5681 - BIOCHEMISTRY AND BIOCHEMISTRY LABORATORY
Academic Year 2018/2019
Free text for the University
ENRICO SANJUST (Tit.)
- Teaching style
- Lingua Insegnamento
|[60/56] INDUSTRIAL BIOTECHNOLOGY||[60/56-00 - Ord. 2016] PERCORSO COMUNE||12||120|
The course of Biochemistry for the Class Biotechnology is aimed to the acquisition of fundamental information and concepts in structural and functional biochemistry. Therefore, it gives basic biochemical knowledge for a first-level degree in Biology, and moreover it is preparatory to further learning courses, implying the knowledge of such fundamentals.
Knowledge and Understanding:
Understanding the Life molecular logic and the primordial abiotic chemistry.
Knowing structures and properties of the main biological compounds such as proteins, glucides, and lipids. Being able to graphically represent their molecules.
Understanding the structural and functional features of those compounds and their localization in cells and tissues.
Knowing and understanding structure/function relationship of the proteins, and their modulation: the case of respiratory proteins.
Knowing the modes of production, storage, use of metabolic energy, and understanding the related thermodynamics.
Knowing the main metabolic pathways and their molecular tools: enzymes, coenzymes, cofactors, effectors, inhibitors.
Understanding metabolic omeostasis and the general integration of metabolic pathways.
Applying Knowledge and Understanding:
The students will be able to:
- take advantage from structure formulas getting the main reaction mechanisms applied to biochemistry for both catalysis and metabolic sequences;
- correctly put the various metabolic pathways within the frame of physiological and eventually pathological mechanisms to get conclusions about material and energy balances;
- apply the acquired knowledge to the study of Molecula Biologu, Genetics, Physiology, Microbiology.
Students will be able to:
- distinguish among main and secondary topics, finding the logics lying under the study of Biochemistry, and avoiding a memory-based study;
- autonomously find and explain retrohinibition, cycles, and futile cycles, anaplerotic reactions;
- individuate the basic concepts to apply them to other disciplines along the Biology course.
The students will be able to discuss the topics of the course by using the language and vocabulary typical for Biochemistry. They will interact with the teacher arguing the main points of the study program with the required detail. Owing to the expert tutorage they will also positively interact with their colleagues, possibly forming study groups.
Student/teacher interaction, auto-evaluation tests, slides and booklets will give the students the required tools for profitable and aware learning.
The students must have a sound basic knowledge of both Inorganic and Organic Chemistry. In particular:
Atomic and molecular orbitals;
Molecules and ions, polarity and polarizability;
Chemical bonds: ionic, covalent, dative, coordinative, hydrogen;
Acids and bases following the various definitions, pH, pKa;
Chemical equilibrium, equilibrium constants, mass action law, Le Chatelier principle;
Oxidizers and reductants, electrochemical potential;
Speed of chemical reactions, order and molecularity of reactions, speed constants;
Basic organic chemistry, aliphatic and aromatic compounds, mesomerism, I and M effects; nucleophilic, electrophilic, and radical substitutions, and their mechanisms.
Main functional groups and their reactivity.
Course divided in two learning units: 1) Structural Biochemistry, and 2) Functional Biochemistry.
Structural Biochemistry (22 hours):
- The molecular logics of the Life, abiotic biochemistry: 2 hours;
The chemical constituents of the living organisms. Abiotic chemistry and biochemistry.
- Aminoacids and peptides, fibrous proteins: 8 hours;
Aminoacids: definition and classification, main physicochemical features. Aminoacids as electrolytes. Essential aminoacids. Peptides. Protein classification and structural levels. The main fibrous proteins.
- Globular proteins: 4 hours;
Main physicochemical features. Globular proteins as amphoionic polyelectrolytes. Glycoproteins. Immunoglobulins. Stability and denaturation of globular proteins.
- Glucides: 4 hours;
Definition and classification, main physicochemical features. Anomerism, mutarotation, glycosides. Mono-, oligo-, and polysaccharides.
- Lipids: 4 hours;
Definition, classification, main physicochemical features. Simple and complex lipids, lycolipids, phospholipids, sphingolipids. Isoprenoids.
Functional Biochemistry (42 hours);
Porphyrins, metalloporphyrins, heme, hemoproteins and their main physicochemical features.
Myoglobin and hemoglobins, oxygenation and deoxygenation, cooperativity and its logical models. Other respiratory proteins.
- Enzyme kinetics: 4 hours;
Catalysis and biocatalysis, enzymes. Molecularity and reaction order of enzymatic reactions. Michaelis and Menten hypothesis and its developments. Enzyme activity inhibition and modulation.
- Enzymatic catalysis: 4 hours;
Physicochemical background of affinity and catalysis. Theory of the activated complex and enzymatic catalysis. Biocatalytic mechanisms.
-Vitamins and coenzymes: 6 hours;
Definition and classification. Hydrosoluble vitamins, coenzymes and cofactors.
- Metabolism and energy metabolism: 4 hours;
Definition and concepts: metabolism, anabolism, catabolism, biosynthesis, biodegradation. Aerobic and anaerobic metabolism. Metabolic paths and cycles, energy production, transportation, storage of metabolic energy: ATP and analogues.
Glucidic metabolism: 8 hours;
General concepts, glycolysis, lactic and alcoholic fermentations. Citrate and glyoxylate cycles.
- Lipid metabolism: 4 hours;
General concepts, beta-oxidation, particular cases, AcetylCoa fate, ketogenesis.
- Protein metabolism: 2 hours;
General concepts, transamination, deamination, ammonia fate, ornithine cycle.
- Respiratory chain: 2 hours;
General concepts, constituents, dioxygen reduction logics, consequences and purposes.
- Oxidative phosphorylation: 2 hours;
general concepts, ATP synthase and its mechanism, respiratory control, uncoupling agents.
Methods (22 hours):
Aminoacids titration (2 hours).
Enzymology of lignin and fungal laccases (2 hours).
Protein purification: cell lysis, fractionated precipitation, centrifugation, dialysis, filtration (4 hours).
Chromatography. TLC, IEC, SEC, IMAC, RP, HIC, HPLC (6 hours).
Spectrophotometric analysis: UV-visible. Total protein quantification, and enzymatic assays (4 hours).
Electrophoresis: native PAGE, SDS-PAGE, staining, IEF, blotting (4 hours).
Laboratory (50 hours):
Pipettes and micropipettes (5 hours)
Preparation of buffer solutions and culture media (5 hours).
Liquid cultures of Pleurotus pulmonarius, with the addition laccase inducers (5 hours).
Laccase activity assay (5 hours).
Purification of laccase with calcium phosphate-based ionic exchange (5 hours).
Ion exchange chromatography of the clarified laccase (5 hours0
Zymogram with Native PAGE. Determination of laccase MW by SDS-PAGE (5 hours).
Total protein determination with Bradford method. Aminoacids TLC (5 hours).
GEl filtration of human hemoglobin.
Lactase immobilization and production of high lactose-reduced milk (5 hours).
Antioxidant evaluation of plant extracts (5 hours).
The course stands along 12 weeks with 48 hours of lectures (4 hours/week) and 72 hours in lab (5 hours/week). Lectures are carried out by using Power Point slides, also taking advantage from animations to visualize reaction mechanism and 3D molecular models.
Verification of learning
1.3. Final decision
Examinations are aimed to a correct and unbiased evaluation of the preparation degree reached by students, i.e. the achievements of the formative targets required in the course.
Individual colloquium, also requiring the use of graphics, equations, schemes, flux diagrams. From general to particular, the student will move among basic concepts to proceed in depth within the topic.
1.3. Final judgement
The final grade takes into account several factors:
Quality of the knowledge, skills, competences:
a) appropriateness, accuracy and consistency of knowledge
b) appropriateness, accuracy and consistency of abilities
c) appropriateness, accuracy and consistency of ability to apply knowledge and understanding
a) Ability of expression;
b) Proper use of the specific language of the discipline;
c) Logical skills and inherent consequentiality in communicating;
d) Manual and practical skills;
e) Ability to connect different subjects by finding the common points and establish a consistent overall design, i.e. taking care of structure, organization and logical connections of speech;
f) Ability to summarize also through the use of specific symbolism of each discipline and graphic expression of ideas and concepts, for example in form of formulas, schemes, equations.
Availability to exchange and interact with the teacher during the interview.
a) critical thinking;
b) ability of self-evaluation.
Consequently, the judgment can be:
a) Sufficient (from 18 to 20/30)
The candidate demonstrates little acquisition of theoretical knowledge, superficial level, many gaps. Modest communicative abilities, but still sufficient to support a coherent dialogue, logical capacity and consequentiality in fitting the subjects of elementary level; poor capacity of synthesis and rather stunted ability of graphical expression, scanty interaction with the teacher during the interview.
b) Moderate (21 to 23)
The applicant demonstrates a moderate acquisition of knowledge but lack of expatiation, a few gaps; communicative abilities more than sufficient to support a coherent dialogue; acceptable mastery of the scientific language, logical capacity and consequentiality in fitting the subjects of moderate complexity, good enough capacity of synthesis and acceptable ability of graphical expression.
c) Good (24 to 26)
The candidate demonstrates a rather large wealth of knowledge, moderate in-depth, with small gaps; satisfactory mastery of the communicative abilities and meaningful scientific language; dialogical ability and critical thinking well detectable, good capacity of synthesis and more than acceptable ability of graphical expression.
d) Outstanding (27 to 29)
The candidate demonstrates a very extensive wealth of notions, high in-depth, with marginal gaps; remarkable ability in communicating and high mastery of scientific language; remarkable dialogical capacity, good competence and relevant aptitude for logical synthesis, high capacity of synthesis and graphical expression.
e) Excellent (30)
The candidate demonstrates a wealth of very extensive and in-depth knowledge, irrelevant gaps, high capacity and high mastery in communicating through the scientific language; excellent dialogical ability and marked aptitude to make connections among different subjects, excellent ability to synthesize and very familiar with the graphical expression.
The praise is attributed to the candidates clearly above average, and whose notional, expressive, conceptual, logical limits, if any, as a whole are completely irrelevant.
Indications always refer to the most recent editions.
1) Abeles, Frey, Jencks. Biochimica. Piccin
2) Baynes, Dominiczac. Biochimica per le discipline biomediche. Ambrosiana
3) Berg, Tymoczco, Stryer. Biochimica. Zanichelli
4) Voet, Voet. Pratt. Fondamenti di Biochimica. Zanichelli
5) Campbell, Farrell. Biochimica. EdiSES
6) Garrett, Grisham. Principi di Biochimica. Piccin
7) Nelson, Cox. I Principi di Biochimica di Lehninger. Zanichelli
8) Ninfa, Ballou. Metodologie di base per la biochimica e la biotecnologia. Zanichelli
For deeper insights:
1) Voet, Voet. Biochemistry. Wiley
2) Branden, Nooze. Introduzione alla struttura delle proteine. Zanichelli
3) Leuzzi, Bellocco, Barreca. Biochimica della nutrizione. Zanichelli
Slides of the lectures and booklets on specific topics are freely available to the students.