1. NUTRIENT ACQUISITION :
DIGESTION & TRANSPORT

2. DISACCHARIDES
Examples: Sucrose, maltose and cellobiose

3. May be transported into fungus:
 Intact or
 Hydrolyzed before being transported.
 E.g., In S. cerevisiae, sucrose was converted
to glucose and fructose at the cell surface and
the monosaccharides were transported.

4. • Isolated cell walls of S. cerevisiae contained
most of the invertase.
• Invertase was solubilized by treated with snail
digestive enzyme in mannitol.
• Protoplast were unable to ferment sucrose, but
could ferment glucose
• Maltose was not located in wall, but retained in
the protoplast.
• Yeast cannot ferment maltose unless the have
been grown on maltose.

5. CELLULOSE
Structure : microfibrillar substance of linear molecules
packed into crystalline arrays interspersed with
amorphous regions.•Non- ordered structure
•Helps in β-linkage to adapt to
microenvironment
Native cellulose : Insoluble
: Comminuted to produce fine
particulate suspension

6. Modified, soluble cellulose
derivatives
Carboxymethyl cellulose ( CMC) and
Hydroxyethyl cellulose ( HEC )
- Thickener in Food
Umbelliferyl cellodextrins
• Chromogenic substances
• Enzyme activities measured based on the
colored products

7. Cellobiohydrolase Endoglucanases
Digest
ONLY Amorphous Region Crystalline Arrays and Amorphous
Region

8. • CBHI and EGI have greater than 50% nucleotide
sequence similarity and about 45% amino acid sequence
similarity.
• CBHII and EGIII were unrelated to each other or the first
pair.
Two reasons for the expression of the genes for the
enzymes in Saccharomyces cerevisiae:
• Since S. cerevisiae has no known exocellular cellulases,
expression of the genes individually resulted in single-
enzyme activities with no cross contamination.
• Since cellulose substrates are highly variable, conversion
of cellulose to glucose may not be optimal with the native
mix from Trichoderma reesei.

9. • Expression of the four cloned cellulase genes of T. ressei in S. cerevisiae
succeeded by using cDNA clones from the mRNAs to eliminate the introns
that were not correctly spliced by yeast, and by providing suitable yeast
promoters.
• The recombinant enzymes from S. cerevisiae were active toward the
natural substrates, barley β-glucan and lichenin, and several artificial
substrates.
• The specific activity and binding of the recombinant CBHII were reduced
in comparison with the natural enzyme, suggesting that the
hyperglycosylation affected activity.

10. GROUP 2

11. STARCHES AND HEMICELLULOSE

12.  β-glucans have β-1,3 and β-1,6 linkages
 Starches and glycogens have α-1,4 and α-
1,6 linkages
 Produced by fungi, algae, vascular plants,
animals
 Sources of carbon and energy
 Fungal walls usually contain β-1,3 and β-
1,6 glucans (β-glucans) which are
associated with wall fractions or the
periplasmic space
 Many glucanases have a nutritional role

13.  The hydrolysis of α-1,4 linkages of starch and
glycogen is catalyzed by α- and β-amylases and
glucoamylases
 Amylases are endoglucanases-yield maltose
and glucose, also limit-dextrins with α-1,6-
branching(debranching enzyme required)
 Glucoamylases are exoglucanases-hydrolyze
glucose
 Many fungi produce amylases and
glucoamylases which cleave α-1,4-bonds to
yield glucose
 Fungi lacking glucoamylase require maltase

14.  Hemicellulose – composed of mixtures of
polysaccharides that have different sugar
monomers-glucose, galactose, mannose,
arabinose, xylose, glucuronic acid
 These are alkali-soluble extractable
polysaccharides of plant cell walls
 Mostly heteropolymeric-have 2 or more sugar
monomers, linear or branched, and acetylated
 Enzymes required to utilize these materials-
xylanase, debranching xylanase, mannanase,
galactanase, arabinosidase, glucuronidase,
acetly esterase

15. LIGNIN COMETABOLISM
• Lignin is the most abundant carbon in
aromatic form.
• Lignin is a branched polymer of cinnmyl
olcohol-derived monomers.
• Lignin impregnating material in wood
which protects polysaccharides
component from enymatic attack.

16.  Aspects of lignin decompositions:
Fungi are the only organisms that
extensively degrade lignin to C02 .
Degradation occurs by predominantly
oxidative without releasing its monomer
into solution.
Does not provide primary source of
carbon and energy for fungal growth.

17.  Decay fungi are grouped into three principal
types according to form decay;
1)White rot
 Basidiomycetes and few Ascomycetes.
 Capable of complete mineralization of lignin.
2)Brown rot
 Basidiomycetes
 Degrade cellulose and hemicellulose preferentially
with limited degradation of lignin.
3)Soft rot
 Ascomycetes and Deuteromycetes.
 Various lignolytic abilities on lignin. ( either no,
little or great atttack).

18.  The requirement for primary source of
carbon energy for degradation of
another compund is known as
cometabolism.
 Addition of glutamate, glutamine or
histidine suppressed ligninase activity.

19.  Exoenzymes involve in lignin degradation:
1)Lignin peroxidase and Manganese (Mn)
peroxidase.
 Contain Fe-heme prosthetic groups.
 Coooperative action of these leads to exocellular
“combustion” that depolymerizes lignin.
2)Laccase
 Cu-containing exoocellular benzenediol oxidase
and other phenol oxidase.
 May acts synergistically with other enzymes.
 Able to generate H2O2.

20. PROTEIN
 Source of:
 Nitrogen
 Sulphur
 Large size polymer
 Digested wt exocellular protease into amino
acid/peptides
 Protease regulation through:
 Induction(presence of protein)
 Catabolite repression(when protein appear as
sole
C/N/S source )

21.  Protease classification:
 Exohydrolases : cleave peptide terminally
Aminopeptidase from N terminus
Carboxypeptidase from C terminus
 Endohydrolases: cleave peptide internally
 Together endohydrolases create short peptide
substrates availability for exohydrolases rapid
protein digestion

22. Source: http://maptest.rutgers.edu/drupal/?q=node/27

23. Source: http://www.uwplatt.edu/~sundin/354-7/l547-46.htm
Source:
http://upload.wikimedia.org/wikipedia/commons/9/93/Tetrapeptide_%26
_Aminopeptidase_V.1.svg

24. EXOENZYME OF PHYTOPATHOGENS
 Exocellular enzymes contribute in fungi pathogenic
process.
 Enzyme activities digest insoluble host materials
that are then taken up by the fungus for growth.
 Examples:
- Cutinases
- Pectinases
- Hemicellulases, cellulases and lipases

25. CUTINASES
 Are esterases.
 Penetrate waxes, cutin, and suberin
protective barrier on outer surface of plants.
 Inducible by cutin monomers.

26. PECTINASES
 Complex polysaccharide composed of -1,4-
galacturonic acid with rhamnose side chains.
 Attack pectic material of the middle lamellae and
primary walls of plant cells which are responsible for
cementing the host cells and wall components
together.
 Enzyme activites:
I. Release methyl groups from carboxyls(Pectin methyl
esterase).
II. Hydrolyze -1,4-galacturonosyl bonds (Hydrolases).
III. Lyse 𝛼-1,4-galacturonosyl bonds by rearrangement
of the hydrogens ( Transeliminases or lyases).

27. HEMICELLULASES, CELLULASES, AND
LIPASE
 Hemicellulases consist of galactanases,
arabanases, xylanases, and mannanases.
 Cellulases :
- Enzyme activity:
I. Cleave cellobiose unit from non reducing ends
of cellulose ( Cellobiohydrolase).
II. Hydrolyze internal bond of amorphous cellulose,
cellodextrins and cellulose derivatives (
Endoglucanase).
III. Hydrolyze cellobiose and oligocellodextrins to
glucose ( Cellobiase)
- Exhibit multiple forms.
- Glycoprotein.
- Very resistant to denaturation.

28.  Lipases :
- Attack plasmalemma.
- Lipases A1 and A2 acted on
diacylphospholipid.
- Lysophospholipases L1 and L2 deacylate
monoacyl product of A enzyme.
- Phospholipase B cleaves both fatty acids,
but it may be a mixture of A and L lipases.

29. EXOENZYMES OF ZOOPATHOGENS
 Enzymes:
 Proteases:
 leucine aminopeptidase
 elastase
 collagenase
 carboxypeptidases
 Lipases
 Acid and alkaline phosphatases
 Plasmacoagulase
 Neuraminidase

30.  Production: In vivo
 Function: Attack and digest host
 Toxin which consist of ezymes
Eg: canditoxin of Candida albicans
 Correlation of enzyme to fungal
pathogenicity
 Eg: phospholipase reponsible for C.
albican pathogenicity
 However phospholipase activity
repressible by presence of glucose,
sucrose, galactose

32. TRANSPORT MECHANISM
 Basic mechanism for communication of cells.
 Plasmalemma: semipermeable layer of hyphal surface
that regulate flow of material.
 2 types of mechanism: passive and active transport
 Passive transport : movement of substances affect by
gradient concentration.
 Active transport : substances moves against concentration
gradient which required energy(ATP).
 Substrate accumulate at higher concentration on one side
of membrane.
 Accumulation inside hyphae can occur through other than
active transport.

33.  Insoluble sink, immobile binding sites or
metabolic sinks may reduce the transport
activity( effective concentration) causing net
directional movement.
 Example: plant cell synthesis oxalic acid causing
crystallization of calcium oxalate resulting high
accumulation of calcium in cells.
 This movement well described in Donnan
equilibrium involves formation of immobile ions.
 Rapid change such as phosphorylation is the
mechanism for accumulation occur in passive
transport.

34.  Most substances that move across
membrane required help of carrier
molecule.There are 3 carrier theory that has
been developed.
1. Without carrier molecule, the rate of
transport to increase proportionally to
increased external concentration.
2. Carrier-mediated transport are inhibited
with some degree of specificity by
substance active in low concentration
3.Similar substances often competitively
inhibit transport of the substrate

35.  Countertransport of these substrate may
occur when they are previously loaded into
hyphae.
 Countertransport is the outward movement
of compund in response to the inward
movement of substance.

36. ENERGY COUPLING IN ACTIVE
TRANSPORT
 Energy coupling in active transport are partially
understood.
 However, coupling of ATP hydrolysis to proton
transport is generally agreed in which it generates
driving force for solute transport [47].
 2 types of energy coupling which are direct and
indirect

37. DIRECT- COUPLING MODELS
 Energy of ATP hydrolysis is utilized in the
movement of binding proteins or in the
dissociation of substrate.
 As ATP-dependent conversion of substrate
occur, metabolically driven transport occurs by
maintain of low internal concentration of
substrate.
 This is the passive diffusion that behaves like
directly coupled active transport since these
mechanism required ATP synthesis.

38. INDIRECT- COUPLING MODELS
 These models based on Mitchell hypothesis.
 These hypothesis stated that, motive force involves
an ATP-generated proton gradient across
membrane.
 It can be accomplished by a substrate-carrier-proton
complex diffusing across the membrane or by
molecular gate.
 Molecular gate involves protein conformation
changes, coupled with proton movement between
outer and inner faces of plasmalemma.

39. GROUP 5

40. MEMBRANE POTENTIALS AND PH
CHANGES

41. MEMBRANE POTENTIALS
 Means the differences in charge across the
plasma membrane.
 Can be affected by:
Transportable substrate (potassium,
glucose, or amino acid)
caused transient depolorization
Metabolic inhibitors (cyanide)

42. PH CHANGES
 Attributed to the operation of proton pump.
 External pH acidic (pH 6 or less).
 Internal pH (6.5 to 6.8)
 provide proton gradient.
 Transport system of fungi  inhibited pH > 7.
 Affected by
 Uncoupling inhibitors (eg. Azide)
 Transportable substrate
 Fungal hyphae grow measured by vibrating
microelectrode.

43. ELECTROGENIC MEMBRANE
ATPASES

44. MEMBRANE ASSOCIATED ATPASES
 Different:
 In cellular locations
 Ion translocating abilities
 Organisms that have been found
 3 types found in fungi
 H +- ATPases located in plasmalemma
 H +- ATPases on vacuole
 H +- ATPases of mitochondrion
TRANSPORT FUNCTIONS
ATP SYNTHASE FUNCTION

45.  Plasmalemma H +- ATPases
 A single monomeric peptide of 100 kDa
 Very abundant (20-40%)
 Forms an outwardly directed H+ pump (membrane potential)
 Vacuolar ATPase
 Translocated protons out of the cytoplasm but into vacuolar
contents
 Heteromultimeric protein similar to the mitochondrial ATPase
 Both coupled ATP hydrolysis to proton translocation > pH
gradients
 Both involved in transportation

46. ROLE OF PLASMALEMMA ATPASE
 Developed through the use of inhibitors and
isolated ATPase in artificial membrane vesicles.
 Ionophores
 Collapse the membrane potential simultaneously
with collapse of H+/ion gradients.
 Stimulated ATPase, inhibit transport
 Inhibitors of ATPase function
 Several types
 Differed in- vivo and in- vitro experiments

47. ATPASE MUTANTS
 Selected in S. cerevisiae and Schizosaccharomyces
pombe
 Resistance to several drugs
 Mutants with reduced H+-ATPase activity
 Proton efflux activity decreases
 Growth rates reduce
 More acidic intracellular Ph
 Cell proliferation increases
Conclusions:
ATPases play important role in proton pumping and
intracellular pH regulation

48. CATION TRANSPORT
 Saccharomyces cerevisiaeand Neurosporacrassa have
been studied for absorping of mineral ions.
 Extensively studied of by using radioisotopes.
 Due to methods problem, the study of ammonium ions have
been hinder even though it plays important roles in
metabolism and metabolic regulation.
 Divalent cations in transport mechanisms have been
examined to far lesser extent.

49. GROUP 6
NUR AMIRAH BINTI SIDEK (164673)
SYUKRIYAH BINTI MAT DAUD (162549)
SITI NUR ALIA BINTI RAMLI (162568)
CHOO KIN YAN (163668)
LEE SIEW YI (163649)
SHEIK ABDUL MUIZZ BIN SHEIK IBRAHIM (162386)
MUHAMMAD ADIB AMIN BIN AZHAN (164865)
Potassium
Ammonium
Divalent cations and iron
Anion transport

50. POTASSIUM

51. POTASSIUM
1. Mechanism transport for Potassium.
• Active transport by using permease.
• Direct participation of H+-ATPase.
• Passive transport by involving Ion channel.
2. Active transport by using permease.
• Active absorption of potassium occurred
depending on the presence of fermentable
or respirable substrate.
• The absorption kinetics demonstrated
substrate saturation at high concentration
following the Mechealis-Menten equation
indicating the occurrence of a potassium
permease.

52. 3. Direct participation of H+-ATPase.
 Other alkali metal ions were transported but with less
affinity for the carrier in the order K+>Rb+>Cs+>Na+>Li+
with relative ratios 100:42:7:4:0.5.
 H+ and K+ were about equal in the inward transporting
system but the outward transporting system
discriminated against K+ in favor of H+ or Na+
4. Passive transport by involving the iron channel.
 The presence of K+ channels and ion channels sensitive
to mechanical stimuli provide additional mechanisms for
K+ transport.
 Ion channels are a class of plasmalemma proteins that
function as gated pores that allow ions to flow down
electrical or chemical gradients.

53. • Ion channels that appear in the membrane
patches in response to mechanical
deformation of the membrane are called
mechanosensitive or stretch-activated
channels.
• Strectch activated channels have also been
proposed to function in tugor regulation.
• Ion channels did not function in active
transport, but their presnce adds another
factor to consider in the overall transport

54. AMMONIUM

55. AMMONIUM
 Ammonium ion has been studied by using
methylammonium labeled wit 14C
otherwise there was no convenient
method for studying this ion.
 The use of the methylammonium was
difficult due to its toxicity to most fungi.
 Both of the ions are using the same
transport system because the observation
shows that ammonium ion was a potent
competitive inhibitor of methylammonium
transport.

56.  In S. cerevisiae ,methylammonium
transport occur by a typical carrier-
mediated, active transport.
 In P. chrysogenum and A. nidulans
indicated the presence of a repressible
high –affinity system for
methylammonium transport that was
competitively inhibited by ammonium
but unaffected by amino acid which is
contrast in the S. cerevisiae.

57. DIVALENT CATIONS
AND IRON

58. DIVALENT CATIONS
 Divalent cations( Mg²+, Ca²+, Mn²+) will be taken
up by carrier-mediated transport system.
 Transport occur:
1. Simultaneously, with phosphate up take.
2. Starved cell will firstly interact with glucose and
phosphate.
 Potassium will greatly simulate the transport.
 Dinitrophenol, azide, and arsenate inhibit it.

59. FE³+ TRANSPORT
 Involve siderochromes;
 Excreted under iron-deficient condition.
 Involved in active transport.
 Aspergillus, Neurospora, Ustilago and bacteria.
 Against concentration gradient.
 Competitive inhibition occur between related
siderochromes as a means to stop the intake of
iron when it became saturated.

60. DIFFERENT FUNCTIONS OF
FERRICHROME AND FERRICHROME A
FERRICHROME FERRICHROME A
 Found intracellular in
Ustilago maydis.
 Both iron-sufficient
and iron deficiency
conditions.
 Secreted outside.
 Iron deficiency only.
 Solubilizing agent, to
solubilize iron.

61. ANION
TRANSPORTPhosphate, sulfate, nitrate, and
organic acids.

62. ANION TRANSPORT
1. Phosphate
 Carrier-mediated.
 Depend on exogenous fermentable
substrate.
 Phosphate transport acts as hydroxide
exchange system based on observation of
the pH change in the medium.
 Inhibit by arsenate.
2. Sulphate
 Study in filamentous fungi.
 2 system

63. ANION TRANSPORT
 3. Nitrate
 Lack study due to the of suitably sensitive
measuring techniques.
 Diffuse through the membrane in response to
concentration gradient created by nitrate
reductase.

64. GROUP 7

65. SUGAR AND AMINO ACID
TRANSPORT

66. ADENOSINE TRIPHOSPHATE (ATP)
 The main energy source for active transport of
sugar.
 The energy cost is one ATP per sugar molecule.
 Fermentation of sugar, e.g: glucose produces ATP.

67. DIFFERENCES BETWEEN ACTIVE AND
FACILITATED TRANSPORT
Active transport Facilitated difussion
Required ATP for
transportation of molecule.
Does not required ATP, but
used the concentration
gradient (external > internal)
to transport molecule.
**Similarity :
Both mechanism use carrier proteins to
transfer molecules to and from the cell.

68. SUGAR TRANSPORT
 The fungi that utilised facilitated diffusion:
1. Saccharomyces cerevisae
2. Neurospora crassa
 The fungi that used active transport:
1. Rhodotorula gracilis
2. Aspergillus nidulans

69. • The glucose transport in Saccharomyces
cerevisae is controlled by two systems:
 Low affinity constitutive system
 High affinity repressible system
• The inhibitor of glucose transport in S. cerevisae:
 Uranyl ion, (UO2)2+.
**Others inhibitors which affect transport of some
sugar:
 2,4-dinitrophenol
 Azide
 Carbonylcyanide-m-chlorophenylhydrazone (CCCP)

70. IN SACCHAROMYCES CEREVISAE
 Glucose and galactose transport systems required kinase
activities and transport proteins.
 Kinase activities: phosphorylation
 A glucose transporter was associated with SNF 3 locus
while a galactose transporter with the GAL 2 locus.
 The SNF 3 and GAL 2 proteins were found in the
plasmalemma.
Glucose transport Galactose transport
The enzymes involve in
the kinase activities:
1. Hexokinase 1
2. Hexokinase 2
3. Glucokinase
The enzyme:
1. Galactokinase

71. AMINO ACID TRANSPORT
• Stage of development
• Age of cultures
• Medium contents

72. MULTIPLE TRANSPORT SYSTEM WITH
OVERLAPPING SPECIFICITY
Table below summarizes the amino acid
transport systems of Neurospora crassa
System Amino Acid Specificity
1 Aromatic and aliphatic
2 Aromatic, aliphatic and
basic
3 Basic
4 Acidic
5 Methionine

73. SPECIFIC TRANSPORT SYSTEM FOR
SINGLE AMINO ACID
 This system has been identified in P. chrysogenum
and Achlya.
 In S. cerevisiae, it has single general amino acid
permease (GAP) with broad affinity, basic amino
acid transporter, and a series of specific
transporters for individual amino acids.
 Fungi seem to be quite individualistic in the
organization of their amino acid transport abilities.

74.  Amino acid transport of fungi is an active
process.
 Anoxia and cyanide are the transport
inhibitors in most fungi, showing that
endogenous respiration is necessary.
However, S. cerevisiae is capable of
anaerobic transport.
 In P. chrysogenum, uncoupling agents
inhibit amino acid transport, but arsenate
(phosphate competitor) does not inhibit the
transport.
Transport Inhibitors

75. MOLECULES INVOLVED IN THE
TRANSPORT PROCESS
 Several kinds of evidence suggest that amino acid
binding molecules involved in this process.
 These include the demonstration that:
1. The molecules are associated with the cell surface
or part of the membrane.
2. They have the same specificities as the transport
systems.
3. Their removal results in decreased transport ability
of the fungus.
4. They are reduced, absent, modified in transport-
deficient mutants.

76. THE END