PMID- 32087608
OWN - NLM
STAT- MEDLINE
DCOM- 20210125
LR  - 20210125
IS  - 2045-8827 (Electronic)
IS  - 2045-8827 (Linking)
VI  - 9
IP  - 4
DP  - 2020 Apr
TI  - Streamlined production, purification, and characterization of recombinant 
      extracellular polyhydroxybutyrate depolymerases.
PG  - e1001
LID - 10.1002/mbo3.1001 [doi]
LID - e1001
AB  - Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases 
      (PhaZs) has been of interest for over 30 years, but implementation is sometimes 
      difficult and can limit the scope of research. With the constant development of 
      tools to improve recombinant protein production in Escherichia coli, we propose a 
      method that takes characteristics of PhaZs from different bacterial strains into 
      account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas 
      testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas 
      stutzeri, and Ralstonia sp. were successfully produced with varying expression, 
      solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were 
      more amenable to heterologous expression in all aspects; however, using the 
      E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions 
      for expression and purification (variation of IPTG concentration and use of size 
      exclusion columns) helped circumvent low expression and purity for the other 
      PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB 
      plate-based method, adapted to test for various pH and temperatures. rPhaZ from 
      M. algicola presented the highest activity at 15 degrees C, and rPhaZs from Cupriavidus 
      sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed 
      herein can be used to test the production of soluble recombinant PhaZs and to 
      perform preliminary evaluation for applications that require PHB degradation.
CI  - (c) 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
FAU - Martinez-Tobon, Diana I
AU  - Martinez-Tobon DI
AD  - Department of Chemical and Materials Engineering, University of Alberta, 
      Edmonton, AB, Canada.
FAU - Waters, Brennan
AU  - Waters B
AD  - Department of Chemical and Materials Engineering, University of Alberta, 
      Edmonton, AB, Canada.
FAU - Elias, Anastasia L
AU  - Elias AL
AD  - Department of Chemical and Materials Engineering, University of Alberta, 
      Edmonton, AB, Canada.
FAU - Sauvageau, Dominic
AU  - Sauvageau D
AUID- ORCID: 0000-0002-1995-5523
AD  - Department of Chemical and Materials Engineering, University of Alberta, 
      Edmonton, AB, Canada.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200222
PL  - England
TA  - Microbiologyopen
JT  - MicrobiologyOpen
JID - 101588314
RN  - 0 (Recombinant Proteins)
RN  - EC 3.1.1.- (Carboxylic Ester Hydrolases)
RN  - EC 3.1.1.- (poly(3-hydroxyalkanoic acid) depolymerase)
RN  - Marinobacter algicola
SB  - IM
MH  - Bacteria/*enzymology/genetics/metabolism
MH  - Bioreactors/microbiology
MH  - Carboxylic Ester Hydrolases/*genetics
MH  - Comamonas testosteroni/enzymology/genetics/metabolism
MH  - Cupriavidus/enzymology/genetics/metabolism
MH  - Escherichia coli/enzymology/genetics/metabolism
MH  - Marinobacter/enzymology/genetics/metabolism
MH  - Pseudomonas stutzeri/enzymology/genetics/metabolism
MH  - Ralstonia/enzymology/genetics/metabolism
MH  - Recombinant Proteins/genetics
PMC - PMC7142370
OTO - NOTNLM
OT  - Escherichia coli vectors
OT  - extracellular PHB depolymerases (PhaZs)
OT  - poly(3-hydroxybutyrate) (PHB)
OT  - polymer degradation activity
OT  - recombinant expression
COIS- None declared.
EDAT- 2020/02/23 06:00
MHDA- 2021/01/26 06:00
PMCR- 2020/02/22
CRDT- 2020/02/23 06:00
PHST- 2019/10/11 00:00 [received]
PHST- 2020/01/05 00:00 [revised]
PHST- 2020/01/07 00:00 [accepted]
PHST- 2020/02/23 06:00 [pubmed]
PHST- 2021/01/26 06:00 [medline]
PHST- 2020/02/23 06:00 [entrez]
PHST- 2020/02/22 00:00 [pmc-release]
AID - MBO31001 [pii]
AID - 10.1002/mbo3.1001 [doi]
PST - ppublish
SO  - Microbiologyopen. 2020 Apr;9(4):e1001. doi: 10.1002/mbo3.1001. Epub 2020 Feb 22.