Research Overview ( 0304 )

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12 Φεβ 2013 (πριν από 4 χρόνια και 4 μήνες)

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1

Research Overview ( 0304 )

Research Group:

Research and Application of Microbial Enzymes

Research Area:

Our group is primarily engaged in enzyme technology, structure and function of
enzymes. Our aim is e
nhancing enzyme activity, modifying enzyme propert
ies through
modern biotechnology (gene engineering, metabolic engineering, DNA shuffling,
pro
t
oplast mutation, ion beam mutation etc); exploiting new enzymes;

and enlarging
fields of enzyme application
.


Group Leader:

Prof.

Shijun Qian


1962
-
1967
Department of chemistry
,
Fudan University

1968
-

Ins
titute of microbiology, Chinese Academy of Sciences

1980.10
-
1981.10

International Post
-
Graduate University Course in Microbiology,
Osaka University, Japan

1988.3
-
1990.4

School of Medicine, University of Washington,

USA

2000.10
-
2000.11 University of

Ce
rgy Pontoise, France

Research group Members:


Assistant Professor: Dr.

Chao Yapeng
,

MS.

Shi Jiaji, MS.

Zhang Guoqing

Technician:

Yan
g

J
ing

Graduate student:

Sun Ya
n
, Liu Weixiao, Liu Xiaoqiu, Yang Xiuqing, Song Liya,
Xie Fuhong, Hu Meirong, Liu Shuz
hen, Bao Hongbo,
Zhang
Chuanbao,
Zhang Lianhui, Lu Jinghua, Tan Huifang

Background and Significance:

In the recent surge of interest in industrial biotechnology,

considerable emphasis
has been placed upon advance in enzymes,

which is
a
key factor in biocon
version and
biocatalysis.

The enzymes have been applied in every
aspect

of

the national economy in
China with great social and economic benefit.

Our research focuses on the exploitation, improvement and new
application

of

2

enzymes related to textiles, food,

medicine, glycotechnology and environment
protection,

etc. We have been studying

laccase,

cellulase, transglutaminase,

heparinase,

polychlorinated biphenyl
-

degrading enzymes,
in an effort
to expand new
application, overcome low biocatalytic capabilities

and
high
production
cost,
and
improve adaptability in
the
reaction process of
these
enzyme
s
.

Moreover, we also have been studying

on

structure, function and mechanism of
enzymes.

Major Achievements:

During the past 4 years, we have got many funds supported

by the government,
Chinese Academy of S
c
iences and company etc. The research items are as following:

1.
National Tenth Five years Plan Key
T
echnologies R&D program of China:
The Study and application of neutral
-
cellulas
e

2. Innovative program of Chinese
A
cademy
S
ciences
:
The enzymes relateing
to
oligosaccharide
s

and
their

application
s


3. The National Natural Sciences Foundation of China: The
r
elationship of level
of DNA repair enzymes and telomerase with oesophagus cancer and lung
cancer

4.

National

High

Technology R&D program of China (86
3
program)
:
Construction of alkaline xylanase
-
secreting and laccase
-
secreting engineered
strain with high activity and their application
i
n paper processin
g

5. National High Technology R&D program of China (863 program)
:

The
exploitation and application of biodegradation enzyme gene resources


6. The National Key Basic Research Program of China (973 program)
:

Methodology and catalytic mechanism of nitrile hydration and hydrolysis
reaction

7. Innovative program of Chine
se
A
cademy
of S
ciences: The single ion beam
microirradiation and its study of molecular and cellular effect

8. The National Natural Science Foundation of China: The analysis of fine
structure of the heparin
oligosaccharide
s

with
bioactivity


3


9. The Nati
onal Major Natural Science Foundation of China:

The study of
chiralblock biosynthesis on chiral drug


10. Collaborative research project of Institute of microbiology, Chinese Academy
of

Sciences
-

P&G :
Intelligent screening for new enzyme activity


11.

Research project of
I
nstitute of microbiology: The new technology and new
method of molecular enzyme engineering


12

A France
-
Chinese
c
ollaborative research project
:

Comparison of
v
arious
t
ransglutaminases to catalyze the sol
-
gel transition of protein

Major achievements:

1.

Neutral Cellulase

We got a high yield strain for producing neutral cellulase by screening,

mutagen
esis (low
-
energy

ion bean,

c
hemical and protoplast mutation),

and
optimization of culturing condition
s
. Now,

we have finished pilot scale test in
20
-
ton fermenter, and got enzyme preparation by filter press,

ultra
-
filter
membrane concentration and spray dr
ying,

which

fill
ed

in the gaps in the
enzyme preparation in China.

We made neutral cellulase to test in the textile
industry
and got good result
s
.

2.

Laccase

Laccases have potential uses in environment protection,

the textile
industry,

organic synthesis and b
iosensor etc.

We screened a White rot fungus strain producing Laccase and purified
the enzyme. cDNA encoding a laccase was isolated from the white rot fungus
by RT
-
PCR. It contained an open reading fram
e

of 1157bp. The cDNA was
cloned into the vector pGAPZ
A, and expressed in the
Pichia pastoris.
The
activity of laccase secreted by the transformant was three times as high as
that of the native strain. The optimal secreting time of laccase was
significantly shorter than the native strain. Moreover,

we treated

laccase gene
with ethyl methane
-
sulfonate to generate
a
mutant library.

Then, the mutant
gene
s
were
cloned into a

vector

to

construct recombinants, which were
transfometed to
Pichia pastoris.
We got a clone from 5000 clones by agar

4

plate and 96
-
well plate

screening
,

with a laccase activity 3 fold
s

of
the
original strain

3.

Uracil N
-
Glycosylase



By RT
-
PCR, human Uracil N
-
Glycosylase

(UNG) coding sequence is
cloned onto pET
-
21(a), and expressed in
E.coli

after induction
.

Taking the
recombinant plasmi
d, whose
copy
number of
UNG

is already known, as
quantification standard, we examined the number of copies of
UNG
mRNA
in the esophagus cancer biopsy samples by
r
eal
-
time quantitative RT
-
PCR,
and determined that there is direct positive correlation between

high
-
level
expression of UNG and
the presence of
esophagus cancer.

4.

Heparinase





Heparinase is of great importance in the fine structural analysis of
heparin /heparan sulfate as well as development
of

heparin oligosaccharides;
moreover, heparinas
e itself showed pharmaceutical potentials.



Heparinase was produced by a novel species of
Sphingobacterium.

It
was purified to apparent homogeneity by a combination of SP
-
Sepharose and
Source 30S chromatographies. MALDI
-
TOF mass spectrum of the purif
ied
heparinase gave a molecular weight of 75KDa of the native enzyme. Peptide
mass spectrum showed
low homology

with the database in the peptide bank.
Inhibition of enzyme activity
by
N
-
acetylimidazole indicated that tyrosine
residues were necessary for en
zyme activity. The heparinase showed similar
activity on both heparin and heparan sulfate except
for
the heparin from
bovine lung.

Porcine intestinal mosca heparin was degraded into a series of
oligosaccharides by the purified heparinase
.

Heparin disaccha
rides were
successfully separated by HPLC. Conformation and
c
harges of disulfated
disaccharides were analy
z
ed by
c
omputer models.

Electrospray ion
-
mass spectrometry ( ESI
-
MS ) was used to identify the
structures of the disaccharides obtained by HPLC abo
ve. The mass
spectrometry of peak I mainly gave the non
-
sulfated disaccharide with the

5

mass of 379. Peak II and III indicated two major monosulfated disaccharides
with different mass, and peak III occupied majority. Both of Peak IV and V
showed the same ma
ss. No tri
-
sulfate modified disaccharides were detected
in the mixture of the heparin digest though they were abundant in the heparin
structures. The results revealed that the heparinase may specifically cut the
low sulfated sites of heparin.

Functional a
nalysis of the heparin oligosaccharides revealed that it can
bind with the cytokine
-
granulocyte
-
colony stimulating factor by
capillary
electrophoresis and mass spectrometry.

5.

Cloning and functional analysis of genes involved in
polychlorinated
biphenyls (
PC
Bs
)

degradation by
Rhodococcus

sp. R04
:

PCB is known as one of 12 persistent contaminants in the world,

PCB
biodegradation by microorganism is not only cheap and efficient,

but also
free of

second
ary

pollution.

A novel PCBs degrading strain
Rhodococcus pyr
idinovorans

was
isolated from oil field in northern
C
hina, and the degradation pathway of
biphenyl was investigated. The studies on capability of degrading and
dechlorination of PCBs revealed that R04 strain was a strong PCB degrader.

A plasmid library wa
s constructed, and approximately 40 positive
clones were obtained
.
The sequence of the positive clones indicated that its
inserted fragment contained six ORFs. Based on the homology, these six
ORFs are presumed to be
bp
hB

(dihydrodia dehydroxygenase) (nucl
eotide
positions 581 to 1397),
bp
hC

(2,3
-
dihydroxybiphenyl dioxygenase) (1426 to
2313)
bp
hA1

(large α
-

subunit) (3278 to 4627),
bp
hA2

(small β
-
subunit)
(4671 to 5198),
bp
hA3

(ferredoxin) (5532 to 5921),
and
bp
hA4

(ferredoxin
reductase) (6374 to 7612) in tu
rn. By PCR amplification, a fragment
contained possible
bp
hD

was obtained. The
bp
hB
,
bp
hC
,
bp
hA1
,
bp
hA2
,
bp
hA3
,
bp
hA4

and
bp
hD

were organized in a gene cluster (Fig

).

To identify the products of
bp
hB,
bp
hC,
bp
hA and
bp
hD
, the proteins
were analyzed by SDS
-
PAGE. The interest
ed

proteins corresponding to size

6

of 30, 32, 50, 20, 14, 48 and 30 KD were observed. Activities of
bp
hA,
bp
hB,
bp
hC, and
bp
hD were determined by HPLC or photometer,
and
when
necessary, by GC
-
MS. The resulting assays indicated that
bp
hA,

bp
hB,
bp
hC,
and
bp
hD were able to transform
biphenyl
2,3
-
dihydro
-
dihydroxybiphenyl
2,3
-
dihydroxybiphenyl and 2
-
hydroxyl
-
6
-
oxo
-
6
-
pheylhexa
-
2,4
-
dienoic acid
separately.



Fig. The organization of
pb
h

gene clusters (
bp
hB
,
bp
hC
,
bp
hA1
,
bp
hA2
,
bp
hA3
,

bp
hA4

a
nd
bp
hD
)

in R04 strain

6.

Nitrile hydratase

Nitrile hydratase has wide application in the synthesis of carboxylic acid and
amide, as well as chiral bioconversion of nitrile compoundes.

The new culture medium and conditions of
Rhodococcus
sp. AJ270 was
found,
which would help us obtain doubled cell density; Nitrile hydratase was
purified by sonication, ammonium sulfate fractionation,

DEAE
-
Sephacel
column chromatography,

second DEAE
-
Sephacel column chromatography,
Phenyl Sepharose column chromatography and
Sepha
cryl S200

column
chromatography.
The molecular mass of α subunit and β subunit of the enzyme
were 22957Da and 23493Da by mass spectrum. The enzyme could hydrate
varieties of aliphatic nitriles and aromatic nitriles. Some substrates could be
enantioselective
ly

hydrated to the corresponding

amides; Crystal of the enzyme
was obtained by hanging
-
drop vapor
-
diffusion method at 4

. After X
-
ray
diffraction and data collection the phase calculation was carried out by
molecular replacement.

7.

Transglutaminase



Transglutimase
has

a
n

applic
able prospect in the food and textile industry.

The whole gene of transglutaminase
was

obtained

from
Streptomyces


7

fraedia through genome walking. The whole gene was clone
d

into pGEM
T
-
easy vector and transformed into BL21(DE3), the protein was expresse
d
with
a yield
500mg/L , but it exist
ed

in the form of inclusion body.


The
Streptomyces

was irradiated by ion beam. The energy and dosage of
ion beam were 10kev and
30meq
-
50meq

respectively
. We investigated the
transglutaminase gene of the mutants by pol
ymerase chain reaction (PCR),
nonradioisotopic single strand conformation polymorphism (SSCP)

and
DNA
sequencing
.
We have already got 21 strains with change in transglutaminase
gene.

8.

Other enzymes



Endo
-
type inulinase attacked inulin endo
-
wise
producing inulo
-

oligosacchorides. An endoinulinase produced by
Chaetomium

sp. C34 was
purified to electrophoretic homogeneity.
Some of its

properties
were
investigated.



Mn
-
Peroxidase is key
-
enzyme for lignin degradation.

We screened a
Tramet
e
s
ve
rsicolor
strain producing Mn
-
peroxidase and purified the enzyme.



Keratinase is used for feed industry and detergent. We screened a strain for
degradation of human skin flake, then isolated and purified

the

keratinase
.

Future Research Plan:

1.
Exp
loitation and application of new enzymes
:

Enzymes related to the tex
t
iles
industry
including cellul
a
se,

laccase,
transglutaminase,

catalase and pectinase will be exploited. Our aim is to
produce two of these enzymes in pilot plant.



Enzyme
s

relat
ed to bioconversion of biomass including laccase
Mn
-
peroxidase and lignin peroxidase will be applied
in

test of conversion of
biomass.

2.

Improv
e
ment of enzymes:



Transglutaminase,

l
accase and neutral proteinase will be improved by in
vitro directed

evolution and site directed mutagenesis to enhance enzyme
activity and to change enzyme properties.


8

3.
Study of structure and function of

the Nitrile hydratase and the amidase.

Publications

1.
Yinda Wang,Shijun Qian

,Guangzheng Meng and Shuzheng Zhang. C
loning
and expressing of L
-
asperaginase gene in
Escherichia
. coli. Applied
Biochemistry and Biotechnology.2001, 95(2):93
-
101.

2.

Shi Jiaji, Cui Fumian,
The immobilization of penicillin G acylase on
γ
-
alumina by glutaraldehyde cross
-
linking, Chinese Journal of antibiotics,
2001

26

5
):
334
-
336
.

3.

Shi Jiaji, Cui Fumian, Ge Meng, The epoxidation of cis
-
propenylphophonic
acid to fosfomycin by
pencillium

sp., Acta Microbiology Sinica, 2001

4

3
):
353
-
356


4.

Shi Jiaji, Cui Fumian, Selection of
β
-
glucanase
-
producing
trichoderma
K
ō
ningii

T199 and its fermentation conditions, Acta Microbiology Sinica,
2001

41

6
):
750
-
752
.

5.

Chao Yapeng, Qian Shijun
,

Fungal laccase and its application, Journal of
Chinese biotechn
ology, 2001, 21(5): 23
-
29
.

6.

Liu Jianguo, Wang Jinfang, Li Gaoxiang, Culture conditions for creatininase
formation by
pseudomonas
sp. K9510, Microbiology, 2001, 28(2): 7
-
11
.

7.

Zhu Xianjun, Liu Jianguo, Li Gaoxiang, Cloning and expression of urate
oxidase

an
d its application in serum uric acid analysis, Chinese Journal of
Biotechnology, 2001, 17

1
):
68
-
72
.

8.

Zhu Yuwen, Liu Jianguo, Li Gaoxiang, Cloning and expression of MGMT
cDNA and analysis of the DNA repair activity of the recombinant protein,
Chinese Jour
nal of Biotechnology, 2001

17

4
):
396
-
399.

9.

Sun and S.Qian
,
Flux control analysis for biphenyl metabolism by
Rhodococcus

pyridinovorans R04. Biotechnology Letters,2002,24(18):

1525
-
1529.

10.

Liu Shuzhen, Qian Shijun, Purification and properties of laccase

from
Basidiomycete
, Acta Microbiology Sinica, 2003, 43(1): 73
-
78
.


9

11.

Bao Hongbo, Qian Shijun
,

Cloning of human uracil N
-
glycosylase and its
detection in cancer tissues by quantitative RT
-
PCR, Chinese Joural of
Biotechnolgy, 2003, 19(5): 561
-
565
.

12.

Sun
Yan, Qian Shijun, Study on the degradation pathway of biphenyl by
Rhodococcus pyridinovorans R04
, Acta Microbiologica Sinica, 43(5):
653
-
658
.

13.

Weixiao Liu, Shijun Qian
,

Molecular cloning and characterization of a
laccase gene from the Basidomycete
Fome
lignosus

and expressionin
Pichia
postoris
, Appl Microbiol. Biotech., 63

2

174
-
181

2003.

14.

Chao Yapeng, Qian Shijun
,

Rapid purification characterization and
substract specificity of Heparinase from a novel species of
Sphi
ng
obacterium

sp
, J. Biochem., 134

3

365
-
371, 2003
.


15.

Qian Shijun
,

progress in study on enzyme preparation, Chinese Joural of
Bioprocess Engineering, 1(1): 28
-
31, 2003
.

16.

Liu Xiaoqiu, Qian Shijun, Transglutaminase in medical research, Journal of
Chinese biotechnology, 2003, 23(11)

32
-
36
.

17.

Bao Hongbo, Wang Jinfang, Qian Shijun, Advances in research of uracil
N
-
glycosylase, Journal of Chinese biotechnology, 2003, 23

1

43
-
47
.


18.

Cui Dafu, Zhang Guoqing, Qian Shijun
,

et al.
Detection of
O
6
-
methylguanine
-
DNA methyltransferase activity

using surface plasmon
resonance biosensor, Progress in Natural Science, 13(8): 874
-
876, 2003
.

19.

Wei xiao Liu, Yapeng Chao, Xiuqing Yang,Hong bo Bao ,Shijun Qian
,

Biodecolorization of azo,anthraqninonic and triphenylmethane dyes by
white rot fungi and a
Laccase
-

secreting engineered strain J.Ind Microbiol
Biotechnol, 2004, 31: 127
-
132.

20.

Xiuqing Yang, Yan Sun, Shijun Qian.Biodegradation of Seven
polychlorinated biphenyls by a newly isolated aerobio bacterium
(
Rhdodcoccun

Sp RO4). J.Ind Microbiol Biotech
nol,2004, 31 : 415
-
420.

21.

Yapeng Chao, Shaoxiang Xiong, Xiulan Cheng, and Shijun Qian
,

Mass
Spectrometric Evidence of Heparin discchrides for the

Catalytic

10

Characterization of A Novel Endolytic Heparinase.Acta Biochimica et
Biophysica Sinica, 2004, 36( 1
2 ):840
-
844.

22.

Liu Weixiao, Chao Yapeng, Qian Shijun
,
Construction and Culture
Conditions of Laccase
-
secreting Engineered Strain, Acta Microbiologica
Sinica 2004,44(5):596
-
599
.


23.

Chao Yapeng, Xu Guanzhu, Cheng Xiulan, Qian Shijun, Preparation and
characteristics of the immobilized endolytic heparinase,
Acta
Microbiologica Sinica,
2004,44(5):689
-

691
.

24.

Liu Weixiao, Qian Shijun
,

Methods of the directed molecular evolution of

enzyme
in vitro
, Microbiology
,

2004, 31(2): 100
-

104
.

25.

Liu Weixiao, Chao Yapeng, Qian Shijun, Construction of laccase
-
secreting
engineering strain and decolorization of RBBR by purified laccase,
Chinese Journal of bioprocess engineering, 2004, 2(1): 21
-

24
.

26.

Sun Yan, Yang Xiuqing, Qian Shijun,Cloning and expression of poly
chlorobiphenyl/biphenyl degrading gene from
Rhodococcus Pyridinovorans
,
Chinese Environmental Sciences,
2004,24(6): 734
-
737
.

27.

Zhang Guoqing, Cui Fumian, Yang Xiuqing, Qian Shiju
n
,

Purification and
Properties of endoinulinase from
Chaetomium
sp. ,Acta Microbiology
Sinica, 2004, 44(6): 785
-
788
.


28.

Sun Yan, Qian Shijun, Characterization and identification of a biphenyl
degrading strain, Microbiology, 2004,31(6):23
-

26.

29.

Aiye Li
ang

Yapeng Chao

co
-
authored
),
Xiaojun Liu

Yuguang Du

Keyi Wang

Shijun Qian and
Bingcheng Lin

,
Heparin oligosaccharides
separation and interaction with Granulocyte
-

colony stimulating factor by
capillary electrophoresis. Electrophoresis.(Accepted)

30. Zh
angLian
-
hui Yang Xiu qing GeKeshan , Qian Shijun, Purification
and Properties of Manganese Peroxidase from
Tramates

verscolor, Acta
Microbiology Sinica,2005, ( in press )