Technical University Braunschweig

German Research Centre forBiotechnology, Braunschweig

WP6, WP7

Screening isolates for enzymatic activities (WP6 and WP7)

Work objectives:to explore the diversity of DHABs:

to isolate, hyperexpress and characterize novel enzymesand other products (DL27 – M34; DL29 - M34, DL30 – M26, DL 31 – M34)

Two approaches: analysis of expression libraries andmicrobial isolates

Focus on enzymes from metagenomic expression libraries (WP7)

Metagenomic expression library in lambda phage

Lambda arms treated with phosphatase

Transduce, select for antibiotics resistans and score for white phages n X-Gal

DNA size-fractionated,partially digested with Sau3A

Ligation

Package in vitro

Library of dozens of thousands phage particles with 0-12 kbp inserts

The ZAP Express vector allows bouth eukaryoticand prokaryotic expression and accomodates DNA insert from 0 to 12 kb in length.

„Oil“ library = 1,8 x 106 phage particles. Average insert size - 7.5 kbp

Clones in the ZAP Express vector can be screend with either DNAprobes or antibody probes

Phage expression system

Screening hydrolases using a pH indicator method:

Insert cloned into the ZAP Express vector excised out of the phage in the form of the Km-resistant pBK-CMV phagemid vector

Screening of ca. 10000 phage clones yields ca. 20 positives

Excision

Selected clones clustered

500 600 700 800 900 1000 1100

20

40

60

80

100 518.3

569.2598.1

637.2

673.4711.3

775.3

826.0

867.0925.5

994.71035.4

Expression

MALDI-TOF

Purification

Sequencing of selected clones

Product, enzymology

From phage library to enzyme

Subcloned DNA fragments from positives

oil2<45% similarity,<30% identity

Plac

oil8Plac

<40% similarity,<30% identityoil7Plac

yafH (29 %)pp-kinase (65 %)

<45% similarity,<30% identity

1 kb

44 520 kDapI 10,88

32516 kDapI 10,25

32627 kDapI 9,26

O02bolAPlac

Rhodanese domain

putative para-nitrobenzyl/carboxyl esterase,Ca. 500 aa, < 35 % seq. similarity

O08Plac

COG0657, Aes, Esterase/lipase Ca. 180 aa 29% similarity

Put. esterase, ca. 130 aa <30% smlr.

Conserved hypoth. protein, proteobact. ca 70 % a.a. sim

Consvd. membrprot, 65 %pfam02517, Abi, CAAX amino terminal protease family

Conserved hyp. Ca. 150 aa, 60 % sim. Esterase motif, Low similarity

O09

Consvd. membr

prot, 65 %,

pfam02517, Abi, CAAX

Plac

COG0657, Aes, Esterase/lipase Ca. 280 aa low similarity

COG1514, LigT, 2'-5' RNA ligase < 50 %

COG0523, Putative GTPases (G3E family)

molGC 68 %

molGC 57 %

molGC 56 %

O04

Plac

COG0247, GlpC, Fe-S oxidoreductasePart of 980 aa, 50% simil.

Cholesteroloxidase pecursor

bolAConserved HypotheticalProtein ca. 70 %

molGC 59 %

New clones coding for hydrolytic enzymes

O12

PlacCOG0657, Aes, Esterase/lipase [Lipid metabolism], <50 % similarity

Enoyl CoAhydratase

Plac

COG0523, COG0523, Putative GTPases (G3E family)pfam02492, cobW, Cobalamin synthesis protein/P47K.

O14

Plac

VPS29-like phosphoesterase-related, 172aa long hypothetical protein [Pyrococcus horikoshii] <30 %

O16Hypothetical,Low homology

No ORFs

COG1514, LigT, 2'-5' RNA ligase 58 %

Plac

Putative membrane proteinLow homology <30 % O21

Hydroxyacyl dehydrogenase

molGC 33 %

molGC 50 %

Cons. hypotheticalCons. hypothetical, low homology

molGC 57 %

New clones coding for hydrolytic enzymes (cont‘d)

Enzyme purification:

Purification: Cationic exchange on MonoSHydrophobic interaction (Phenylsuperose)Gel filtration (Superose 12)

Native gel electrophoresis,development with -naphtylbutyrate

Enzyme reaction products

TG

1,3-DG1(3), 2-DG

MG

O

O

O

O

O

O

oil2 oil8oil7

Features of the enzymes

Temperature (°C)

20 40 60 80

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tivity

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Temperature (°C)

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Temperature-dependent activity

Oil 2 Oil 7Oil 8

Temperature optima

Time (min)

0 30 60 90 120 150 180

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OIL 2 OIL 7 OIL 8

30 °C

40 °C

50 °C

THERMOSTABILITY Thermostability

µm

ol/m

in/µ

g pr

otei

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C2 C3 C4 C6 C8 C12C14C16

OIL 2 OIL 7

OIL 8 O.4

O.5 O.8

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Carbon atoms

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C2 C3 C4 C6 C8 C12C14C16

Carbon atoms

O.9 O.10

Specific activities with p-nitrophenol derivatives

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C2 C3 C4 C6 C8 C12C14C16

Carbon atoms

O.11

O.13

O.16

O.23

O.12

O.14

O.21

O.2

Specific activities with p-nitrophenol derivatives

BIOCHEMICAL PROPERTIESpH

optimum

Temperature

Optimum (°C)

Na+/K+ optimum Stable at

pH1

Stable at

Temperature (°C)2

O.4 8.0 60 25-75 mM 10.5 (95%) 60

O.5 9.0-9.5 40 Inhibited at > 25 mM 10 (95%) 46

O.8 8.0 60 25-75 mM 10 (95%) 60

O.9 8.5-9.0 50 Inhibited at > 25 mM 10 (95%) 54

O.10 8.0 50 1.5-2.0 M 10 (60%) 58

O.11 9.0 50 1.0-1.5 M 10 (70%) 56

O.12 8.0 60 25-75 mM 10 (30%) 60

O.13 8.0 40 25-75 mM 10 (40%) 45

O.14 8.0 40 Inhibited at > 25 mM 10 (60%) 45

O.16 9.0 50 2.0-4.0 M 10 (80%) 59

O.21 8.5 60 3.5 M 10 (54%) 59

O.23 8.0 50 25-75 mM 10 (80%) 53

Oil2 8.5 40 3.5 M 10 (20%) 48

Oil7 8.5 50 0.8 M 10 (80%) 56

Oil8 8.0 50 4.0 M 10 (25%) 49

O.2 8.0 60 Inhibited at > 25 mM 10 (30%) 671 In pharenthesis the remaining activity after 24 h incubation at the indicate pH2 Half life of the enzyme more than 3 h

EFFECT OF ORGANIC SOLVENT ON STABILITY OF ENZYMESAdditive Porcentage Activity (%)

% O.2 O.4 O.8 O.12 O.13 O.14 O.16 Oil21 O.23

None 100 100 100 100 100 100 100 100 100n-propanol 10 117.9 86.2 87.1 99.5 60.1 66.0 73.6 92.9 15.8

30 171.6 97.2 6.6 13.9 120.2 7.8 31.0 44.2 6.0Ethanol 10 223.8 100.8 35.3 47.3 39.8 22.5 98.0 29.8 4.8

30 171.3 100.8 66.9 34.9 54.5 54.0 55.2 87.3 81.8t-amylalcohol 10 149.5 99.6 28.2 32.6 50.7 23.5 83.3 139.4 42.6

30 152.3 99.4 171.3 181.8 235.2 195.9 71.0 134.2 123.6Acetonitrile 10 115.1 100.0 180.2 171.5 211.2 237.4 86.6 99.1 123.4

30 47.3 101.2 13.7 14.5 90.5 28.8 8.6 59.4 70.7DMSO 10 119.3 99.4 113.7 86.4 163.8 181.6 71.0 155.4 147.0

30 101.7 99.9 28.4 20.7 62.0 76.2 46.5 27.5 44.2

Additive Porcentage Activity (%)% O.5 O.9 O.10 O.11 Oil2 Oil7 Oil8

None 100 100 100 100 100 100n-propanol 10 92.9 93.0 78.3 75 70 72

30 44.2 93.4 96.7 74 68 72Ethanol 10 29.8 93.7 168.8 131 84 101

30 87.3 98.5 37.9 83 83 133t-amylalcohol 10 139.4 97.1 204.8 66 68 65

30 134.2 98.2 46.5 26 36 21Acetonitrile 10 99.1 98.5 128.7 98 76 92

30 59.4 93.9 46.7 168 67 124DMSO 10 155.4 95.0 96.0 59 89 96

30 27.5 98.7 41.2 54 99 99

2 mL Iso-octane20 mg E. coli esterase clones

ENANTIOMERIC RESOLUTION OF 1-PHENYLETHANOL BY TRANSESTERIFICATION WITH VINYL ACETATE

+

1 M 1-phenylethanol 1 M Vinylacetate

RESOLUTION OF 1-PHENYLETHANOL

Substrate 1-phenylethanol

Esterase % c % e.e. E Stereo-preference

SK2-71 36.2 33.7 2.4 S

Oil71 37.8 37.4 2.7 S

Oil81 42.2 78.4 14.7 R

O.22 26.2 98 42.8 R

O.42 30.8 98 116.2 R

O.82 8.71 2.5 3.8 S

O.122 4.33 2.12 1.1 R

O.132 3.4 2.38 1.1 R

O.142 7.71 8.64 1.20 R

O.162 10.06 39.36 2.40 S

O.212 39.3 88.6 29.7 S

O.232 29.0 74.46 9.2 R

1 Reaction time 24 hours2 Reaction time 7 hours

E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio)

e.e. > 70 INDUSTRIAL POTENTIAL (Enantiomeric excess)

HYDROLYTIC ACTIVITIES AND ESTIMATED ENANTIOSELECTIVITIES TOWARDS CHIRAL ESTER LIBRARY: primary or secondary alcohols

Esterase Hydrolytic activity

Units/g (Interval)1

E (Stereo-preference)

Solketal

butyrate

2-methyl-

glycidyl

1-phenylethyl

butyrate

Menthyl

acetate

Oil7 5445.6 – 7904.0 2.9 (R) 4.4 (S) 2.7 (S) 26.7 (S)

Oil8 4424.8 – 7782.0 9.0 (R) 3.9 (R) 14.7 (R) 29.6 (R)

O.22 268.0 – 747.0 14.4 (R) 9.8 (R) 42.8 (R) 27.6 (R)

O.4 8987.6 – 12209 9.3 (R) 9.6 (R) 116.2 (R) 39.9 (R)

O.8 5972.1 – 7002 2.9 (S) 2.9 (S) 3.8 (S) 23.9 (S)

O.12 7966.0 – 12622 1.3 (R) 3.5 (R) 1.1 (R) 21.7 (R)

O.13 3950.0 – 5585 1.1 (R) 2.2 (R) 1.1 (R) 21.0 (R)

O.14 4497.9 – 7782 1.7 (R) 1.4 (R) 1.20 (R) 27.9 (R)

O.16 864.1 – 992.1 1.0 (S) 2.7 (S) 2.40 (S) 39.9 (S)

O.21 13102.1 – 18577.0 8.6(S) 25.5 (S) 29.7 (S) 39.5 (S)

O.23 8885.0 – 15613 5.6 (R) 39.8 (R) 9.2 (S) 21.5 (R) 1 Reaction time 15 seconds: estimation for the average hydrolysis rate for all compounds tested E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio) SUMMARY:

i) Oil7, O.8, O.12, O.13, O.14 and O.16 are more specific for aromatic

chiral compounds.

ii) Oil8, O.2, O.4, O.21 and O.23 highly potent for industrial

resolutions, of both aromatic or non-aromatic compounds.

HYDROLYTIC ACTIVITIES AND ESTIMATED ENANTIOSELECTIVITIES TOWARDS CHIRAL ESTER LIBRARY: esters of chiral carboxylic acids (stereocenter alpha to carbonyl)

Esterase Hydrolytic activity

Units/g (Interval)1

E (Stereo-preference)

Methy 3-hydroxy-2-

methylpropionate

Alanine

methyl ester

Tryptophan methyl

ester

Methyl lactate N-benzyl ethyl

ester

Oil7 6806 – 7700 n.d n.d n.d 9.9 (S) 9.4 (S)

Oil8 545 – 625 10.1 (S) 28.0 (R) 7.5 (R) 3.7 (S) 5.8 (S)

O.2 5530 – 3900 2.9 (R) 7.6 (S) 3.7 (R) 9.6 (S) 3.7 (R)

O.4 < 500 6.9 (S) 2.7 (R) 39.8 (R) 3.9 (R) 4.9 (R)

O.8 < 500 n.d n.d n.d 8.9 (S) 6.7 (S)

O.12 < 500 n.d n.d n.d 6.2 (R) 9.6 (R)

O.13 < 500 n.d n.d n.d 5.2 (R) 3.8 (R)

O.14 885 – 982 n.d n.d n.d 28.6 (R) 27.4 (R)

O.16 6506 – 9961 n.d n.d n.d 39.5 (R) 59.6 (S)

O.21 18109 – 18343 9.9(S) 8.8 (S) 4.6 (S) 19.3 (S) 21.5 (R)

O.23 4220 - 5060 9.3 (R) 29.8 (R) 20.0 (S) 2.6 (S) 2.8 (R) 1 Reaction time 15 seconds: estimation for the average hydrolysis rate for all compounds tested E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio) SUMMARY: i) O.14 and O.16 useful for resolution of aromatic compounds containing chiral carboxylic acid with a stereocenter

to carbonyl. ii) O.4, Oil8, O.21 and O.23 potentially useful for industrial resolutions of chiral carboxylic acid with a stereocenter

to carbonyl

HYDROLYTIC ACTIVITIES AND ESTIMATED ENANTIOSELECTIVITIES TOWARDS CHIRAL ESTER LIBRARY: esters of chiral carboxylic acids (stereocenter to carbonyl) and lactones

Esterase Hydrolytic activity

Units/g (Interval)1

E (Stereo-preference)

Methyl 3-

hydroxybutyrate

pantolactone Dihydro-5-hydroxymethyl-

2(3H)-furanone

Oil7 150 n.d n.d n.d.

Oil8 730 13.0 (S) 15.0 (R) 64 (R)

O.2 1200 1.3 (R) 11.0 (R) n.d.

O.4 450 20.0 (S) n.d. n.d.

O.8 52 n.d. n.d. n.d.

O.12 30 n.d. n.d. n.d.

O.13 40 n.d. n.d. n.d.

O.14 28 n.d. n.d. n.d.

O.16 25 n.d. n.d. n.d.

O.21 580 6.5 (S) 8.3 (S) 28.0 (S)

O.23 1800 3.1 (R) 4.4 (S) 4.0 (S) 1 Reaction time 15 seconds: estimation for the average hydrolysis rate for all compounds tested

E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio) SUMMARY: i) 4 lactone hydrolases: potentially good for industrial resolutions of lactones

strain identification homology,% basin protease phosphatase esterase lipase glucosidase

3A Alteromonas marina 98 A 3 37A Alteromonas macleodii 99 A 3 38A Pseudoalteromonas sp. 92 A 3

14A no identification A 1 117A Alteromonas macleodii 99 A 318A Slope str., DIIII I c 97 A 2 3

1B Marinobacter hydrocarbonoclasticus 97 B 2 33B Marinobacter hydrocarbonoclasticus 100 B 3 2 3 34B Idiomarina baltica 99 B 3 3

5B bis Marinobacter hydrocarbonoclasticus 100 B 3 36B no identification B 212B Bacillus licheniformis 98 B 217B Rhodospirilaceae bact 97 B 319B no identification B 3

2D Alteromonas macleodii 99 D 34D Alteromonas macleodii 99 D 25D Alteromonas macleodii 99 D 2 3

Screening of hydrolytic activity: Conisma strains (the others to come)

Substrate: -naphtylacetate, butyrate, laurate, palmitate, phosphate, glucoside, galactoside and Fast Blue RR

Collection of hydrolytic enzymes from expression libraries obtained after oil enrichment, have been characterised

they exhibit novel structures (low homology to the homologs), have a good potential for industrial applications and “tell the stories” about the environment and organisms they were derived from

Conclusions and outlook

Screening/characterisation of enzymatic activities from the isolates will be continued

Screening of biosurfactant producing isolates started for further characterization of novel structures

analysed

DL29- M34 20 % “Structures of novel surfactants etc – (screening stage)”DL30-M26 – 100 % “Clones, hyperexpression clones etc.”DL31-M34 – 100 % * “ Data sets of activities of obtained compounds”* all selected items characterized

Progress estimates:

Best regards from Peter, Manolo and Ken

Publications???