Part A | 8.6
238 Part A Marine Flora and Fauna
ZP 01036720
- - - F A L D F G E L A P Q V I A L D G Q - P - V T P H A P Q D A R G W V V I G P A M R V D L M L D M T G A P G E T V T
[300]
ZP 00964414
- - - F A L D F G D H T P R I I A L D G Q - P - V T P H N P S D - - G L V V L G P A M R V D V I L D C N G E P S A R S Q
[300]
ZP 00961617
- - - F G L D F G D F E P V V V A L D G Q - P - V T P H A P D G - - G V V V I G P A M R V D L A I E M T G K P G E A V T
[300]
YP 002289160
- - - F G L T F E G H A P T I V A M D G H - P - V T P H A P E K - - G Q V V I G P A M R V D L V I D M T A A P G S R F K
[300]
ADV52199
- - - F A L E F Q D H R P M V I A L D G Q - P - V E P H E P A G - - G R V V L G P A M R A D L A F D M T G R A G D G S T
[300]
YP 004619745
- - - F S L G F D G P A P W V V A L D G Q - P - V A P H A P P G - - G R V R L A P G M R C D L V L D A G Q Q A G Q R H A
[300]
ZP 02186341
- - - F A L E F E G H Q P Q I V A L D G Q - P - V A P H A P S G - - G R V M L G P A M R A D L I I D M T G A P G A R F R
[300]
ADM87301
- - - F P V E L E G V E G K V V A L D G M - P I V D P Q E F S G - - - - L I L A P A Q R A D I I A D V - - - - - - - - -
[300]
SNB11
- - - F A L T F Q D L N P W I I A L D G H - P - V E P K S V Q N - - D R I V L G A G Q R T D L I V D V T G K P G E I S S
[300]
SNB12
F S N F P P N L S G L D P W I I A L W T P G R - T E V C S E R S - - N C A R S W T A H R S D C R C D R Q A R D F V R S G
[300]
ZP 01036720
V I D R F F S - D R N Y E L V D L T Y - - S E T P - L R D A - - - - P P N W E M A L A P N P L P E P D I G Q A R R H E V
[360]
ZP 00964414
L V D R A Y R - G N D F R V L D F T Y - - A E T A - L R E A - - - - T P D W P I A L P A N P I P E P D L S S A Q R H E V
[360]
ZP 00961617
V T D V F Y E - G L E Y R L V D L V Y - - G P D R - L R D S - - - - V P D W S M D L P P N P L A E P D M A S A A R H Q I
[360]
YP 002289160
V M D S F Y Q - Q Q E F R L F H M V Y - - A D K P - L R A E - - - - P R T D A I A L P H N P V P E P D L A T A Q R H D V
[360]
ADV52199
V T D P F Y R - G L E Y R L L D L V Y - - E D A P S L R E H - - - - P L D A P M R L A A N T M P E P D L A R A E R H E I
[360]
YP 004619745
V F D D F Y P - R G A F D L L Q R V V - - E G Q P - L R A Q - - - - P L T P P A A L P G N P L A E P D L G A A Q T H E V
[360]
ZP 02186341
V I D G F Y R - G R E Y R L H D V V Y - - T D I P - L R A A - - - - P P D T A I E L P A N P L A E P D V A D A Q R H A V
[360]
ADM87301
I T D A P I - - G F V F P T R D G P Y L L G E I P - V K G A N T T R Q P S E I P A L P P N E V T S P D M G S A V S L T L
[360]
SNB11
V V D S A L G I D F A Y E L M E T R L V R N S D S S L P L H - - - - S R S A P L A L L P N P V A R P D I A N A E R H Q I
[360]
SNB12
Q C P - - L E L T L L T N L C E M E T V R N F D S S L P L H - - - - S R S A P L A L L P N P V A R P D I A N A E R H Q I
[360]
ZP 01036720
I F T G G M M - - G G M V E R Q M G L S G R G G M M G G G M M N R M H D G - G I W F V N G V A A E G - H - - - - - - - -
[420]
ZP 00964414
T F T G G M M - - G M M V E R E M G L E R R G G M M G G G M M G M M H D G R G I W F V N G K A A E G - H - - - - - - - -
[420]
ZP 00961617
V F N G G M M - - G Q M M M - - - - - - - - G G D M G S M M D Q M R E G - - N M W F I N G E A A T G - H - - - - - - - -
[420]
YP 002289160
V L G G G M M - - G Q M A S A Q V - - - - - G G E T M D I R T M L R H G - - L I W A I N G V A S T E - H - - - - - - - -
[420]
ADV52199
A F S G G M M - - G G M M G D G M M G G M E P G M M G G M M R G M Q H S - - G T W A I N G V S A T G - D - - - - - - - -
[420]
YP 004619745
V F Q G G M M - - G S L H R A L L - - - - - D G Q P L P M M G L L R Q G - - K A W A V N G V V S G G - H G E G G A H G R
[420]
ZP 02186341
S F G G G M M - - G G M M G G G M - - - - - G G M M R R M M P G M Q H G - - G I W N I N G V S A T G - H - - - - - - - -
[420]
ADM87301
T M E G G A M - - - - - - - - - - - - - - - - - - S R R M M Q G M M G G - - D I W A F N G Q S G L - - - - - - - - - - -
[420]
SNB11
V F E G G A M E T G G M E T S G A Y L - - - N G E F K T M E T R N L A E L G K L W A I N G K I P G N V H - - - - - - - -
[420]
SNB12
V F E G G A M E T G G M E T S G A Y L - - - N G E F K T M E T R N L A E L G K L W A I N G K I P G N V H - - - - - - - -
[420]
ZP 01036720
I L D P M L V L A R D A S H V I A M T N A T A F H H P I H L H G H S F R V I N R N G V P T A H H E W Q D T V L M A P R E
[480]
ZP 00964414
V L D P F L T L E R G A S H V L A M T N A T A F H H P I H F H G H S F R V V K R D G V N T A H Q E W Q D T V L M A P R E
[480]
ZP 00961617
M M D P L L V L P Q G T S H V F E M D N R T A W H H P I H F H G H S F R V I A R N G Q P T Q Y R E W Q D T V L M A P E E
[480]
YP 002289160
V H E P M A T L K R G T S H I L A M E N E T A W W H P M H L H G H A F R V L S R N G V P T R H R E W R D T V L V A P R E
[480]
ADV52199
I M E P F L T L Q R G R S Y V L A M H N H T A W H H P M H L H G H V F R V I A R D G R P T R Y Q E W Q D T V L M A P H E
[480]
YP 004619745
H E A P I V T L R R G R S Y V L A L H N D T R W H H P I H L H G H S F R V L R R N G R E T A H R E W R D T V L L D P G E
[480]
ZP 02186341
V M D P M L T L A Q G R S Y L L A L T N D T A F H H P I H L H G H S F R V L S R N G V P T R R R E W Q D T V L M D P R E
[480]
ADM87301
T D T P L H S F E R G Q T A R I R L V N D T R F P H G I H L H G H H F F E V G A D G - - - N L G A L R D T T L V D A G E
[480]
SNB11
T E P P L L T L E L G K S Y I L E L I N R T A F E H P I H L H G H S F Q V I R N N D R Q L N N P P I R D T V L L Q P G E
[480]
SNB12
T E P P L L T L E L G K S Y I L E L I N R P A F E H P I H L H G H S F Q V I R N N D R Q L N N P P I R D T V L L Q P G E
[480]
ZP 01036720
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A G G M M G V I R V A - - - - - - [539]
ZP 00964414
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R V A - - - - - - [539]
ZP 00961617
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R V D P G T T K T [539]
YP 002289160
R V E V A F V A D N P G D W M - - F H C H V L E H Q - - - - - - - - - - - - - - - - A G G M M G T V R V I - - - - - - [539]
ADV52199
R V E I A F V A D N P G D W M - - F H C H L L E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [539]
YP 004619745
R A D I A F V A D N P G D W M - - L H C H V L E H Q - - - - - - - - - - - - - - - - E G G M M G V F R V A - - - - - - [539]
ZP 02186341
R V D I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R I A P E T S R T [539]
ADM87301
T R D I V C V F D N P G N W L - - L H C H M L G H Q - - - - - - - - - - - - - - - - A A G M K T W V E V A - - - - - - [539]
SNB11
N K E I A F V A D N P G Q W M E T F H C H I L D Q I I R P Q A S R A A S S R P H V W G P E L R G R F Q - - - - - - - - [539]
SNB12
N K E I A F V A D N P G Q W M E T F H C H I L D Q I I R P Q A S R A A S S R P H V W G P E L A A A V S G V - - - - - - [539]
Fig. 8.8 Partial amino acid alignment of bacterial laccase genes including gene sequences derived from metagenomic studies
(ADV52199 – drained peat soil metagenome, ADM87301: South China Sea marine microbial metagenome, SNB11 and SNB12:
marine sponge metagenome). Histidine-rich copper binding domains are indicated by vertical boxes. ZP 01036720: multicopper oxidase domain protein Roseovarius sp. 217, ZP 00964414: multicopper oxidase domain protein Sulfitobacter sp. NAS14.1,
ZP00961617: multicopper oxidase domain protein Roseovarius nubinhibens, YP 002289160: CumA Oligotropha carboxidovorans OM5, ADV52199: laccase uncultured bacterium, YP 004619745 cueO Ramlibacter tatouinensis TTB310, ZP 02186341:
alpha proteobacterium BAL 199, ADM87301: laccase uncultured bacterium, SNB11 and SNB12: sponge metagenome-derived
sequences
using either hybridization probes or PCR primers for
these genes. This involves the design of DNA probes or
PCR primers typically derived from conserved regions
of already well-characterized genes or protein families.
Using the homology-based approach, PCR amplification is performed, following which the putative gene
is sequenced and then subsequently cloned into an appropriate expression vector where the heterologously
expressed protein is then purified and biochemically
characterized to ascertain its function. While such ap-
238 Part A Marine Flora and Fauna
ZP 01036720
- - - F A L D F G E L A P Q V I A L D G Q - P - V T P H A P Q D A R G W V V I G P A M R V D L M L D M T G A P G E T V T
[300]
ZP 00964414
- - - F A L D F G D H T P R I I A L D G Q - P - V T P H N P S D - - G L V V L G P A M R V D V I L D C N G E P S A R S Q
[300]
ZP 00961617
- - - F G L D F G D F E P V V V A L D G Q - P - V T P H A P D G - - G V V V I G P A M R V D L A I E M T G K P G E A V T
[300]
YP 002289160
- - - F G L T F E G H A P T I V A M D G H - P - V T P H A P E K - - G Q V V I G P A M R V D L V I D M T A A P G S R F K
[300]
ADV52199
- - - F A L E F Q D H R P M V I A L D G Q - P - V E P H E P A G - - G R V V L G P A M R A D L A F D M T G R A G D G S T
[300]
YP 004619745
- - - F S L G F D G P A P W V V A L D G Q - P - V A P H A P P G - - G R V R L A P G M R C D L V L D A G Q Q A G Q R H A
[300]
ZP 02186341
- - - F A L E F E G H Q P Q I V A L D G Q - P - V A P H A P S G - - G R V M L G P A M R A D L I I D M T G A P G A R F R
[300]
ADM87301
- - - F P V E L E G V E G K V V A L D G M - P I V D P Q E F S G - - - - L I L A P A Q R A D I I A D V - - - - - - - - -
[300]
SNB11
- - - F A L T F Q D L N P W I I A L D G H - P - V E P K S V Q N - - D R I V L G A G Q R T D L I V D V T G K P G E I S S
[300]
SNB12
F S N F P P N L S G L D P W I I A L W T P G R - T E V C S E R S - - N C A R S W T A H R S D C R C D R Q A R D F V R S G
[300]
ZP 01036720
V I D R F F S - D R N Y E L V D L T Y - - S E T P - L R D A - - - - P P N W E M A L A P N P L P E P D I G Q A R R H E V
[360]
ZP 00964414
L V D R A Y R - G N D F R V L D F T Y - - A E T A - L R E A - - - - T P D W P I A L P A N P I P E P D L S S A Q R H E V
[360]
ZP 00961617
V T D V F Y E - G L E Y R L V D L V Y - - G P D R - L R D S - - - - V P D W S M D L P P N P L A E P D M A S A A R H Q I
[360]
YP 002289160
V M D S F Y Q - Q Q E F R L F H M V Y - - A D K P - L R A E - - - - P R T D A I A L P H N P V P E P D L A T A Q R H D V
[360]
ADV52199
V T D P F Y R - G L E Y R L L D L V Y - - E D A P S L R E H - - - - P L D A P M R L A A N T M P E P D L A R A E R H E I
[360]
YP 004619745
V F D D F Y P - R G A F D L L Q R V V - - E G Q P - L R A Q - - - - P L T P P A A L P G N P L A E P D L G A A Q T H E V
[360]
ZP 02186341
V I D G F Y R - G R E Y R L H D V V Y - - T D I P - L R A A - - - - P P D T A I E L P A N P L A E P D V A D A Q R H A V
[360]
ADM87301
I T D A P I - - G F V F P T R D G P Y L L G E I P - V K G A N T T R Q P S E I P A L P P N E V T S P D M G S A V S L T L
[360]
SNB11
V V D S A L G I D F A Y E L M E T R L V R N S D S S L P L H - - - - S R S A P L A L L P N P V A R P D I A N A E R H Q I
[360]
SNB12
Q C P - - L E L T L L T N L C E M E T V R N F D S S L P L H - - - - S R S A P L A L L P N P V A R P D I A N A E R H Q I
[360]
ZP 01036720
I F T G G M M - - G G M V E R Q M G L S G R G G M M G G G M M N R M H D G - G I W F V N G V A A E G - H - - - - - - - -
[420]
ZP 00964414
T F T G G M M - - G M M V E R E M G L E R R G G M M G G G M M G M M H D G R G I W F V N G K A A E G - H - - - - - - - -
[420]
ZP 00961617
V F N G G M M - - G Q M M M - - - - - - - - G G D M G S M M D Q M R E G - - N M W F I N G E A A T G - H - - - - - - - -
[420]
YP 002289160
V L G G G M M - - G Q M A S A Q V - - - - - G G E T M D I R T M L R H G - - L I W A I N G V A S T E - H - - - - - - - -
[420]
ADV52199
A F S G G M M - - G G M M G D G M M G G M E P G M M G G M M R G M Q H S - - G T W A I N G V S A T G - D - - - - - - - -
[420]
YP 004619745
V F Q G G M M - - G S L H R A L L - - - - - D G Q P L P M M G L L R Q G - - K A W A V N G V V S G G - H G E G G A H G R
[420]
ZP 02186341
S F G G G M M - - G G M M G G G M - - - - - G G M M R R M M P G M Q H G - - G I W N I N G V S A T G - H - - - - - - - -
[420]
ADM87301
T M E G G A M - - - - - - - - - - - - - - - - - - S R R M M Q G M M G G - - D I W A F N G Q S G L - - - - - - - - - - -
[420]
SNB11
V F E G G A M E T G G M E T S G A Y L - - - N G E F K T M E T R N L A E L G K L W A I N G K I P G N V H - - - - - - - -
[420]
SNB12
V F E G G A M E T G G M E T S G A Y L - - - N G E F K T M E T R N L A E L G K L W A I N G K I P G N V H - - - - - - - -
[420]
ZP 01036720
I L D P M L V L A R D A S H V I A M T N A T A F H H P I H L H G H S F R V I N R N G V P T A H H E W Q D T V L M A P R E
[480]
ZP 00964414
V L D P F L T L E R G A S H V L A M T N A T A F H H P I H F H G H S F R V V K R D G V N T A H Q E W Q D T V L M A P R E
[480]
ZP 00961617
M M D P L L V L P Q G T S H V F E M D N R T A W H H P I H F H G H S F R V I A R N G Q P T Q Y R E W Q D T V L M A P E E
[480]
YP 002289160
V H E P M A T L K R G T S H I L A M E N E T A W W H P M H L H G H A F R V L S R N G V P T R H R E W R D T V L V A P R E
[480]
ADV52199
I M E P F L T L Q R G R S Y V L A M H N H T A W H H P M H L H G H V F R V I A R D G R P T R Y Q E W Q D T V L M A P H E
[480]
YP 004619745
H E A P I V T L R R G R S Y V L A L H N D T R W H H P I H L H G H S F R V L R R N G R E T A H R E W R D T V L L D P G E
[480]
ZP 02186341
V M D P M L T L A Q G R S Y L L A L T N D T A F H H P I H L H G H S F R V L S R N G V P T R R R E W Q D T V L M D P R E
[480]
ADM87301
T D T P L H S F E R G Q T A R I R L V N D T R F P H G I H L H G H H F F E V G A D G - - - N L G A L R D T T L V D A G E
[480]
SNB11
T E P P L L T L E L G K S Y I L E L I N R T A F E H P I H L H G H S F Q V I R N N D R Q L N N P P I R D T V L L Q P G E
[480]
SNB12
T E P P L L T L E L G K S Y I L E L I N R P A F E H P I H L H G H S F Q V I R N N D R Q L N N P P I R D T V L L Q P G E
[480]
ZP 01036720
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A G G M M G V I R V A - - - - - - [539]
ZP 00964414
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R V A - - - - - - [539]
ZP 00961617
R V E I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R V D P G T T K T [539]
YP 002289160
R V E V A F V A D N P G D W M - - F H C H V L E H Q - - - - - - - - - - - - - - - - A G G M M G T V R V I - - - - - - [539]
ADV52199
R V E I A F V A D N P G D W M - - F H C H L L E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [539]
YP 004619745
R A D I A F V A D N P G D W M - - L H C H V L E H Q - - - - - - - - - - - - - - - - E G G M M G V F R V A - - - - - - [539]
ZP 02186341
R V D I A F V A D N P G D W M - - F H C H I L E H Q - - - - - - - - - - - - - - - - A A G M M G V I R I A P E T S R T [539]
ADM87301
T R D I V C V F D N P G N W L - - L H C H M L G H Q - - - - - - - - - - - - - - - - A A G M K T W V E V A - - - - - - [539]
SNB11
N K E I A F V A D N P G Q W M E T F H C H I L D Q I I R P Q A S R A A S S R P H V W G P E L R G R F Q - - - - - - - - [539]
SNB12
N K E I A F V A D N P G Q W M E T F H C H I L D Q I I R P Q A S R A A S S R P H V W G P E L A A A V S G V - - - - - - [539]
Fig. 8.8 Partial amino acid alignment of bacterial laccase genes including gene sequences derived from metagenomic studies
(ADV52199 – drained peat soil metagenome, ADM87301: South China Sea marine microbial metagenome, SNB11 and SNB12:
marine sponge metagenome). Histidine-rich copper binding domains are indicated by vertical boxes. ZP 01036720: multicopper oxidase domain protein Roseovarius sp. 217, ZP 00964414: multicopper oxidase domain protein Sulfitobacter sp. NAS14.1,
ZP00961617: multicopper oxidase domain protein Roseovarius nubinhibens, YP 002289160: CumA Oligotropha carboxidovorans OM5, ADV52199: laccase uncultured bacterium, YP 004619745 cueO Ramlibacter tatouinensis TTB310, ZP 02186341:
alpha proteobacterium BAL 199, ADM87301: laccase uncultured bacterium, SNB11 and SNB12: sponge metagenome-derived
sequences
using either hybridization probes or PCR primers for
these genes. This involves the design of DNA probes or
PCR primers typically derived from conserved regions
of already well-characterized genes or protein families.
Using the homology-based approach, PCR amplification is performed, following which the putative gene
is sequenced and then subsequently cloned into an appropriate expression vector where the heterologously
expressed protein is then purified and biochemically
characterized to ascertain its function. While such ap-