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Figure legends

Figure 1. Screening results of hydrolysis activity of bacterial CFE against Nma-His-Pro-Lys(Dnp).

12

360

NBRC 3022 (Bacillus subtilis) and NBRC 13719 (Bacillus amyloliquefasciens) are positive controls. Each

361

ΔRFU·sec-1 during 4500 s and 15300 s was listed below. 200527-1, 0.021; 200527-5, 0.022; 200527-14, 0.054;

362

200527-13, 0.21; 191211-2, 0.038; NBRC 3022, 0.026; NBRC 13719, trace.

363

Figure 2. Enzyme purification from the CFE of ther strain 200527-13. SDS-PAGE analysis of the each

364

fraction obtained by Superdex 200 Increase 10/300 GL column chromatography (A) and the hydrolysis activity

365

of the each fraction toward the fluorogenic substrate (B).

366

Figure 3. Sequence analysis of EntCP. (A) Sequence alignment of EntCP and selected sequences of the M32-

367

CAP family proteins retrieved from the PDB database. The conserved motif HEXXH is in solid line square, while

368

others are in dashed line square. (B) Phylogenetic tree analysis by the maximum likelihood method. B38-CAP,

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BsuCP, PfuCP, FisCP, DnrCP, and TthCP are carboxypeptidase from Paenibacillus sp. B38, Bacillus subtilis,

370

Pyrococcus furiosus, Fervidobacterium islandicum, Deinococcus radiodurans, and Thermus thermophilus,

371

respectively. DsbA, disulfide oxidoreductase from Vibrio cholerae, serves as an outgroup.

372

Figure 4. Expression, purification, and characterization of the recombinant EntCP. (A) SDS-PAGE results

373

of CFE of E. coli harboring empty plasmid or plasmid with EntCP gene. The recombinant EntCP is indicated by

374

the arrows). (B) SDS-PAGE results of each fraction obtained by HisTrapTM column chromatography. (C) Ang II

375

cleavage product of human ACE2 and purified recombinant EntCP detected by positive mode of the LC-MS

376

system. Monoisotopic molecular weights of Ang II, Ang 1-7, and phenylalanine is 1045.5, 898.5, and 165.1,

377

respectively.

378

379

Figure 5. ACE2 and EntCP catalyzing reactions . (A) Nma-His-Pro-Lys(Dnp) or (B) Ang II as the substrate.

13

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Table 1. Quantification of specific activity during protein purification

Step

Vol Total protein

(ml)

Cell free extracts

HiPrep Q XL 16/10

Superdex 200 Increase 10/300 GL

100

(mg)

110

Specific activity

Purification fold

(RFU·sec−1·L·mg−1 protein)

(%)

3300

2.5

10

6.2

8200

0.38

79000

Yield

24

100

14

1.7

Supplementary data

Intensity [µV]

Ang II + Buffer

ACE2 + Ang II

CFE NBRC 3022 + Buffer

CFE NBRC 3022 + Ang II

CFE NBRC 13719 + Buffer

CFE NBRC 13719 + Ang II

Retention time [min]

Intensity [µV]

Ang II + Buffer

ACE2 + Ang II

CFE 200527-13 + Buffer

CFE 200527-13 + Ang II

Retention time [min]

Figure S1. HPLC analysis of Angiotensin II (Ang II) hydrolysis products. (A) by three different positive

control samples, (B) by the CFE of the strain 200527-13. Control samples of the CFE from NBRC 3022:

Bacillus subtilis and NBRC 13719: Bacillus amyloliquefasciens. Retention times of Ang II and Ang1-7 were

21.0 min (20.5 min (B)) and 12 min (10.0 min (B)), respectively, in the results of ‘Ang II + Buffer’ and ‘ACE2 +

10

Ang II’.

11

12

100000

95000

1: 1046. 50( +)

1: 899. 50( +)

1: 166. 10( +)

90000

85000

80000

75000

70000

65000

60000

55000

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

100000

95000

10

20

30

40

50

60

70

80

90

100

110

min

10

20

30

40

50

60

70

80

90

100

110

min

10

20

30

40

50

60

70

80

90

100

110

min

1: 1046. 50( +)

1: 899. 50( +)

1: 166. 10( +)

90000

85000

80000

75000

70000

65000

60000

55000

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

100000

95000

1: 1046. 50( +)

1: 899. 50( +)

1: 166. 10( +)

90000

85000

80000

75000

70000

65000

60000

55000

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

100000

95000

( )

1: 1046. 50( +)

1: 899. 50( +)

1: 166. 10( +)

CFE 200527-13 + Buffer

90000

85000

80000

75000

70000

65000

60000

55000

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

13

10

20

30

40

50

60

70

Retention time [min]

80

90

100

110

min

1046.5 (+)

899.5 (+)

166.1 (+)

14

Figure S2. LC-MS analysis of Angiotensin II (Ang II) hydrolysis products. (A) by human ACE2, (B) by the

15

CFE of Bacillus subtilis NBRC 3022, and (C) by the CFE of the strain 200527-13. The analysis of the CFE of the

16

strain 200527-13 without Ang II (D) indicated endogenous phenylalanine in the CFE of the strain 200527-13.

17

...