Satoshi Koikeda · PDF fileSatoshi Koikeda 9th American Biotechnology Congress . 2 What we are Amano Enzyme Amano Enzyme USA China Amano Enzyme Europe Amano Enzyme Japan Amano Enzyme

www.amano-enzyme.co.jp

New concept of prebiotics :Prebiotic Enzyme Transglucosidase

Satoshi Koikeda

9th American Biotechnology Congress


2

What we are

Amano Enzyme

USA Amano Enzyme

China

Amano Enzyme Europe Amano Enzyme

Japan

Amano Enzyme

Mexico Amano Enzyme

Asia Pacific Office


3

World Markets for Enzymes (Estimated) 2015 Food and Industrial Use 4,490 Medical Use 2,430 (US＄ Million)

Research

Use

1,060

Digestive Enzyme (including Supplement)

220

Biotrans-

formation

230

Research &

Biotechnology

Enzyme

Replacement

Therapy

Thrombolysis

710

Other

Pharmaceutical

710

Total 7,980

Ｒｅｆｅｒｅｎｃｅ : World Enzymes (Freedonia, 2011)

Others 50

Fat & Oil 30

Protein 200

Brewing 270

Dairy 425

Flavor 70

Textile 120

Baking 625

Starch 300

Detergent

1,080

Fruit &

Juice 120

Waste

Treatment

25

Animal

Feed 545

Pulp

15

Leather

15

Bioethanol

600

Diagnostics

560


4

4

Jōkichi Takamine,

1854 – 1922

Water extraction

Powderization

Digestive aid enzyme has a long history

Aspergillus oryzae

Wheat bran

Produced by Parke-Davis (1895)

Invented by Jōkichi

Takamine (1894)

http://www.google.co.jp/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCL-WwK-wu8cCFQVvPgodcQcLMg&url=http%3A%2F%2Fproduto.mercadolivre.com.br%2FMLB-681189558-vidro-farmacutico-antigo-de-taka-diastase-_JM&ei=yrrXVb-JDYXe-QHxjqyQAw&psig=AFQjCNHXmPHEDWUVyM7MYCgAynbgUQmQfw&ust=1440287817181250

http://www.google.co.jp/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCN_t1q6zu8cCFUQ4PgodE8oB7g&url=http%3A%2F%2Faltover.trustpass.alibaba.com%2Fproduct%2F130845861-103508186%2FTakadiastase.html&ei=7b3XVZ_DMMTw-AGTlIfwDg&psig=AFQjCNGJgxuOOXrYXiO-AsSyvS0k8tyA6A&ust=1440288620699325


5

5

Expected function of digestive aid enzyme

ＥｎｚｙｍｅＥｎｚｙｍｅ

Regulating Nutrition Absorption Regulating Nutrition absorption

and Microbiota System

Novel digestive enzyme

Microbiota


6

Enzymatic reaction by transglucosidase

OH

CH2OH

OH O HO HO CH2

OH HO HO

OH

CH2OH

OH

HO OH

Glucose

Carbohydrate degradation

Transglucosidase reaction

CH2OH

OH O

HO HO CH2

OH

HO HO

CH2OH

OH

HO OH O

panose

CH2OH

OH O HO

HO CH2

OH HO HO

OH

O CH2

OH

HO HO

CH2OH

OH O HO HO

CH2OH

OH

HO OH

Maltose

Isomaltotriose

Transglucosidase is bifunctional enzyme

Isomaltose

Starch Alpha Glucosidase


7

Bifidobacteria ↑ Lactobacillus ↑ Bacteroides↑

Food Starch

Prebiotic enzyme concept on Transglucosiadase

Carbohydrate

Prebiotic Enzyme

(Transglucosidase)

Oligosaccharide

Slow digestion Energy absorption

Blood glucose↓

Microflora

Digestive Enzyme

Weight management


8

Transformed Oligosaccharides by Transglucosidase

Adjust pH 5.5

Add TG 90ku

37 ℃, up to 90 min

HPLC Analysis

Stir and Dissolution with amylase

0

5

10

15

20

25

0

5

10

15

20

25

0 30 60 90 min 0 30 60 90 min

Olig

osacch

ari

de (

g/m

eal)

Pasta (100g) Carbohydrate 70g

Noodle (100g) Carbohydrate 70g

In vitro analysis


9

Prevention of Postprandial Hyperglycemia and

Hyperinsulinemia in Healthy Volunteer

450

500

550

600

P < 0.05

P < 0.05

( n = 17 )

AU

C o

f B

lood S

uga

r （m

g h

/dL）

50

75

100

125

150

P = 0.099

( n = 17 )

0 mg 150 mg 300 mg

AU

C o

f B

lood Insulin

（μ

U h

/mL）

0 mg 150 mg 300 mg

Sasaki M et al. J. Clin. Biochem. Netr., 41, 191-196, 2007

• 522Kcal meal: Protein (14.4g), Fat (2.1g), Carbohydrate (111g)

+ Transglucosidase

• Blood sampling: every 30 min for 2 h


10

Efficacy in diabetes patients Trial Design:

•3 months’ randomized, double-blind, placebo-controlled trial

Inclusion criteria:

•Type 2 diabetes outpatient

•HbA1c is stable from 5.8% to 7.5% (JDS)

6.2% to 7.9% (NGSP)

•Stable dosage of medication for at least 1 month

•Stable diabetes condition for at least 3 months

(change in HbA1c less than 1.0%)

Exclusion criterion:

•Gut resection history

Sasaki M et al. J. Clin. Biochem. Netr., 41, 191-196, 2007


11

Study End Points

Presentation No.124

Primary outcome:

•The change in HbA1c level

Secondary outcomes:

•The change in various other metabolic parameters

Fasting blood glucose (FBG), Glycosylated albumin (GA),

Plasma insulin level, Total cholesterol (T-CHO),

High-density lipoprotein cholesterol (HDL-C),

Low-density lipoprotein cholesterol (LDL-C),

Triglyceride (TG), Free fatty acid (FFA),

High-molecular-weight (HMW) adiponectin

M. Sasaki et al. Diabetes, Obesity and Metabolism, 4, 379–382, 2012


12

Study flow diagram

Presentation No.124

Entrée (n=64)

Placebo

(n=16)

Transglucosidase,

300 mg/d (n=18)

Transglucosidase,

900 mg/d (n=17)

Withdrew from trial (n=3)

• Stopped medication (n=1)

• Deviation from protocol (n=2)

Placebo

(n=19)

Transglucosidase,

300 mg/d (n=23)

Transglucosidase,

900 mg/d (n=22)



•Left study because of

hospitalization (n=1)

•Lost to follow-up (n=1)






13

Primary outcome

Presentation No.124

Change of HbA1c

Calculated by using a two-way ANOVA and Benferroni post-hoc test

-0.1

0

0.1

0.2

0.3

0.4

0M 3M

Hb

A1

c L

evel, %

Placebo

Transglucosidase, 300 mg/d


-0.18% (p < 0.05)

-0.21% (p < 0.01)

***, p < 0.001 v.s 0M

***

Mean ±SE


14

Secondary outcomes (1)

Presentation No.124

Calculated by using a two-way

ANOVA and Benferroni post-hoc test

#, p < 0.05 v.s placebo

##, p < 0.01 v.s placebo

*, p < 0.05 v.s 0M

**, p < 0.01 v.s 0M

-15

-10

-5

0

5

10

15

0M 3M

FB

S L

ev

el,

mg

/dL

Fasting Blood Glucose

Pacebo



-16 # * Mean ±SE

-10

-5

0

5

10

15

0M 3M

LD

L-C

HO

Lev

el, m

g/d

L

LDL-Cholesterol

Placebo



*

-9 ##

-7 #

Mean ±SE

-0.5

0

0.5

1

1.5

0M 3M

Gly

co

alb

um

in L

ele

v,

%

Glycoalbumin

Placebo



**

-0.9 ## Mean ±SE

-2.8 #

-3.6 ##

*

-3

-2

-1

0

1

2

3

4

5

0M 3M

Insu

lin

Leb

el, m

U/m

L

Insulin

Pacebo



Mean ±SE

-30

-20

-10

0

10

20

30

0M 3M

TG

Lev

el, m

g/d

L

Triglyceride

Placebo



-21 # *

Mean ±SE


15

Secondary outcomes (2)

Presentation No.124

-6

-4

-2

0

2

4

6

8

10

0M 3MD

iasto

lic B

P L

ev

el, m

mH

g

Diastolic Blood Pressure

Placebo



*

-8 ## Mean ±SE

*

Mean ±SE -0.1

0

0.1

0.2

0.3

0.4

0Ｍ 3Ｍ

Bo

dy M

ass In

dex L

ev

el, K

g/m

2

BMI

Placebo



** +0.6 #

Mean ±SE -0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0M 3M

Ad

ipo

ne

cti

n,

Le

vel, m

g/m

L

Adiponectin

Placebo



*

Mean ±SE -2

0

2

4

6

0M 3M

AL

T L

ev

el, U

/L

ALT

Placebo



-5 #

Mean ±SE

*

-4

-2

0

2

4

6

8

0M 3M

GG

T L

ev

el,

U/L

GGT

Placebo



*

Mean ±SE -2

0

2

4

6

0M 3M

AS

T L

ev

el, U

/L

AST

Placebo




16

Ley, R.E.et al. Nature, 444, 2006 Sasaki M et al. BMC GastroenterologyJ, 41, 2013

Bacteroidete/Firmicutes Bacteroidete/Firmicutes

Placebo TG 300mg/day

TG 900mg/day

Change of gut microbiota composition by

transglucosidase administration

Weight loss


17

Possible action of prebiotic enzyme

Bacteroidete/Firmicutes↑

Energy ↓

Food starch

Oligosaccharide

Gut microbiota

Slow digest↓

BMI ↓

SCFA↑ Bifidobacteria ↑

Lactobacillus ↑

Transglucosidase

?

• LDL-Cholesterol ↓

• Triglyceride ↓

• ALT, AST, GGT ↓


18

Comparison between prebiotics and probiotics

Probiotics

Convenient intake

Safety of living

bacteria

Prebiotic Enzyme

Convenient intake

Natural diet

Prebiotics

Inconvenient intake

Unfamiliar food

ingredient

Safety in the case of

overdose


19

Conclusin

Presentation No.124

We proposed new enzyme concept: prebiotic enzyme.

Developing Transglucosidase as prebiotic enzyme is on going.

Clinical study resulted in lowering of HbA1c and blood insulin

level, and improvements in metabolic and cardiovascular risk

factors.

Protein engineering makes Transglucosidase more effiently.


20

Aichi Medical UniversitySchool of Medicine

Makoto Sasaki,

Naotaka Ogasawara,

Mari Mizuno,

Akihito Iida,

Kunio Kasugai,

Nagoya City University Graduate School of Medical Sciences

Takeshi Kamiya,

Hiromi Kataoka,

Takashi Joh

Acknowlegements


21

Thank you very much for your attention


22

Production of Transformed Oligosaccharides by

Transglucosidase

Presentation No.124

Corn Starch (DE 5～15)

Glucose (G1) 25%

Maltose (G2) 5%

Isomaltose (G2) 15%

Maltotriose (G3) 2%

Panose (G3) 5%

Isomaltotriose (G4) 8%

not specifiede (G4≤) 40%

58 ℃

pH 5.5

48 hours

With amylase

IMOs (～30％）

Protein

Engineering


23

Amano Enzyme Group History 1899 Pharmaceutical business started.

1948 Production of pharmaceutical enzyme (API) started.

1960 Enzyme R＆D Center inaugurated.

1961 Production of food-processing enzyme started.

1970 Production of diagnostic enzyme started.

1981 Amano International Enzyme Co., Ltd. started in Virginia.

It was transformed to current Amano Enzyme USA Co.,Ltd. in Elgin IL.

1983 European office opened in Frankfurt, Germany. Now Amano

Enzyme Europe Ltd. in Chipping Norton, OX, UK

2003 China office opened in Shanghai. Now Amano Enzyme China Ltd.

2012 Amano Enzyme Asia Pacific office opened in Kuala Lumpur, Malaysia.


24

Putative catalytic site

(D490, D660)

MOE program (Chemical Computing Group). The

corrected aglA sequence from GenBank

(accession number D45356) was aligned against

Maltase-glucoamylase (Protein Data Bank: 2QLY).

Maltose

Structuremodeling of Transglucosidase


25

min

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

V

0.00

0.01

0.02

0.03V

0.00

0.01

0.02

0.03

5.55

2

8556

90

7.04

1

8873

83

9.29

4

4600

32

9.84

6

5802

16

13.0

72

4705

92

15.0

25

2637

16.9

81

3917

8

17.4

50

1267

4

18.1

01

7845

4

19.5

36

3849

検出器 B343M343m

保持時間面積

min

6 8 10 12 14 16 18 20

V

0.00

0.01

0.02

0.03

0.04

V

0.00

0.01

0.02

0.03

0.04

5.54

7

9678

55

7.04

9

2753

43

7.58

9

7275

50

9.87

2

7273

82

10.6

05

2497

20

13.1

63

1443

38

14.2

74

1893

19

16.5

75

845

17.0

13

639

18.6

92

6156

18.9

87

5941

19.6

91

2066

検出器 BWildwild

保持時間面積

WT

W343M

α-1,4-linkages

Substrate：25%maltose

CH2OH

OH O

HO HO

CH2OH

OH

HO OH

OH

CH2OH

OH O

HO HO CH2

OH

HO HO

CH2OH

OH O

HO HO

CH2OH

OH

HO

CH2OH

OH

HO OH O

CH2OH

OH O

HO HO CH2

OH

HO HO

CH2OH

OH

HO OH O G

lucose

Malt

ose

Is

om

alt

ose

Panose

M

alt

oto

riose

N>>4

Isom

alt

trio

se

Glu

cose

Malt

ose

Is

om

alt

ose

Malt

oto

riose

N>>4

Isom

alt

trio

se Group1

Panose

Isomaltose （a-1,6）

Maltose （a-1,4）

Panose （a-1,4, a-1,6）

Maltotriose （a-1,4, a-1,4）

Group 1 mutant


26

min

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

V

0.00

0.02

0.04

0.06

0.08

V

0.00

0.02

0.04

0.06

0.08

5.226

2708

758

7.112

18776

57

10.0

54

82826

7

14.5

18

2163

04

RID10AWT ISOWT ISO

• ÛŽ �Ž žŠ Ô– Ê� Ï

min

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

V

0.00

0.01

0.02

0.03

0.04

0.05

0.06

V

0.00

0.01

0.02

0.03

0.04

0.05

0.06

5.22

3

1874

893

7.11

2

1892

512

10.0

56

1239

790

14.5

68

3143

68

RID10A495G ISO495G ISO

保持時間面積

Glu

cose

Malt

ose

Isom

alt

ose

Panose

M

alt

oto

riose

N>>4

Isom

alt

trio

se

WT

α-1,6-linkages

Group 2

OH

CH2OH

OH O

HO HO CH2

OH

HO HO

CH2OH

OH O HO

HO CH2

OH HO HO

OH

O CH2

OH

HO HO

Isomaltose （a-1,6） Isomaltotriose

（a-1,6, a-1,6） S495G

Glu

cose

Malt

ose

Isom

alt

ose

Panose

M

alt

oto

riose

N>>4

Isom

alt

trio

se

Substrate：25% isomaltose

Group 2 mutant


27

Transglucosidase (original)

Group 1 mutants

(W343X, S496X）

Group 2 mutants

(S495X)

Carbohydrase

Transglycosylation

a-1,4 a-1,6

a-glucosidase

(yeast)

Isomaltase

(Bacteria) a-glucosidase

(mammal)

Improving IMO production capability with

protein engineering

Documents

Satoshi Koikeda · PDF fileSatoshi Koikeda 9th American Biotechnology Congress . 2 What we are Amano Enzyme Amano Enzyme USA China Amano Enzyme Europe Amano Enzyme Japan Amano Enzyme