BBE4302 Midterm 4

enzymes

protein based macromolecular biological catalysts that accelerate chemical reactions

catalysis

facilitating a reaction by helping it overcome its threshold energy (activation energy)

3 advantages of enzymes (over chemicals)

highly selective in what they react with, generally nontoxic, often do not produce by-products

enzyme suffix

usually -ase, denotes the substance it degrades

EC numbering system

categorize enzymes into six main groups

Oxidoreductases

catalyze oxidation-reduction reactions

transferases

transfer functional groups

hydrolases

catalyze cleavage with the addition of water (hydrolisis of bonds)

lyases

cleave bonds by means other than oxidation and hyrolisis

isomerases

catalyze isomerization changes

ligases

catalyze the formation of covalent bonds

active site

a region on an enzyme that binds to a protein or other substance during a reaction.

denaturing

when an enzyme loses its form and stops performing its function

optimal conditions that enzymatic activity

conditions that increase the speed of the reaction and minimize denaturing of the enzyme (proper pH, temp, etc)

lock and key model

The model of the enzyme that shows the substrate fitting perfectly into the active site

induced fit model

Change in the shape of an enzyme's active site that enhances the fit between the active site and its substrate(s)

Kcat

the turnover number, max number of substrate molecules converted to products per enzyme molecule per second

kcat/km

catalytic efficiency, a measure of how efficiently an enzyme converts a substrate into a product

difference between cellulose and starch

cellulose has beta 1-to-4 linkages between glucose units and starch has alpha 1-to-6 and 1-to-4 linkages between glucose units

starch vs cellulose degradation

much easier to degrade starch

fermentation

when glucose is metabolized directly by yeast

amylases

Digestive enzymes that converts the starch into accessible glucose units

hemicellulases

degrade hemicellulose, xylanase/mannase/galactanase

cellulases

enzymes that break down cellulose, usually hydrolytic enzymes, must be able to break beta linked glucose bonds AND penetrate crystalline regions

ligninases

degrade lignin, mostly thru oxidation, lignin peroxidase, manganese peroxidase, versatile perozidase, laccase

endoglucanase

cleaves the middle of polymer chain

exoglucanase

cleaves the end of a polymer chain and leaves a glucose dimer (cellobiose)

endo-exo synergy

the "free ends" are created for exo by endo, and the crystalline regions are opened to endo by exo

beta-glucosidase

converts cellobiose to glucose

amylases degrade _____, cellulases degrade ____

starch, cellulose

mechanism for enzyme improvement

traditional mutagenesis, genetic modification, rational design/directed evolution, optimization of fermentation conditions

environmental advantages of using lignocellulose

causes less carbon emissions, sustainable/renewable carbon source, abundant

five goals for feedstocks

fast growth, quality raw material, low maintenance, environmentally friendly, high digestability

Mn feedstock types

corn stover, switchgrass, aspen, MSW

fractionation

the process of a biorefinery, separating (refining) the individual components of lignocellulose for utilization

biorefinery

facility that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from biomass

uses for cellulose

paper fibers, textile fibers, nanofibers, alcohols

uses for hemicellulose

alcohols, solvent for paper making, industrial chemicals

uses for lignin

burned for energy, thermoplastics, value adding

uses for extractives

industrial chemicals, sap for latex

uses for bark

cork, mulch, charcoal or energy

Bioprocessing

Production of a commercially useful chemical or

fuel by a biological process, such as microbial fermentation or degradation

6 Goals for Biodegradative Organisms/Systems

fast acting, selective/efficient, robust, low cost, compatible with other degradative systems, multifunctional

Bioprospecting

Surveying biological organisms in general for potential utilization (Traditionally referred to surveying plants for medicinal applications); Find a system that works, designed by 'mother nature,' and then speed

up the process

Three ways to 'harness' an organism of interest.

direct utilization, bioengineering, biomimicry

biofuel processing steps

biomass production/delivery, pretreatment, cellulose hydrolysis, sugar fermentation, biofuel processing

cellulosic ethanol vs corn ethanol

both are abundant, but producing cellulosic ethanol is more difficult and expensive (requires more enzymes)

Environmental Remediation

detoxification or removal of contaminants/pollutants from an environmental medium (soil, groundwater, etc.)

four types of environmental remediation

excavation, pump and treat, in situ remediation, vapor extraction

three most common methods of environmental remediation

incineration, thermal desorption, solidification/stabilization

bioremediation price

varies widely

bioremediation definition

the use of microorganisms, plants,

or their metabolites to detoxify or remove pollutants

Phytoremediation

Use of green plants and microbes to clean up soil and groundwater (bioremediation with plants)

Mycoremediation

using fungi (fungal degradation) to remove or degrade dangerous chemicals

advantages of using microbes for remediation

heartless/gutless, reproduce quickly, they go to extremes, may already be present

targets for remediation

organics, organometallics, metals

xenobiotic

Synthetic compound not found in nature

examples of organic targets for bioremediation

hydrocarbons like PAHs and BTEX, penta, TNT, DDT, dioxins

examples or organometallic targets for bioremediation

methyl mercury,

examples or inorganic targets for bioremediation

CCA

Passive in-situ bioremediation

using native organisms

promoted in-situ bioremediation

using biostimulation to promote native organsims activities (adding oxygen, nutrients, etc)

is key for biostimulation

oxygen

inoculation bioremediation

adding enzymes/vegetation, etc

Ex situ bioremediation

Involves removing the polluted materials and treating them off site

Four key advantages to bioremediation with fungi

high surface area:volume ration, hydrophobic extracellular sheath (allows them to form biofilms and improve capture), can tolerate environmental stress & fluctuations, can break down (biodegrade) many pollutants

Co-metabolism

Co-metabolism - The enzymes secreted to metabolize

compound #1 also metabolize compound #2.

Mycofiltration

using mycelial mats formed by

fungi as a living filter (this will likely not be pure)

gene

a sequence of nucleotides in DNA or RNA that encodes the synthesis of a gene product, either RNA or protein

DNA structure

DNA consists of two long chains of nucleotides twisted into a double helix and joined by hydrogen bonds between the complementary bases adenine and thymine or cytosine and guanine

Chromosome

DNA (deoxyribonucleic acid) molecule with part or all of the genetic material (genome) of an organism

Genome

the total genetic material of an organism

Comparative Genomics

compare genomic features including the DNA sequence, genes, gene order, regulatory sequences, and other genomic structural landmarks

Phylogenomics

the intersection of the fields of evolution and genomics, using genomic information to reconstruct the evolution

DNA replication

DNA unzips into two parts and splits with the cell. In it's new home each side of the DNA strand attack to matching nucleotides to create 2 exact copies. It is important in puberty and other times of growth as it is the reproducing of your cells.

DNA sequencing for fungal identification

1) Extract DNA from pure culture 2) PCR amplification of ITS region / well-conserved sections 3) Sequence the amplified DNA strands using either the Sanger or HTP 4) conduct a BLAST analysis to identify/confirm

why is starch easier to hydrolosize than cellulose

starch has alpha bonds between glucose monomers while cellulose has beta bonds, which are harder to cleave, as well as a robust crystalline composition. Cellulose degradation requires a synergistic mixture of enzymes for degradation while starch mainly relies on amylase

CRISPR-Cas9

used to edit parts of the genome by removing, adding or altering sections of the DNA sequence to create or enhance desired traits... uses a "molecular scissors"

three types of cellulases and their synergy

1. Endoglucanases cleave middle of polymer chain, creating "free ends" 2. Exoglucanse cleaves glucose dimer (cellobiose) from one end of the polymer chain 3. beta-glucosidase turns dimers into monomers

the free ends are created by the endoglucanase for the exoglucanase and the crystalline regions are opened by the exoglucanase for the endoglucanase

What are the 4 methods of enzyme improvement?

additive, chemical modification, immobilization and protein engineering

What are the goals of bioprocessing?

fast, selective, efficient, robust, low cost, compatible with other enzymes, multifunctional

What are the top 3 remediation methods?

Incineration, thermal desorption, solidification

What is the difference between lateral/horizontal and vertical gene transfer?

lateral is donor to unrelated recipient, vertical is parent to child

how can you identify fungal species using DNA sequencing?

extract dna, amplify via PCR, verify PCR, sequence, search

Why is hydrolysis of cellulose harder than hydrolysis of starch?

there are more hydrogen bonds between glucose units and the crystalline structure

Which EC group are starch degrading enzymes in?

EC 3 hydrolases

True or False, enzymes are only proteins

False, there are also RNA enzymes

What does the number signify in fuel types eg. E-15

The percent ethanol

What makes lignocellulose renewable

reduces carbon emissions, grows back fast

what is the difference between forward and reverse genetics?

forward: a known phenotype is used to discover gene

reverse: a known gene is used to discover phenotype

What processes are used in forward genetics?

Mutagenesis, Positional cloning

What processes are used in reverse genetics?

Gene targeting, Ectopic expression

bacterial transformation

process by which bacterial cells take up naked DNA molecules (plasmids)

bacterial transduction

DNA transferred from one bacterium to other via virus

bacterial conjugation

the direct transfer of genetic material (DNA) from one bacterial cell to another via sex pilus

what is the most prevalent ligninase?

Lignin peroxidase

What are the commercial uses for cellulose?

paper product, cellophane, possibly ethanol

What are the 4 advantages of fungal bioremediation

high surface to volume ratio

slimy sheath

can tolerate desiccation and temperature changes

can break down many pollutants