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Protein engineering and characterization of the blue mussel β-mannanase

Svantesson Sjöberg, Johan (2013) MOBM15 20122
Degree Projects in Molecular Biology
Abstract
Abstract

The β-mannanase MeMan5A from the blue mussel, Mytilus edulis, has a distal sugar-binding
subsite in its active site. Two residues, W240 and W281, are suggested to be involved in the binding
of sugar units in this distal “+2” subsite. In previous work, MeMan5A variants were created in
which W240 and W281 were replaced by alanines, and the genes for the wild-type (WT) sequence
enzyme and the two +2 subsite mutants were transformed into the yeast P. pastoris. In the current
work, both mutations were introduced in one gene construct, and the double-mutant enzyme was
expressed in P. pastoris, where it showed β-mannanase activity in the culture supernatant. The WT
MeMan5A and the two variants (W240A and W281A) were expressed... (More)
Abstract

The β-mannanase MeMan5A from the blue mussel, Mytilus edulis, has a distal sugar-binding
subsite in its active site. Two residues, W240 and W281, are suggested to be involved in the binding
of sugar units in this distal “+2” subsite. In previous work, MeMan5A variants were created in
which W240 and W281 were replaced by alanines, and the genes for the wild-type (WT) sequence
enzyme and the two +2 subsite mutants were transformed into the yeast P. pastoris. In the current
work, both mutations were introduced in one gene construct, and the double-mutant enzyme was
expressed in P. pastoris, where it showed β-mannanase activity in the culture supernatant. The WT
MeMan5A and the two variants (W240A and W281A) were expressed in P. pastoris and purified
using nickel ion affinity chromatography, taking advantage of the high native amount of surfaceexposed
histidine residues. The two enzyme variants were characterized and compared to the WT
MeMan5A with regards to pH optimum and temperature stability. Kinetic parameters for locust
bean gum (galactomannan, LBG), mannotetraose (M4) and mannopentaose (M5) hydrolysis were
determined for the WT MeMan5A and the two variants. The results suggest decreased catalytic
efficicency for both variants towards all three substrates, and a decreased binding affinity towards
LBG for the W240A variant. The preferred binding mode for the WT MeMan5A and the two
variants towards M5 were established by bydrolysis of M5 in H2
18O and MALDI-TOF MS analysis.
The results show that the WT MeMan5A and both variants strongly prefer binding M5 from subsite
-3 to +2, generating M2 and labelled M3 as the main hydrolysis products. (Less)
Abstract
Enzyme engineering for a better future

It is essential for many organisms on this planet to be able to digest plants. One of those
organisms is the blue mussel, which uses certain enzymes in its stomach to break down sugar
fibers in the algae that it eats. But how does this really work, and can we use it for anything?
Plant cells have very strong walls, protecting them from environmental stress. These walls consist
mainly of carbohydrates, different kinds of sugar molecules connected in long chains or fibers. One
type of sugar fiber, called mannan, is common in pine trees, beans, algae, and some other plants.
Mannan consists mainly of a kind of sugar called mannose, and interacts with other sugar fibers to
give the plant cell... (More)
Enzyme engineering for a better future

It is essential for many organisms on this planet to be able to digest plants. One of those
organisms is the blue mussel, which uses certain enzymes in its stomach to break down sugar
fibers in the algae that it eats. But how does this really work, and can we use it for anything?
Plant cells have very strong walls, protecting them from environmental stress. These walls consist
mainly of carbohydrates, different kinds of sugar molecules connected in long chains or fibers. One
type of sugar fiber, called mannan, is common in pine trees, beans, algae, and some other plants.
Mannan consists mainly of a kind of sugar called mannose, and interacts with other sugar fibers to
give the plant cell walls strength. In order to digest these fibers and extract nutrition from the plant
cells, organisms use enzymes that act like scissors and cut the fibers into very small pieces so that
the plant cell walls break. One of the enzymes that digest mannan is called beta-mannanase, and is
produced mostly by various molds and bacteria. Beta-mannanases have several practical
applications. For example, they can produce substances that promote the growth of beneficial
bacteria in our digestive systems, and they could also be used to convert mannan-containing waste
materials into useful products, such as biofuels.
Researchers have recently found a beta-mannanase in the stomach of the blue mussel. Similar betamannanases
exist in several other marine animals. We are interested in these enzymes since they
seem to be slightly different from most beta-mannanases found in bacteria, mold and yeast, and we
want to find out more about how they work and what they can be used for. Therefore, I have studied
the beta-mannanase from the blue mussel, using several biochemical and molecular biology
methods.
To have easier access to the blue mussel beta-mannanase, I have cut out the gene that the mussel
uses to make the enzyme, and put it into a kind of yeast that we can grow easily in our laboratory.
This way, the yeast can produce the blue mussel enzyme for me. I then isolated the enzyme from the
yeast, and studied how quickly the enzyme digests mannan, and which end-products (shorter
mannose chains) are formed in the digestion process. I also altered the enzyme slightly to see if this
gives it different properties, by replacing two of its amino acids, which are the building blocks of
the enzyme. I then studied the digestion of mannan by the altered enzymes and compared them to
the unmodified version.
My results show that the modified enzymes act differently when digesting mannan, compared to the
unmodified enzyme. The digestion process as a whole is slower, which shows that the two amino
acids that I replaced are important for the enzyme to digest mannan efficiently The end-products of
mannan digestion seem to be similar to the unmodified enzyme. This shows that they still seem to
function in the same way as the unmodified enzyme, only slower. The next step is to see if I can use
the enzymes to synthesize new carbohydrates, since there are several other beta-mannanases that
can do this. In the future, I hope that we can engineer the enzymes to use this ability to produce
carbohydrates with e.g. pharmaceutical or health-promoting effects. I would also like to continue
using these enzymes to make biofuel production and other processes more efficient and more
environmentally friendly.

Advisor: Henrik Stålbrand (Department of Biochemstry and Structural Biology)
Master´s Degree Project 30 credits in Cell and Molecular Biology 2013
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Svantesson Sjöberg, Johan
supervisor
organization
course
MOBM15 20122
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3561641
date added to LUP
2013-03-05 09:45:52
date last changed
2013-03-06 14:15:46
@misc{3561641,
  abstract     = {Enzyme engineering for a better future

It is essential for many organisms on this planet to be able to digest plants. One of those
organisms is the blue mussel, which uses certain enzymes in its stomach to break down sugar
fibers in the algae that it eats. But how does this really work, and can we use it for anything?
Plant cells have very strong walls, protecting them from environmental stress. These walls consist
mainly of carbohydrates, different kinds of sugar molecules connected in long chains or fibers. One
type of sugar fiber, called mannan, is common in pine trees, beans, algae, and some other plants.
Mannan consists mainly of a kind of sugar called mannose, and interacts with other sugar fibers to
give the plant cell walls strength. In order to digest these fibers and extract nutrition from the plant
cells, organisms use enzymes that act like scissors and cut the fibers into very small pieces so that
the plant cell walls break. One of the enzymes that digest mannan is called beta-mannanase, and is
produced mostly by various molds and bacteria. Beta-mannanases have several practical
applications. For example, they can produce substances that promote the growth of beneficial
bacteria in our digestive systems, and they could also be used to convert mannan-containing waste
materials into useful products, such as biofuels.
Researchers have recently found a beta-mannanase in the stomach of the blue mussel. Similar betamannanases
exist in several other marine animals. We are interested in these enzymes since they
seem to be slightly different from most beta-mannanases found in bacteria, mold and yeast, and we
want to find out more about how they work and what they can be used for. Therefore, I have studied
the beta-mannanase from the blue mussel, using several biochemical and molecular biology
methods.
To have easier access to the blue mussel beta-mannanase, I have cut out the gene that the mussel
uses to make the enzyme, and put it into a kind of yeast that we can grow easily in our laboratory.
This way, the yeast can produce the blue mussel enzyme for me. I then isolated the enzyme from the
yeast, and studied how quickly the enzyme digests mannan, and which end-products (shorter
mannose chains) are formed in the digestion process. I also altered the enzyme slightly to see if this
gives it different properties, by replacing two of its amino acids, which are the building blocks of
the enzyme. I then studied the digestion of mannan by the altered enzymes and compared them to
the unmodified version.
My results show that the modified enzymes act differently when digesting mannan, compared to the
unmodified enzyme. The digestion process as a whole is slower, which shows that the two amino
acids that I replaced are important for the enzyme to digest mannan efficiently The end-products of
mannan digestion seem to be similar to the unmodified enzyme. This shows that they still seem to
function in the same way as the unmodified enzyme, only slower. The next step is to see if I can use
the enzymes to synthesize new carbohydrates, since there are several other beta-mannanases that
can do this. In the future, I hope that we can engineer the enzymes to use this ability to produce
carbohydrates with e.g. pharmaceutical or health-promoting effects. I would also like to continue
using these enzymes to make biofuel production and other processes more efficient and more
environmentally friendly.

Advisor: Henrik Stålbrand (Department of Biochemstry and Structural Biology)
Master´s Degree Project 30 credits in Cell and Molecular Biology 2013
Department of Biology, Lund University},
  author       = {Svantesson Sjöberg, Johan},
  language     = {eng},
  note         = {Student Paper},
  title        = {Protein engineering and characterization of the blue mussel β-mannanase},
  year         = {2013},
}