Download (H + OH) +

Hydrolytic and Oxidative Enzymes
Pectinases
(Poly)phenol oxidases
Cellulases
Hemicellulases (Xylanases)
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Problem: Fungi cannot
directly utilize complex
organic substrata.
Many of the constituents of the substrata are polymers of
simple molecules, such as sugars, amino acids, etc.
Cellulose is a polymer of D-glucose
Hemicellulose is a polymer of D-xylose
Solution: Use extracellular enzymes to
degrade substratum to simple molecules,
which can be transported easily across cell
walls and membranes. Enzymes can be
hydrolytic or oxidative.
Some examples:
A battery of cellulases (hydrolytic enzymes) degrade various
forms of cellulose to either cellobiose or glucose.
(Poly)Phenol oxidases (oxidative enzymes) thought to be
involved in lignin degradation.
Enzymes are Either Inducible or Constitutive
Ways to Measure Enzyme Activity
** Measure Enzymatic Products
Reducing Sugars, Change in pH, PPC
** Change in Concentration of the Substrate
** Depletion of Co-factors (ATP, NAD, etc.)
Detection of Activity Using Reducing
Compounds (Sugars)
CH2OH
C=O
C-OH
O
6 CH2OH
C
1 O
C-OH
C-OH
Dglucose
1 OH
α
(Alpha
)
H
HO-C
C-OH
H
OH
Carbony
l
Carbon
ANOMERIC
CARBON
CH2OH
O
OH
1H
β
(Beta)
Standard Curves –Beer’s Law
Used to determine unknown concentrations by
comparison to the best fit line (linear regression)
of known concentrations
Beer’s Law. The relationship between absorbance and the concentration of a compound is
linear except at very low and high concentration
and in impure solutions
Do NOT extrapolate values beyond
last data point used to calculate the
line
Making Standards Using Dilutions
Use general formula: C1 x V1 = C2 x V2
C1 = Stock of BSA (1 mg/ml)
C2 = Concentration (mg/ml) desired standard
V1 = Volume (ml) used of C1
V2 = Final volume necessary for C2
Example:
1 mg/ml x 5 ml = 0.2 mg/ml x V2 -- Solve for V2
V2 = (1mg/ml) x 5 ml)/ 0.2 mg/ml
V2 = 1 x 5 ml/ 0.2 = 25 ml = Final vol. for standard
V2 – V1 = ml to add to stock or 25 – 5 = 20 ml of water
LOWERY PROTEIN DETERMINATION
0.1
mg/ml BSA
0.2
0.3
0.4
Linear Regression p. 341-342
Y = mX + B
*
*
*
**
*
*
T
Abs
**
**
Concentration
Abs = log(1/T)
Calculation of Unknown Concentration
Y = mX + b
Y = Absorbance of unknown sample
m = Slope of the calculated line
X= Concentration of compound
b = Y-intercept of line (almost never “0”)
X = (Y/m) - b
CH2OH
O
CH2OH
O
O
1
4
Cellulose
β1
4 (n)
Cellobiose (2 D-Glucose)
β
Insoluble
CH2OCH3
O
CH2OCH3
O
CMC
O
1
4
β
Methyl Ester on
Carbon 6 confers
partial + charge
Soluble
Enzymatic Hydrolysis
CH2OH
O
CH2OH
O
HO
H
O
1
H
4
OH
H2O (H + OH) + “Hydrolase”
CH2OH
O
CH2OH
O
HO
OH
H
H
HO
H
OH
H
Cellulolytic Enzymes (Inducible)
CH2OCH3
O
NR
4
CH2OH
O
O
1
CH2OCH3
O
O
O
4
1 4
CH2OH
O
1
1
4
(n)
n= 15,000
Cx1
C1
Cx2
C1 = 1,4- β- glucan cellobiohydrolase yields cellobiose NR
Cx1 = 1,4- β - exoglucanase yields D-glucose NR
Cx2 = 1,4 - β - endoglucanase yields dextrans
Cx = 1,4 - β – glucosidase acts on cellobiose
2 glucose
Crystalline Cellulose
C1
C1 + Cx
Inhibiton
Swollen Cellulose
CMC
C1
C1
Cx2
Cx2
Cellobiose
Cx3
Cx1
Random Dextrans
Inhibiton ??
Cx3
C1 or Cx
D-Glucose
(Methyl , if CMC)
1. Middle lamella
COOH
2. Ca and Mg ions
O
3. Often methylated
OH
HO
Galacturonic acid -- basic unit of pectin
COOH
CHOCH3
O
O
*
HO
NR
Inducible
COOH
*
O
1
O
4
O
1
4
α 1,4 galacturonic acid or
polygalacturonic acid
OH
(n)
Battery of 9
Hydrolytic
Enzymes
6 and 7
Polygalacturonase (PG) pH = 5.0
COOH
COOH
O
O
*
HO
NR
COOH
*
O
1
O
4
Exo
O
1
4
OH
(n)
Endo
Products are Galacturonic Acid (Changes in pH and
RS) (Exo)and Shorter-Chained Polymers of
Galacturonic Acid (Changes in Viscosity) (Endo).
Pectin Methyl Galacturonase (PMG) pH = 5.0
COOCH3
COOCH3
O
O
*
HO
NR
COOCH3
*
O
1
O
4
Exo
O
1
4
OH
(n)
Endo
Products are Methylated Galacturonic Acid
(Reducing Sugar) (Exo) and Shorter-Chained
Polymers of Methylated Galacturonic Acid (Endo). 8 and 9
Hemicellulose – Pentose Sugar (Xylose Shown)
β - 1,4 configuration
O
Xylose is a reducing sugar
O
O
O
NR
HO
4
1
O
4
Exo
1
4
Endo
OH
1 (n)
Products are Xylose, Xylobiose (Two Sugar Residues)
(Exo) and Shorter-Chain Xylans (Endo). Xylobiase
Hydolyzes Xylobiose to Two Xylose Residues.
Measurement by Reducing Sugars (Exo) and Change
in Viscosity (Endo).
Lignin and (Poly) Phenol Oxidases
Heterogeneous Polymer (Complex) of Aromatic Alcohols
May Be Involved in Lignin Degradation??
May Be Active Against Defense Phenolic Compounds.
Polyphenol Oxidase is a Catch-all Term.
Can Be Used to Distinguish Brown and White Rot
Fungi. Brown Rot Primarily Cellulolytic; White Rot
Ligninolytic and Cellulolytic.
Four Phenol Oxidase Classes of Enzymes
1. Tyrosinase -- Monophenols Such as Monophenolic Amino
Acids.
2. Laccase – Polyphenolic Compounds
3. Peroxidase – Both Mono and Polyphenols
4. Catechol Oxidase -- Both Mono and Polyphenols
CO2H
CO2H
OH
OH
OH
Monophenol
Polyphenol
OH
Inducible
Polyphenol Oxidase
(Presumptive Test for the Ability to Degrade Lignin)
Gallic Acid
Quinones
CO2H
CO2H
OH
O
PPO
O
OH
OH
OH
Quinones are Unstable and Spontaneously
Polymerize to Form Colored Compounds
S..ZINE
GALLIC
CATECH
S.. HYDE
ABTS
Solid Medium
Gallic Acid
PAGE
ABTS Development for Laccase
Development of a Protein Standard Curve
1. Add 5 ml of the cupric sulfate solution to 1 ml of protein
and mix well. Allow to stand for 10 min.
2. Add 0.5 ml of Diluted Folin-Ciocalteau reagent to each of
the tubes and set aside for 30 min. at room temperature.
3. Read absorbances at 500 nm and record results. Use the 0
mg/ml BSA as the “zero” reference.
4. Graph results – absorbance on the Y axis and BSA on the
X axis.
5. Add 1 ml of each standard to a test tube – repeat three
time.