Marine Microbial Enzymes: Biotechnological & Biomedical Aspects

Presented by

Akram Najafi

Introduction:

• With the development of science, enzymes became inevitable for a range ofindustrial processes.

• Enzymes play a key role in “green” industrial processes – they arerenewable, biodegradable, eco-friendly, and nonhazardous for a worker’shealth.

Currently, enzymes are largely used in :

• food processing industry

• detergent industry

• pharmaceutical industry

• organic synthesis

• biotechnology

• biofuels

• biomedicine

• Although many important enzymes have been isolated from plants and animals.

problems:

• large-scale cultivation

• disruption of ecosystem

• depletion of resources

Microbial enzymes:

• diverse biochemical properties

• easy genetic manipulation

• large-scale production

• Marine ecosystem: hot and cold streams, acidic and alkaline water flow, bright

surface, dark deep sea with high barometric pressure, hypersaline hydrothermal

vents, or cold seeps enriched with different minerals/gases and deep sea

volcanoes.

• This complex environment contains extremely diverse microbial populations,

including several extremophiles that can survive in and even may require

extreme physicochemical conditions that are detrimental to most living systems

on Earth.

Marine microorganisms have excellently adapted to diverse environmentalparameters:

• high salt concentration

• extreme temperatures

• acidic and alkaline pH

• extreme barometric pressure

• low nutrient availability

• Marine microbial enzymes are of special interest due to their better stability,activity, and tolerance to extreme conditions that most of the other proteinscannot withstand.

• So far archaea, extremophiles, and symbiotic microorganisms are reported to besources of most of the interesting marine enzymes with distinct structure, novelchemical properties, and biocatalytic activity.

Detergent Industry

• Enzymes are being used in the detergent industry since 1913 and the firstbacterial protease containing detergent was available in 1956.

• Most important enzymes in this industry:

proteases, lipases, cellulase and amylase

• Ideal enzymes for detergent application:

- broad substrate specificity

- activity over a broad range of temperatures and pH

- compatible with other ingredients of detergent formulation

- active in the presence of organic solvents

Chemical and Pharmaceutical Synthesis

• Marine enzymes are of special interest for their unique catalytic properties, novelstereochemical properties, and solvent stability.

• Marine enzymes are reported to produce pure racemic compounds that are notobserved in normal catalysis. Several examples are found in the class of lipase,esterase, and oxidoreductase.

• Lipase B from Candida antarctica is a commercial enzyme with highenantioselectivity and is used in enzymatic acylation of Nelarabin for the

production of its 5’-monoacetate derivative, which is an antileukemic agentwith higher solubility and thus with better bioavailability.

C. antarctica lipase B is active in the presence of organic solvents that facilitate

their use in catalyzing transesterification reactions. It is being studied for its

potential application for enzymatic synthesis of vitamin E acetate by

lipasecatalyzed transesterification

Biotechnological Research

• Enzymes are well-known tools of biotechnological research.

• Two thermostable DNA polymerases from marine microorganisms:

• - Thermococcus litoralis (Vent polymerase, New England Biolab)

- Pyrococcus furiosus (Pfu polymerase, Stratagene)

• DNA ligase from the marine isolate Thermus thermophilus is another

important enzyme in biotechnological research.

• Several restriction endonuclease, including:

AspMD1, DmaI, DpaI, AgeI, HjaI, Hac1, Hsa1, and Hag1, were isolated from

marine bacteria and some of them are already commercially available.

Biomedical Aspects

• Enzymes can be used as therapeutic agents in treating a range of physiological

disorders starting from digestive problem to neoplastic disorders.

• High affinity and specificity of each enzyme to a particular substrate is the majoradvantage for their therapeutic applications.

• Stability at acidic pH of gastrointestinal tract is the biggest challenge for anenzyme to qualify for digestive applications.

• Many plant and animal enzymes were classically used as digestive

enzymes without any remarkable tolerance to acidic pH.

• Now partially replaced by microbial enzymes and recombinant

proteins.

• Acid-stable marine microbial enzymes can be a good replacement

for these traditional digestive enzymes.

• Collagenases have medical applications in wound healing. They are effective forthe removal of dead tissue from wounds, burns, and ulcers, which can speed upthe growth of new tissues and skin grafts.

• Collagenase also has the ability to inhibit the growth of some contaminantpathogens and is used in combination with some antimicrobial agents.

• Two amino acid hydrolyzing enzymes – asparaginase and glutaminase – havepotential application as anticancer agents.

• There is growing interest in screening of these enzymes for their exploitation as

anticancer drugs.

• Pseudomonas fluorescens is a marine bacterium that produces a salt-tolerantLglutaminase reported to have antineoplastic activity.

• Marine microorganisms are well-known sources of these two enzymes.

• RNA is the sole genetic material in severalpathogenic viruses, which can be killed byribonuclease enzymes.

• Several ribonucleases are being studied for theirpotential application in treatment of HIV andother viral infections, but mostly from higherorganisms.

• Microbial ribonucleases can be studied, andscreening of marine microorganisms may open apotential field of antiviral research.

Biomedical applications of enzymes are being restricted by several factors:

• First, enzymes are too large to be distributed within the cells and that is why the

enzyme therapy cannot be used to treat any disease at genetic level.

• Second, in most cases, enzymes are foreign proteins to the human body and are

susceptible to antigenic reactions that may cause mild allergic reactions to severe

life-threatening immune responses.

• Third, enzymes have very short effective lifetime in blood circulation that may not

be enough to complete the enzymatic reactions to treat some disorders.

Entrapment of enzymes in nonproteinaceous materials may help in most cases, but

many of these materials often cause undesired side effects.

Bioremediation and Biofouling

• Bioremediation is the technique for removal of pollutants by biological means,mainly by the use of microorganisms.

• Marine microorganisms have added advantages of adaptability to high salinity,high temperatures, and extreme pH.

• Although almost all large-scale bioremediation techniques deal with intactmicroorganisms, several enzymes were tested for this purpose.

• However, large-scale production and immobilization of enzymes may not befeasible for bioremediation applications.

• Biofouling is attachment and growth of marine biomasses on submergedsurfaces.

• It increases surface roughness, damage surface coating, and enhance corrosion

of surface materials.

• Historically, biofouling is controlled by the use of antifouling coatings on

submerged surfaces that prevent microbial attachment by releasing biocides,

which are toxic to marine wildlife and hazardous to marine environment.

• Extracellular polymeric matrixes reported to play key role in biofouling. These

matrixes are composed of polysaccharides, proteins, glycoproteins, and

phospholipids– hydrolytic enzymes can potentially prevent their deposition and

inhibit biofouling at the very initial step.

• They must be stable/active in highly saline marine environments and over a

broad range of pH and temperature.

• Logically, in this environment, marine microbial enzymes should perform

better over their terrestrial counterparts.

Concluding Remarks and Perspectives

• Hundreds of marine microbial enzymes are already reported, but most reports

are concluded with bioprospecting and physicochemical characterization of the

isolated enzymes.

• A few of them are further studied for cloning and gene overexpression,

laboratory-scale product optimization, and structural profile.

• Enzyme immobilization is a very important technique for repeated catalysis in

industrial processes. It is inevitable in terms of cost- effectiveness of industrial

biocatalysis, but it is rarely practiced in marine biotechnology.

• Future research need to focus on protein engineering, structural profile, and

scale-up and downstream processing of marine microbial enzymes.

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