Philippine Panay Island Bushy-tailed Cloud Rat (Crateromys heaneyi): A Preliminary Behavioural Study of Captive Cloud Rats

Abstract

Panay Island bushy-tailed cloud rats Crateromys heaneyi are nocturnal, arboreal, probably herbivorous Philippinerodents. Apart from limited morphological data, there is very little reported information about them. The aim of thisstudy was to gather preliminary data with a view to developing an ethogram for these taxa, based on a captivepopulation house at ZSL London Zoo. Cloud rats are probably not social rodents and are likely to live in pairs orsolitarily in the wild when not raising offspring. They are intolerant of intruders in their territory and will fight to thedeath when stressed. They spend the majority of their time resting, climbing and feeding. Cloud rats are fastidious intheir habits; defecating and urinating away from their nest boxes and food at a particular constant site and cleaningthemselves methodically after every meal. There are significant gaps in our knowledge of these mammals, which arelisted as endangered on the IUCN Red List.

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Editorial Board
Abdul Khaliq Naveed National University of Science & Technology, Pakistan
Amara Naksathit Mahidol University, Thailand
Anne Brown Canadian Center of Science and Education, Canada
Billy Sinclair University of Cumbria, UK
Da Jia UT Southwestern, USA
Florent Engelmann Institut de Recherche pour le Développement, France
Ignacy Kitowski Maria Curie-Sklodowska University, Poland
Mark Andrew Skidmore The University of Liverpool, UK
Mike Gormally National University of Ireland, Ireland
Mike Watson University of Southern Queensland, Australia
Muhammad Ishtiaq University of Azad Jammu & Kashmir, Pakistan
Naowarat Cheeptham Thompson Rivers University, Canada
Nikolas Zagris University of Patras, Greece
Olga Kukal Queen’s University, Canada
Olga Pantos University of Queensland, Australia
Paramita Basu Indian Institute of Technology Kanpur, India
Patrick Marcel Schaeffer James Cook University, Australia
Peer Schenk The University of Queensland, Australia
Risto Penttinen University of Turku, Finland
S Craig Roberts University of Liverpool, UK
Salil Kumar Bose Nanyang Technological University, Singapore
Shree R. Singh National Cancer Institute, USA
Stuart Craig Smith Deakin University, Australia
Zaini Mohd Zain University Technology MARA, Malaysia

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International Journal of Biology July, 2009
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Contents
Philippine Panay Island Bushy-tailed Cloud Rat (Crateromys heaneyi): A Preliminary Behavioural Study
of Captive Cloud Rats
Root-Gutteridge, H.A.J. & Chatterjee, H.J.
Purification and Characterization of Cold-Adapted Metalloprotease from Deep Sea Water Lactic Acid
Bacteria Enterococcus Faecalis TN-9
Qingzhu Yuan, Atsushi Hayashi, Yoshihisa Kitamura, Takashi Shimada, Ren Na & Xiao Jin
Model Stability Analysis of Marine Ecosystem
Yuejian Jie & Yuan Yuan
Preliminary Study on Mitochondrial DNA Cytochrome B Sequences and Genetic Relationship of Three
Asian Arowana Scleropages Formosus
Yinchang Hu, Xidong Mu, Xuejie Wang, Chao Liu, Peixin Wang & Jianren Luo
Genetic Variation of Six Azadirachta excelsa (Jacks) Jacobs Populations
Hazandy Abdul-Hamid
Investigation and Application Progress of Vero Cell Serum-free Culture
Tian Chen & Keping Chen
Purification and Characterization of the Lipase from Marine Vibrio fischeri
P. Ranjitha, E. S. Karthy & A. Mohankumar
Cloning and Analysis of DNA Sequence of Gene CylA of Enterococci Inducing Sheeps Encephalitis
Guijun Ma, Sujuan Han, Genqiang Yan & Xia Zhou
Biology of Macrolophus caliginosus (Heteroptera: Miridae) Predator of Trialeurodes vaporariorum
(Homoptera: Aleyrodidae)
Mohd Rasdi, Z., Fauziah, I., Wan Mohamad, W.A.K, Syed Abdul Rahman, S.R, Che Salmah, M.R. &
Kamaruzaman, J.
Molecular Cloning and Characterization of a Putative BnHEC3 Gene in Oilseed Rape (Brassica Napus)
Xiaoli Tan, Lili Zhang & Zongwei Xia
A New Species of Aspergillus
Zongzhou Zhang
Transcription and Expression of Major VP Gene of Bombyx Mori Parvo-like Virus
Jinsong Cheng, Qin Yao, Meng Lv, Chen Sun, Yuanqing He & Keping Chen
Effects of Waterlogging on Growth and Physiology of Hopea odorata Roxb
Hazandy Abdul-Hamid, Nor Aini Ab. Shukor, Sapari Mat, Abdul Latib Senin & Kamaruzaman Jusoff
Preliminary Study on the Mechanism for Adaptation of Stipagrosits Pennata to Desert
Baohua Gong, Jianbo Zhu, Hongying Zhao & Yuxing Zhang
Isolation and Characterization of a Silkworm cDNA Encoding a Protein Homologous to the 14kDa Protein
of Bovine Ubiquinol-cytochrome C Reductase
Guangwei Xing, Jinghong Xing & Suhua Wang
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Contents
Expression of Cocoonase in Silkworm (Bombyx mori) Cells by Using a Recombinant Baculovirus and Its
Bioactivity Assay
Jianjun Yang, Wenbing Wang, Bing Li, Yudan Wu, Huiling Wu & Weide Shen
IGF-1 Gene Polymorphism and Weight-Related Analysis
Wei Li, Fangqun Li & Daquan Li
Bioinformatics of NS3 Gene and Inverted Terminal repeats (ITR) of Bombyx Mori Parvo-like Virus (China
Zhenjiang Isolate)
Chen Sun, Qin Yao, Huijuan Yin, Yuanqing He & Keping Chen
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International Journal of Biology July, 2009
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Philippine Panay Island Bushy-tailed
Cloud Rat (Crateromys heaneyi): A Preliminary Behavioural
Study of Captive Cloud Rats
Root-Gutteridge, H.A.J. & Chatterjee, H.J. (Corresponding author)
Research Department of Genetics, Evolution and Environment
Darwin Building, University College London
Gower Street, WC1E 6BT, London, UK
Tel: 44-207-679-4113 E-mail: h.chatterjee@ucl.ac.uk
Abstract
Panay Island bushy-tailed cloud rats Crateromys heaneyi are nocturnal, arboreal, probably herbivorous Philippine
rodents. Apart from limited morphological data, there is very little reported information about them. The aim of this
study was to gather preliminary data with a view to developing an ethogram for these taxa, based on a captive
population house at ZSL London Zoo. Cloud rats are probably not social rodents and are likely to live in pairs or
solitarily in the wild when not raising offspring. They are intolerant of intruders in their territory and will fight to the
death when stressed. They spend the majority of their time resting, climbing and feeding. Cloud rats are fastidious in
their habits; defecating and urinating away from their nest boxes and food at a particular constant site and cleaning
themselves methodically after every meal. There are significant gaps in our knowledge of these mammals, which are
listed as endangered on the IUCN Red List.
Keywords: Cloud rats, Ethogram, Behavioural profile, Endangered, Captive population
1. Introduction
Cloud rats, or cloud runners as they are also called, are medium sized, soft-furred, nocturnal, arboreal rodents, found
exclusively in the Philippine Islands at 10onorth latitude and 125o45’ east longitude. The Panay Island bushy-tailed
cloud rat (Crateromys heaneyi) is the most recent to be identified and London Zoo has the only breeding colony outside
of the Philippines. Classified by the IUCN as endangered, the Zoo’s protective attitude prevented any invasive or
disturbing means of observing behaviour. Almost nothing is known of the genus’ behaviour or ecology and the study
was intended to function as a preliminary observation on which more intensive studies could be based. Behavioural
studies have not been undertaken and at London Zoo the cloud rats are kept according to the principles that govern the
captivity of other nocturnal rats. There has been no proper survey of population size or range for any species of cloud
rat and the threat of extinction has only been estimated (Heaney et al., 1998, 2005; Musser et al., 1981, 1985; Gonzales
and Kennedy, 1996; Nowak and Paradiso, 1983).
All assumptions of behaviour are based on anecdotal evidence gathered by Musser et al. (1985) from Philippine Island
hunters. Detailed descriptions of their teeth by Gonzales and Kennedy (1996) indicate herbivory. The cloud rats scurry
around the tops of oak and pine trees at altitudes of up to 400m and are seen as glimpses amongst the clouds (Heaney et
al, 1998; Oliver et al., 2001). They have yet to have more than one offspring per pregnancy in captivity which is
unusual amongst rodents (London Zoo, 2005).
Humans threaten wild cloud rats as they hunt them for meat and pelts, furthermore deforestation has seen a 20%
reduction in forest cover between 1950 and 1980. As there is no accurate estimate of population sizes, the probable
effect of such habitat loss can only be estimated (Heaney et al., 2005; IUCN, 2006; Heideman et al., 1987; Kummer,
1991).

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Vol. 1, No. 2 International Journal of Biology
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This behavioural study aimed to build upon existing knowledge to improve captive care. Small mammals are
notoriously sensitive to stress and with almost no data about their lives in the wild, it is very difficult to assess the
requirements of cloud rats in captivity.
2. Materials and Methods
All methods were designed to minimise stress and disruption to the cloud rats.
2.1 Captive Habitat Conditions and Group Composition
London Zoo houses ten cloud rats in four enclosures (Table 1). The largest enclosure (Enclosure 1) housed a family
group; the remaining three enclosures were smaller and housed a breeding pair (Enclosure 2) and single individuals
(Enclosures 3 and 4). Each had a layer of woodchips on the base with a number of thick branches acting as climbing
poles; this was the only enrichment. The enclosures’ building was renovated and their move to new accommodation
ended the data collection.
2.2 Observational Data Collection Methods
In October 2005, 60 hours and 8 minutes of observation were undertaken based on the protocol used by London Zoo for
all its ethograms. Behavioural categories were added following a brief pilot survey lasting 3 days to include cloud rat
specific activities. Instantaneous and one-zero samplings at two-minute intervals were combined to record 20-minute
periods of the behaviour of a single focal animal. Data could not be collected by the use of cameras, infra red or
changes in photoperiod as the Zoo did not permit any invasive methods. The cloud rats’ light sensitivity only allowed
observation of activity from 10.45am to 4pm. Table 2 outlines the key behaviours and the definitions were subsequently
used as variables for data collection. The duration of time when no cloud rats were visible was recorded as negative
data.
Diet sheets were provided by London Zoo and food intake details were recorded over two weeks of London Zoo’s
normal husbandry routine. Likely calorie intake was estimated from food provided by the keepers and the remains
measured the next day. Biographical details (Table 3) were estimated from London Zoo’s brief biographical record
sheets.
3. Results
Preliminary observations based on the above protocol suggest that the captive cloud rat population at London Zoo spend
most of their visible waking time climbing, feeding and resting (Figures 1 - 4). Other activities such as grooming and
playing account for significantly less total activity time (Table 4). Their waking time is predominately spent in repose
and the only possible activities in the nest boxes are self grooming, allogrooming and resting. This suggests that the
cloud rats are either a) generally inactive or b) inactive in captivity. Data for cloud rats in enclosures 2, 3 and 4 were
limited and focus here is towards the family group in enclosure 1.
The data suggest that there was little correlation between age and time spent in company outside the nest box with all of
the cloud rats spending at least 200 minutes outside of the nest box with another cloud rat. Proximity was usually
0.5-0.75m apart (Table 4). How significant the results are for behaviour is not known as the cloud rats had little choice
in proximity due to the enclosure size (Figure 5).
Biographical data is summarised in Table 3. The second youngest specimen at London Zoo, aged some 2 months when
the study started, appeared to have a light coloured soft natal coat which was shed at age approximately 10 weeks to be
replaced by a darker adult coat. The diet data was analysed and showed distinct food preferences on the part of the
cloud rats: Fruit was always favoured while seeds, nuts, rodent pellets and bread were most commonly rejected (Figure
6). The average calorie intake was calculated to be 176.985 calories per day per cloud rat (Calorie King, 2006; Zoo Plus,
2006).
4. Discussion
London Zoo’s captive breeding program is part of an international effort to prevent the extinction of threatened species.
As far as possible, the effects of enforced time limits were curbed and the resulting data were tested and shown to be
statistically significant under Dytham’s rule (1999); even rough estimates of likely gestation time and age of sexual
maturation are an important advance.
4.1 Negative Data
There were more negative data when no observations of cloud rat activity were recorded than positive data when there
were observations (4680 minutes compared to 1600); this was probably because the cloud rats spent most of their time
in nest boxes. The family group was the most active and had the largest enclosure so it seems more likely that the cloud
rats were idle because of lack of stimulation. The cloud rats were sensitive to light and would not emerge in either
artificial or natural light conditions in any of the enclosures.

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International Journal of Biology July, 2009
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4.2 Feeding and Play Behaviour
The cloud rats showed a preference for fruit and with the high metabolic demand of a small homeothermic body it is
likely that they preferentially feed on the high-energy sugary food. The calorie intake was higher than would normally
be predicted (Barnett, 1963), but there was a nursing female who would have an elevated calorie intake. Spatial
constrictions did not permit whole group feeding so no conclusions about sociality and food can be made and this is a
distinct limit to the research (Montgomery and Gurnell, 1985). As the family only spent 1% of their time allogrooming,
close social bonds cemented by altruistic behaviour are unlikely (Figure 3), but the juveniles did follow the adults
across branches in play behaviour that could teach agility and territorial boundaries.
4.3 Escape and Reintroduction
In the final week of the study, the juvenile male CRJ1 showed increasingly distinctive behaviour typified by standing on
the nest box on the uppermost ledge and clawing / chewing at the ceiling. This formed a hole through which CRJ1,
CRJ2 and CRJ3 escaped. Following a temporary separation of approximately 72 hours, they were reintroduced to their
family group in the new enclosure. This presented antagonistic behaviour within the group resulting in some physical
injury to the two offspring males (CRJ1 and CRJ3) one of which was killed. It may be that cloud rats are territorial and
once the males have left the natal group they are not accepted back, which is true in rats (Barnett, 1963). Why the cloud
rats had not previously reacted in this way is not known, but juvenile mice produce urine that has a distinctive smell
which reduces the aggressiveness shown by elders towards them (Poole, 1985). The clouds rats were often sociable in
pairs and were observed feeding or playing in groups of two or three. There was some correlation between age, sex and
sociability.
5. Conclusion
This preliminary study has presented novel findings regarding the captive behaviour of the world’s only breeding
population of cloud rats. Observations made over a longer time period are required. The most important future research
would be a simple continuation of the ethogram over a greater range of daily times and also for a longer length of
observation, ideally including nest box cameras and infra red. A continuation of the present study was not possible due
to concerns expressed by London Zoo regarding within group aggression and stress induced by on-site observers;
infra-red cameras and nest box cameras would help alleviate these stresses. Dietary changes, increasing male aggression
with age, offspring dispersal, the effect on behaviour of changes in temperature, and daylight length and intensity,
would be interesting subjects for further study. Critically, further field studies of C. heaneyi and other cloud rats are
required to understand their behaviour in the wild. This is particularly important in light of the significant extinction risk
facing these enigmatic Philippine rodents.
Acknowledgements
We are extremely grateful to Andy Hartley of the Zoological Society of London, Marie Whatmough and all the small
mammal keepers of London Zoo, Lawrence Heaney of the Chicago Field Museum, for whom the Panay Island
bushy-tailed cloud rat is named and William Oliver, the Director of the Philippines Biodiversity Conservation
Programme.
References
Barnett, S. (1963). The Rat. Chicago & London: University of Chicago Press.
Calorie King. (2006). Calorie king food search:
http://www.calorieking.com/ (January 26, 2009)
Dytham, C. (1999). Choosing and using statistics: a biologist’s guide. Blackwell Science.
Gonzales, P.C. & Kennedy, R.S. (1996). A new species of Crateromys (Rodentia: Muridae) from Panay, Philippines.
Journal of Mammalogy, 77, 1, 25-39.
Heaney, L.R., Balete, D.S., Dolar, M.L., Alcala, A.C., Dans, A.T.L., Gonzales, P.C., Ingle, N.R., Lepiten, M.V., Oliver,
W.L.R., Ong, P.S., Rickart, E.A., Tabaranza, Jr. B.R. & Utzurrum, R.C.B. (1998). A synopsis of the mammalian fauna
of the Philippine Islands. Fieldiana Zoology new series, 88, 1-61.
Heaney, L.R., Walsh, Jr. J.S. & Townsend Peterson, A. (2005). The roles of geological history and colonisation abilities
in genetic differentiation between mammalian populations in the Philippine archipelago. Journal of Biogeography, 32,
229-247.
Heideman, P.D., Heaney, L.R., Thomas, R.L. & Erickson, K.R. (1987). Patterns of faunal diversity and species
abundance of non-volant small mammals on Negros Island, Philippines. Journal of Mammalogy, 68,884-888.
IUCN. (2006). IUCN red list of threatened species. [Online] Available: http://www.iucnredlist.org (January 26, 2009)
world’s biggest database. [Online] Available:

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Krebs, J.R. & Davies, N.B. (1987). An introduction to behavioural ecology. Blackwell Scientific Publications 2nd
edition.
Kummer, D.M. (1991). Deforestation in the postwar Philippines. University of Chicago Geography Research paper
234:39-76.
London Zoo. (2005). Biographical Records for the Captive Cloud Rats. Unpbl. material.
Montgomery, W.I & Gurnell J. (1985). The behaviour of Apodemus. In J.R. Flowerdew, J.Gurnell, & J.H.W. Gipps
(Eds.), The Ecology of Woodland Rodents, Bank Voles and Wood Mice (p 89-115). Zoological Society of London
Symposia 55: Oxford, Clarendon Press.
Musser, G.G. & Gordon, L.K. (1981). A new species of Crateromys (Muridae) from the Philippines. Journal of
Mammalogy, 62, 513-525.
Musser, G.G., Heaney, L.R. & Rabor, D.S. (1985). Philippine rats: a new species of Crateromys from Dinagat Island.
American Museum Novitates, 2821, 1-25.
Nowak, R.M. & Paradiso, J.L. (1983). Walker’s mammals of the world. 4th Ed. Baltimore and London: Hopkins
University Press.
Oliver, W.L.R., Lindsay, N. & Kloes, H. (2001). Philippine cloud rats’ conservation programme – a report on progress
to June 2001. Unpbl. Courtesy of William Oliver, FFI-Philippines Programme, Manila, Philippines.
Poole, T.B. (1985). Social behaviour in mammals. 1st Ed. Blackie, USA: Chapman and Hall, New York.
Zoo Plus UK. (2006). Nutrition analysis of Eukanuba dog food. [Online] Available: http://www.zooplus.co.uk (26
January, 2009)
Table 1. Captive habitat conditions and group composition
Enclosure
1
Description of Enclosure
2 x 2 x 2m
Concrete enclosure with one glass wall
exposed to the public
Reverse lighting conditions
2 nest boxes
Captive Population
CRA1: Adult male (2 years of age)
CRA2: Adult female (2 years of age)
CRJ1: Juvenile male (8 months)
CRJ2: Juvenile female (5 months)
CRJ3: Juvenile male (2 months)
CRJ4: Juvenile female (< 5 days old)
CRA3: Adult male (age unknown)
CRA4: Adult female (age unknown)
2 1 x 1 x 1m
Concrete enclosure with one glass wall
exposed to the public
Reverse lighting conditions
1 nest box
1m depth x 1m width x 2m height
Wire cage enclosure, closed to the public
Normal lighting conditions
1 nest box
1m depth x 1m width x 2m height
Wire cage enclosure, closed to the public
Normal lighting conditions
1 nest box
3 CRA5: Adult male (age unknown)
4 CRA6: Adult female (age unknown)

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International Journal of Biology July, 2009
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Table 2. Definitions of the behaviours
Activity Definition
Feed Inactive: feeding at rest or if combined with foraging feeding during movement.
Forage Active: searching for food, not eating it
Rest Inactive: sitting or lying down without other activity.
Play Active: usually walk / climb behaviour with another cloud rat as in “follow my
leader” game or wrestling.
Walk / climb Active: movement that was not part of a game with another cloud rat or in
search of food.
Groom self Active: grooming self always done in sitting position and involved a small
amount of gymnastic movement.
Groom other Active: grooming another cloud rat, sometimes moving around or over the other
individual to do so. Also known as allogrooming.
Neighbour ID The identity of the nearest neighbour (if any) to the focal animal.
Distance to nearest neighbour The estimated distance between the focal animal and its nearest (if any)
neighbour.
Hide Any period when the cloud rat was not visible during the observation time, not
definitely at rest but due to the arrangement of the enclosure, usually resting,
feeding or grooming were the only activities possible.
Other Any behaviour that is not covered by the above. Described further in notes;
included mating behaviour, creating a hole in the ceiling and other social
activities.

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Table 3. Life history data for C. heaneyi and P. pallidus
C. heaneyiP. pallidus
Average female sexual maturity10 months8 months
Average male sexual maturity 10 months12-18 months
Gestation time parameters 1-2 months69-95 days
Birth interval 2-5 months8 months
Number offspring per pregnancy 1 1
Weight of young at birth 124g 100-150g
Estimated weaning age 2 months 5 months
Oestrus cycle Unknown 10-15 days
Lifespan Unknown 13-15 years in captivity
C. heaneyi values calculated from data given by London Zookeepers (2006).
P. pallidus values from Minnesota Zoo website (2006).
Table 4. Total behaviour times for individuals and total behaviour times overall (positive and negative data)
(time in minutes)
ID Feed Forage Rest Play Climb Groom self
Groom
Other Hide OtherID Distance
Positive
Data
Negative
Data
CRA1 78 96 116 12 174 54 2 170 0 0.707 340 468
CRA2 154 86 32 42 174 44 2 66 0 0.455 360 468
CRA3 10 10 4 2 28 0 0 8 60 B 2.75 80 380
CRA4 12 10 58 2 26 0 0 2 10 A 2.75 80 380
CRA5 0 0 40 0 0 0
N/A 0 0 N/AN/A 40 310
CRA6 0 0 0 0 0 0
N/A 0 0 N/AN/A 0 310
CRJ1
182 74 202 46 94 50 8 130 0 0.647 480 468
CRJ2 104 88 50 42 230 52 2 92 4 0.5 340 468
CRJ3 86 58 60 50 116 16 6 54 0 0.278 240 468
CRJ4 0 0 0 0 0 0 0 0 0 0 0 468
TOTAL 532 422 562 209 842 216 20 522 74 1600 4188

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International Journal of Biology July, 2009
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Total Positive Behaviour Tim es
Feed
16%
Forage
12%
Rest
17%
Play
6%
Clim b
25%
G room self
6%
Other
G room
1%
Hide
15%
Other
2%
Figure 1. Pie chart showing % behaviour times for all study individuals.
Total Behaviour Times
0
Feed
500
1000
1500
2000
2500
3000
3500
4000
4500
Forage
Rest
Play
Climb
Groom 1
Groom 2
Hide
Other
Negative Data
Behaviour
Minute
Figure 2. Time spent by individuals in different types of behaviour.

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Total Fam ily Behaviour Tim es
Feed
19%
Forage
13%
Rest
14%
Play
6%
Clim b
24%
G room self
7%
Other
G room
1%
Hide
16%
Other
0%
Figure 3. Pie chart showing % behaviour times for the family group.
Figure 4. Time spent by the family group in different types of behaviour.

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International Journal of Biology July, 2009
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Figure 5. Time spent by the focal animal within <1m to its nearest neighbour.
Food Outtake
0
5
10
15
20
25
30
35
40
Rodent pellet
Poultry mix
Sunflower seed
Orange
Carrot
Whole Nut
Egg
Apple
Pear
Banana
Grape
Prickly pear
Plum
Sweet potato
Sweet-corn
Mango
Melon
Whole lychee
Papaya
Bread
Spinach
Cucumber
Kiwi
Foodtype
%
Figure 6. Percentage of uneaten food removed from enclosures. Figures are collated from across the whole study period.

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Purification and Characterization of Cold-Adapted Metalloprotease from
Deep Sea Water Lactic Acid Bacteria Enterococcus Faecalis TN-9
Qingzhu Yuan (Corresponding author) & Atsushi Hayashi
Central Research Laboratories, Nichinichi Pharmaceutical Co., Ltd.
Mie 518-1417, Japan
Tel: 81-595-48-0201 E-mail: yqz5012001@yahoo.com.cn
Yoshihisa Kitamura & Takashi Shimada
Central Research Laboratories, Nichinichi Pharmaceutical Co., Ltd.
Mie 518-1417, Japan
China-Japan Collaborating Centre for Probiotics Research
Nanjing Medical University
Nanjing, Jiangsu 210029, China
Ren Na
Development & Service Centre of Cow Industrialization
Xilinhaote, Inner Mongolia 026000, China
Xiao Jin
Animal Hygiene Supervision Place of Inner Mongolia
Huhehaote, Inner Mongolia 010015, China
Abstract
This paper investigated a 3-step purification and characterization of a protease from Enterococcus faecalis TN-9, a
bathypelagic lactic acid bacteria. The purification procedure includes precipitation with (NH4)2SO4, then ion-exchange
chromatography with DEAE-Sephadex A-25 and DEAE Cellulofine A-500. Native PAGE analysis indicates a single
protease band. The molecular weight is 30 kDa by SDS-PAGE analysis, and 69 kDa by gel chromatography analysis. It
proves that the optimal temperature for protease reaction is 30 ºC, and the optimal pH is 7.5-8.0. The reaction is stable
while pH is 6.0-9.5 and temperature is under 45 ºC. The relative activity is 6.1% at 0 ºC. The enzyme is totally
deactivated with heat treatment at 60 ºC or over. The protease is partially inhibited by EDTA-2Na, Hg2+, Cu2+, Ni2+,
Ag2+, Co2+ and Pepstatin A. Zn2+ shows obvious activation to the protease. Km and Vmax of purified protease acting on
azocasein are 0.098 % and 72 mg/(h.mg) respectively. This protease is one of gelatinase with N-terminal sequence of
VGSEVTLKNS, and shows characteristics of a cold-adapted metalloprotease.
Keywords: Deep sea water, Lactic acid bacteria, Cold-adapted enzyme, Metalloprotease, Azocasein
1. Introduction
The cold-adapted enzymes from cold-adapted organisms or other microbial life in extreme environments have been
widely studied in recent years. Reports are often on cold-adapted enzymes from marine microorganism. Shi et al (2005,
p. 258-263; 2006, p. 72-75) separated and purified a strain of cold-adapted enzyme from a psychrophilic bacteria
Bacillus cereus SYP-A2-3 acquired from samples collected from glacier. Nushin Aghajari et al (2003, p. 636-647) and
Denner E B et al (2001, p. 44-53) isolated a strain of psychrophilic bacteria from samples of Antarctic and studied on
the cold-adapted metalloprotease. Miyamoto K et al (2002, p. 416-421) acquired and purified two types of cold-adapted

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International Journal of Biology July, 2009
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metalloprotease (MprI and MprII) from marine microorganism Alteromonas sp. strain O-7. However, we have not
found any report on the cold-adapted protease isolated from marine lactic acid bacteria.
Study described in this paper isolated 25 strains of lactic acid bacterium from deep sea water at Toyama Bay of
Japanese Sea. Among them Enterococcus faecalis strain TN-9 (Atsushi Hayashi, 2007, p. 58-64) shows liquefying
gelatin, fermented and solidified litmus milk, ?-galactosidase activity and other characteristics. Oral administration has
been proved safe by sub-acute toxicity study of this bacteria strain in rats (Chie Motonaga, 2007, p. 191-196). Therefore,
E. faecalis TN-9 is expected promising in the research and development of health foods containing lactic acid bacteria.
Based on above-mentioned results, the study in this paper isolated, purified and characterized the protease from lactic
acid bacteria E. faecalis TN-9.
2. Materials and methods
2.1 Materials and equipments
Bacteria stain: lactic acid bacteria E. faecalis strain TN-9, stored and provided by Nichinichi Pharmaceutical Co., Ltd;
Lactobacilli MRS Broth from USA; Hinute SMP provided by Fuji Oil Co. of Japan; Azocasein from Sigma of USA;
DEAE-Sephadex A-25 from Pharmacia of Sweden; DEAE-Cellulofine A-500 and Toyopearl HW-55s provided by
Chisso of Japan; standard protein provided by Wako of Japan; and other commonly used reagents made in Japan. Key
instruments: U-2000 spectrophotometer from Hitachi of Japan and KUBOTA 7780 centrifuger from Kubota of Japan.
2.2 Methodology
2.2.1 Culture of strain and collection of culture media
(1) Culture medium
Take MRS agar plate as solid culture media. Liquid culture media is a mixture of A, B and C, where A is composed of
1.5 g Hinute SMP, 2.4 g D-Glucose, 0.2 g NaCl and 70 ml distilled water; B is composed of 18.0 g K2HPO4 and 100 ml
distilled water; C is composed of 0.05 g FeSO4.7H2O, 0.58 g MgSO4.7H2O, 0.03 g MnSO4.5H2O, 0.10 g ZnSO4.7H2O
and 100 ml distilled water. Begin by formulating A and B, in turn autoclaving at 121 ºC for 15 min, then sterilize C by
sterilizing filter (DISMIC-25CS/0.45 μm, made in Toyo of Japan), finally mix A, B and C at the ratio of 7:2:1 in aseptic
environment, ready for culture of strain.
(2) Culture of strain and collection of culture media
On the MRS medium, cut marker lines on the -80 ºC stored bacteria strain, culture the slides at 30 ºC for 48 h. Select
and inoculate the independent bacterial colonies into 50 ml the above-mentioned liquid medium. After static culture at
30 ºC for 18 h, transfer it into 800 ml the same liquid medium to have the second static culture at 30 ºC for 18 h.
Centrifuge at 4 ºC (12,000×g, 10 min) to collect supernatant as crude enzyme solution for isolating and purifying
protease.
2.2.2 Determination of protease activity
Protease activity is measured by the TCA-azocasein assay based on the methods proposed by Hagihara B et al (1958, p.
185-194) and Thomas J B et al (1986, p. 139-145). 100 ?l protease solution with specific concentration is added into
400 ?l 1.25% (w/v) phosphate-buffered saline (100 mM Na-K phosphate buffer with pH of 7.5, called buffer A); react at
30 ºC for 10 min (shaking at 170 times per minute) in water bath; adding 1 ml 10 % (w/v) trichloroacetic acid (TCA) to
stop the reaction; centrifuge (16,000×g, 20 min, 10 ºC) the resulting product to remove undigested azocasein. The
absorbance (A335) of the obtained supernatant is measured at 335 nm with a spectrophotometer (Hitachi, U-2000).
Protease activity is calculated from the amount of L-tyrosine, which is achieved from A335. One unit of protease
activity is defined as the amount of enzyme for 1 ?g of L-tyrosine which is released from the substrate per minute under
the above conditions.
2.2.3 Determination of protein
Protein is measured by the Lowry process (Lowry O H, 1951, 265-275), with bovine serum albumin (Sigma of USA) as
criterion.
2.2.4 Polyacrylamide gel electrophoresis (PAGE)
Referring to the method proposed by Reisfeld R A et al (1962, p. 281-283), Native-PAGE is carried out under the
following conditions: 2.5% (w/v) stacking gel, 7.5% (w/v) separation gel (pH 4.0), alanine-acetic acid (pH 4.5) as
electrolyte bath, anode/cathode reversely-connected NA-1311 Electrolyte Bath (NIHON EIDO Co. Ltd. Tokyo), and
electrophoresis at a constant current of 2.5 mA for 3.5 h per column. Referring to the method proposed by Weber K and
Osborn M (1969, p. 4406-4412), SDS-PAGE is carried out under following conditions: 7.5% (w/v) polyacrylamide gel
and 0.10% (w/v) SDS-0.10 M phosphate buffer (pH 7.2) as electrolyte bath, electrophoresis at a constant current of 6.0
mA for 3 h per column. Use BSA (79,000), Aldolase (42,000), Carbonic Anhydrase (30,000), Trypsin Inhibitor (20,000)
and Lysozyme (14,000, from Wako of Japan) as reference standards for molecular weight. Stain for 1 h with 0.25% (w/v)