GH76
56 LAN_11_156 Endo-β-1,3-glucanase (
M. larici-populina
) 2e-06 GH16
57 LAN_11_306 Cytosolic endo-beta-N-acetylglucosaminidase (
Morus notabilis
) 4e-49 GH85
58 LAN_11_346 Galactose mutarotase (
Lactococcus lactis
) 2e-05 GH-type carbohydrate
binding subgroup domain
59 LAN_11_456 Chitinase (
M. larici-populina
) 5e-14 GH18
60 LAN_11_482 Oligo-1,6-glucosidase (
R. toruloides
) 1e-105 GH13
61 LAN_11_485 Endo-1,4-beta mannanase (
R. toruloides
) 2e-55 GH5
62 LAN_11_506 Endo-beta-mannanase (
R. toruloides)
7e-48 GH5
63 LAN_12_102 Trehalase (
R. toruloides)
2e-06 GH37
Table 1 continue...

170 GLYCOSIDE HYDROLASES FROM THE PSYCHROPHILIC YEAST Glaciozyma antarctica PI12
64 LAN_12_113 Chitinase (
R. toruloides)
5e-47 GH18
65 LAN_12_116 Galactose mutarotase (
R. toruloides)
6e-05 GH16
66 LAN_12_147 Endo-beta-D-1,4-mannanase (
R. toruloides)
5e-80 GH5
67 LAN_12_265 Alpha-glucosidase (
R. toruloies)
2e-06 GH13
68 LAN_12_427 Alpha-1,2-mannosidase ((
R. toruloides)
1e-150 GH47
69 LAN_12_521 Alpha-mannosidase (
R. toruloides)
1e-128 GH38
70 LAN_13_036 Alpha-amylase (
Cryptococcus neoformans
) 0 GH13
71 LAN_14_046 Chitinase (
Puccinia triticina
) 7e-54 GH18
72 LAN_14_077 Glucoamylase (
M. larici-populina)
2e-54 GH15
73 LAN_14_140 Expansin (
R. toruloides
) 5e-06 GH45
74 LAN_14_164 Cellulase (
R. toruloides)
9e-11 GH5
75 LAN_14_165 Endo-beta-1,3-glucanase (
R. toruloides)
9e-05 GH81
76 LAN_14_244 Beta-1,3-xylanase (
R. toruloides)
1e-35 GH26
77 LAN_14_270 Chitin deacetylase (
R. toruloides)
6e-09 GH10
78 LAN_14_284 Allantoicase (
Glarea lozoyensis
) 5e-84 Galactose-binding
domain like
79 LAN_14_333 Galactose mutarotase (
R. solani
) 3e-37 GH31
80 LAN_15_276 Chitin deacetylase (
R. toruloides)
GH18
81 LAN_15_282 Chitin deacetylase (
Puccinia graminis)
6e-11 GH10
82 LAN_15_284 Chitin deacetylase (
P. graminis
) 2e-13 GH10
83 LAN_15_297 Chitin deacetylase (
P. graminis)
1e-06 GH10
84 LAN_16_072 Polyphosphoinositide phosphatase (
R. solani
) 0 GH31
85 LAN_16_363 Beta-1,3-xylanase (
Dacryopinax sp
.) 8e-76 GH26
86 LAN_16_504 Hexose-6-phosphate mutarotase (
R. toruloides)
3e-32 GH10
87 LAN_16_507 Glucan synthase (
Metarhizium anisopliae)
3e-30 GH23
88 LAN_16_574 N-acetylhexosaminidase (
Postia placenta
) 8e-132 GH20
89 LAN_16_632 Expansin (
R. toruloides)
1e-41 GH45
90 LAN_16_634 Expansin (
R. toruloides)
8e-34 GH45
91 LAN_16_648 Alpha-1,6-mannanase
(R. toruloides)
6e-30 GH76
92 LAN_16_668 Alpha-amylase (
M. larici-populina
) 0 GH13
93 LAN_16_703 Alpha-amylase (
Microbotryum violaceum
) 0 GH13
94 LAN_16_718 Endo-beta-1,3-glucanase (
M. larici-populina
) 8e-06 GH16
95 LAN_16_736 Cold-induced thioredoxin domain-containing protein (
C. neoformans
) 0 GH45
96 LAN_16_754 Exo-alpha-1,6-mannosidase (
Elizabethkingia anophelis
) 4e-21 GH126
97 LAN_16_887 Chitinase (
Coniophora puteana
) 5e-58 GH18
*The identification number assigned to each gene in the
G. antarctica
genome database (http://27.126.156.144/glaciozyma_antarctica/)
Table 1 continued...
Fig. 1. Distribution of glycoside hydrolase families in G. antarctica.

GLYCOSIDE HYDROLASES FROM THE PSYCHROPHILIC YEAST Glaciozyma antarctica PI12 171
Table 2. Summary of
G. antarctica
glycoside hydrolases that contain
signal peptides
Number of
Family Predicted activities proteins with
signal peptides
GH45 Endoglucanase 9
GH10 Xylanase 7
GH16 Endo-1,3(4)-β-Glucanase 5
GH5 Endo-1,4-β-D-Mannanase 4
GH76 α-1,6-mannanase 3
GH31 α-glucosidase 3
GH26 Lichenase 3
GH18 Chitinase 3
GH55 Exo-β-1,3-glucanase 2
GH88 β-glucuronyl hydrolase 1
GH72 β-1,3-glucanosyltransglycosylase 1
GH63 α-glucosidase 1
GH47 α-mannosidase 1
GH23 Lysozyme 1
GH20 β-hexosaminidase 1
GH13 α-amylase 1
Fig. 2. Venn diagram of the three bioinformatics software
programmes used to predict the signal peptide sequence in
the glycoside hydrolases of G. antarctica. A total of 46
proteins were predicted to contain a signal peptide in at least
two software programmes.
is an extremely important component of the primary
cell wall of plants, while laminarin is a storage
glucan in brown algae and lichenan is a complex
glucan identified in certain lichen species. The
secreted enzymes might facilitate nutrient
scavenging by G. antarctica. Studies have
previously shown that fungi isolated from the
interior structural woods at Ross Island, Antarctica
secrete endoglucanases into the environment
(Duncan et al., 2006). These enzymes are active at
low temperatures, as indicated through the cellulose
degradation observed at 4ºC and 15ºC.
Thus, in the present study, we classified 97
putative genes from G. antarctica into 23 different
glycoside hydrolase families from the 133 CAZy GH
families available. These groups primarily comprise
extracellular enzymes, including endoglucanases,
xylanases and chitinases, which are important for
nutrient utilisation for the survival of these
microorganisms. The optimal activity of glycoside
hydrolases at low temperatures facilitates the study
of cold active enzyme mechanisms and the potential
application of these enzymes in biotechnology.
Indeed, extracellular cold enzymes have useful
functions in food, detergent and biofuel industries.
Therefore, it is important to elucidate the
biochemical properties of these annotated glycoside
hydrolases to understand the unique properties of
these enzymes.
ACKNOWLEDGEMENTS
The authors would like to thank Malaysian Genome
Institute (MGI) for providing access to the
Glaciozyma antarctica Genome Database. This study
was financially supported through the Ministry of
Science and Technology, Malaysia (MOSTI), grant
no. 02-05-20-SF0007 and 10-05-16-MB002.
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