1. Career achievement

2. Research during Master’s de-gree

Han Ji Hee2016.May

Nanobrick / SPM-Bio

Biochemical characterization of L-asparaginase in NaCl-tol-erant Staphylococcus sp.OJ82 isolated from fermented food.

Division: SPM-Bio Role : Material development and commercialization

SPM nanoparticle? Superparamagnetic Iron oxide nanoparticle (100um~1um) - Nanoparticle for Bio-purification, separation - Efficient separation method of purification (Solution for time consuming, labor intensive purification equipment)

◆ Product Benefits Uniformly dispersed Particle High magnetization Value High concentrated functional group coating Easy and efficient separation of Biomolecule

SPM ! magnetized and dispersed Product Property

◆ SEM image, XRD, Hysteresis analysisBiotinylated coupling, antibody purification etc.

Product Property

Magnetite based SPM exert excellent separation property with its monodispersed particle and sufficient magnetization up to 80emu/g.

Application

MRI Imaging agent

DNA/RNA

Separa-tion

Anti-body

Detec-tion

Biosen-sor

ProteinPurifica-

tion

DrugDeliverySystem

WaterTreat-ment

SPM

Product Property

Core Technology

• The main concept of MTX (Magnetically Color Tunable Photonic Crystal) was

first inspired from the nature; Beetle shells, Morpho butterflies and etc.

• The exact mechanism of the structural color is used in MTX; production of

color is made by microscopically structured surfaces in combination with pho-

tonic crystals, fine enough to interfere with visible light.

• M-Tag is the world’s first and only magnetically color-changeable anti-counter-

feiting solution.

▶ Principle of MTX : Highly ordered array of nanoparticles

Magnetically Color-changeable Photonic Crystal (MTX)Nature

Rub-ber Cell-

phone

Product Property (Data Nanobrick)

FDA approved commercial products

Porton products, UKErwinase (Erwinia chrysanthemi)

Research

Extracellular Asparagine Aspartate+Ammonia

Oxaloac-etate

As-paragineSyn-thetase

ER

No intracel-lular synthe-sis

L-Asparagi-nase

Extracellular Asparagine Aspartate+Ammonia

Aspar-tate

Oxaloac-etate

As-paragine

As-paragineSyn-thetase

ER

L-Asparagi-nase

(Intracellular)Normal CellIn ER, Asparagine synthetase pro-duce Asparagine

Tumor Cell

No as-paragine syn-thetase

Anti-Leukemia therapy

Introduction

What’s the function of L-asparaginase in cell?

Introduction

Toxic Acrylamide

Toxic Acrylamide in Food: Burnt Sugar + Asparagine during Maillard reaction

What’s the role of the L-Asparaginase ?

<120℃

Asparaginase reduce acrylamide by socking, spraying, mixing with flours

Potato chips French Fries

Bread, Bis-cuit

Chocolate, coffee

Emphasis of study

- Homology identity level was low - Phylogenetically distant. (Most of them are Gram negative sources)

Optimal conditions of SoAsn and EcAsn are similar - Temperature and pH

The purified SoAsn holds 61.2% activity in 2M NaCl, which is higher than EcAsn.

Major cofactor of SoAsn is cobalt while others are enhanced by Mg or Zn

Substrate specificity improved by PEG and further enzyme engineering

1

2

3

4

5

Active sites

Dimer

L-asparaginase 3D structure

Results & Discussion

Cloning of L-asparaginase from Staphylococcus sp. OJ82

L : 1kb ladder1 : SOJ_15970 PCR

Eco R I Xho I Purified each step 2 : pET28(a)+ Eco R I Xho I purified each step

L 1 2 L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 P N

L : 1kb ladder1~15 : BL21 Colony(Asparaginase)P : Positive controlN : Negative control

T7 promoter forward & Reverse Slightly (160bp) upper band

Liga-tion

Transforma-tion

(Top10,BL21)

Results & Discussion

L-asparaginase activity assay

(NH4)2SO4 as standard. Ammonia is released.UV-Spectrometer 436nmTris buffer( pH8.6 ), 37℃

Step 2Nessler’sreage

nt

NH3 Re-

leased

International Units = umoles of am-

monia / minute,ml

Step 1E-S

Reac-tion

Results & Discussion

Purification step L-asparaginase activity IPTG induction and Purification of enzyme SoAsn Increases the SoAsn specific activity and its fold change

Results & Discussion

Coenzyme factor (2mM metal in Tris buffer pH8.6) Most effective metals are different SoAsn : cobalt (263.9% increase)

EcAsn : Magnesium, Zinc (Normally Mg is the major cofactor of L-as-

paraginase)

Metal(cofactor) dependent L-asparaginase Assay

Results& DiscussionResults & Discussion

SoAsn maintains 61.2% of its activity under high NaCl

But, EcAsn drastically dropped to 48%SoAsn is Tolerant to Salt(NaCl) Stress

0.9% NaCl is humane fluid salinity.

Contorl

0.2M 0.5M 1M 1.5M 2M40

50

60

70

80

90

100

110

120

61.2

44.7Rel

ativ

el L

-Asn

Act

ivit

y (%

)Salt(NaCl) stress dependent L-Asparaginase As-

say

Results& DiscussionResults & Discussion

SolutionPEG(Polyethyleneglyco

l)

2.5%(v/v) PEG improve Substrate specificity

By lowering Glutamine degradation to 31%

Substrate specificity testResults& Discussion

SoAsn has low specificity than EcAsn(Tris pH8.6, 37℃, 30min)

SoAsn

EcAsn

0 10 20 30 40 50 60 70Glutamine degradation (%)

SoAsn

EcAsn

0 10 20 30 40 50 60 70Glutamine degradation (%)

Results & Discussion

-PEG-Asparaginase ; Increase stability

-Immobilization of the enzyme by polyacrylamide increased its stability todenaturation and proteolysis (Galaev, Chuplygina and Klement’Eva, 1981).

-Immobilization of L-asparaginase into a biocompatible poly ethylene glycol albumin hydrogel has been reported.

Enzyme modification Technol-ogy

Further study

Protein engineering of enzymepolyethylene can be improved with modifying reagent Methoxy-p-nitrophenyl carbamate of polyethylene glycol. Conjugation of oxidized inulin with L-as-paraginase affects pharmacokinetic and immunological properties of this anti-leukemic enzyme The conjugated enzymes had longer shelf life, higher thermostabil-ity, and more resistance to trypsin digestion, also the optimum pH range increased

The enzyme conjugated with oxidized inulin in 2:1 ratio showed de-creased antibody (IgG) titer and immunogenicity after repeated injection as compared to native enzyme.

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