Session 3: Clean Technologies and Biomaterials
Lectures
L3.2
L3.1
Biotechnological methods in
the modern cosmetology
Directed evolution of
glycosylating enzymes
Tom Desmet, Manu De Groeve, Wim Soetaert
Ghent University, Center for Industrial Biotechnology and Biocatalysis,
Belgium
e-mail: Tom Desmet <[email protected]>
Glycoside phosphorylases (GPs) are a group of enzymes
with characteristics in between those of hydrolases and
transferases. Their physiological role is the degradation of
the glycosidic bond in disaccharides with the use of inorganic phosphate. However, GPs can also be used for the
synthesis of glycosidic bonds in vitro. For that, they only
need a glycosyl phosphate as donor substrate, which is
much cheaper than a nucleotide-activated donor. To broaden the synthetic applications of GPs, we have modified
both the donor and acceptor specificity of cellobiose phosphorylase (CBP) by means of enzyme engineering.
As new glycosyl donor, we have chosen galactose-1-phosphate, which differs from the natural donor only in the orientation of the C4-hydroxyl group. The corresponding disaccharide substrate is lactose, which has been used for the
selection of mutant libraries on a minimal medium. Random mutations were introduced by means of error-prone
PCR, revealing two aminoacid positions that influence the
donor specificity of the enzyme. In that way, an enzyme
was created that efficiently phosphorolyses lactose, an activity that has never before been observed in nature [1].
To modify the acceptor specificity of CBP, we have followed a more rational approach by examining the enzyme’s
crystal structure [2]. Several aminoacid residues were identified in the active site that interact with the acceptor substrate. The corresponding positions were submitted to
site-saturation mutagenesis, generating activity towards a
number of alkyl and aryl glucosides. Combining the modified donor and acceptor specificity of CBP allowed us to
develop a biocatalytic production process for methyl lactoside, a compound that is known to inhibit metastasis of
lung tumor cells in mice.
References:
1. De Groeve M et al. (2009) Protein Eng Des Sel 22: 393–399.
2. Van Hoorebeke A et al. (2010) Acta Cryst F 66: 346–351.
Jacek Arct, Katarzyna Pytkowska
Academy of Cosmetics and Health Care in Warsaw, Warsaw, Poland
e-mail: Jacek Arct <[email protected]>
The presentation is an attempt to present the connection
between the oldest human needs – that of looking better
and younger and one of the most important new technology which is biotechnology. Last twenty years the application of biotechnological methods has started the new era
in cosmetology.
The use of biotechnological methods and technologies in
cosmetology could be divided into three main areas:
— Skin biotechnology including cell and tissue cultures;
— Skin bioengineering;
— Production of cosmetic raw materials via fermentation
and biocatalysis processes.
The best method for the preliminary investigations of
cosmetic actives are keratinocytes and fibroblasts cultures.
Using appropriate biochemical tests a lot of the of possible influences on the skin could be predicted. Last years
such a methods were successfully used for the evaluation
of biologically active LMW peptides. Reconstituted dermis,
epidermis and epithelia are widely used for in vitro cosmetological tests like irritation, pigmentation, depigmentation,
photoprotection and percutaneous absorption as replacement of the banned animal tests.
Modern methods of the in vivo evaluation of cosmetic
products are based on the skin engineering technology.
They concerns mainly noninvasive tests done on the human skin in vivo. The data obtained give precise information about skin condition like barrier properties, level of
moisturisation, mechanical properties, relief and colour,
bioavailability of cosmetic actives etc.
Biotechnological processes have a very broad use in production of cosmetically valuable biopolymers like hyaluronic acid, chitosan, xantans, fucose-rich oligosaccharides and polypeptides of different activity. Also many low
molecular weight actives unavailable by chemical synthesis
are produced by fermentation. Examples are LMW peptides, GLA containing triglycerides, carotenoids, dihydroxyacetone, ubiquinone and many others. Microbial and
enzymatic methods are the excellent tools for transformation of natural products from renewable sources into important cosmetic components. One of the most important
are LMW peptides obtained from milk and soy proteins.
Biotechnological methods are also used for enriching plant
oils in valuable fatty acids, increasing bioavailability of plant
extracts and in many other cosmetic technologies.
The main impact of biotechnology is directed into medicine and health care but the knowledge gained on this field
could be used to create new methods and products crucial
for the development of modern cosmetology.
Eurobiotech 2010
Oral Presentations
O3.2
O3.1
The influence of surfactants and
hydrolyzed proteins on keratinocytes
viability and elasticity
Fungal laccase — blue enzyme
for green biotechnology
Anna Jarosz-Wilkołazka, Jolanta Polak
Biochemistry Department of Maria Curie-Skłodowska University, Lublin,
Poland
e-mail: Anna Wilkołazka <[email protected]>
Laccases are a third group of extracellular oxidoreductases
whose exploitation has been quite neglected until few years
ago despite the fact that they are quite abundant in nature,
particularly in fungi (Navarra et al., 2010). Laccases are ideally “green” catalysts, as they occur naturally, use air and
produce water as a by-product during oxidation of broad
spectrum of substrate molecules to corresponding reactive
radicals. Synthetic exploitation of laccases can follow two
different strategies: the direct oxidation of the substrates
or the oxidation of mediator molecules that then oxidize
the other target substrates (Mikolasch et al., 2009). Of the
multi-copper oxidases, the fungal laccases court considerable industrial interest. But even today few data are available on preparative scale laccase-catalyzed oxidation of
different compounds. Most reactions involve the modification of natural compounds, as for example, the synthesis
of antibiotics (actinocine, penicillin G), stilbene derivatives
(resveratrol), or steroid compounds (Polak et al., 2007).
In our group, fungal laccase and fungal biomass of the
strains with a well-known ability to secrete laccase were
used for biotransformation of specific precursors to dyes.
These precursors are commercially available chemicals and
can be oxidized into coloured compounds (Forte et al.,
2010). Obtained dyes displayed low toxicity and good dyeing properties of wool fibres. From the dyes and additives
used to dye fibres, to production processes and polluted
wastewater, the colour industry can have a significant impact on the health of customers, as well as on the environment.
References:
Forte S et al. (2010) J Mol Cat B: Enzym 63: 116–120.
Mikolasch A et al. (2009) Appl Microbiol Biotechnol 82: 6-5–624.
Navarra C et al. (2010) J Mol Cat B: Enzym 65: 52–57.
Polak J et al. (2007) Biotechnologia 4: 82–94.
Acknowledgements:
This work was partially supported by SOPHIED project (NMP2CT-2004-505899) and the Foundation for Polish Science Ventures Programme co-financed by the EU European Regional Development Fund.
53
T. Kobiela1, K. Leleń2, M. Stepulak2, M. Lekka3,
M. Malejczyk4, J. Arct2, S. Majewski2,4
1Warsaw University of Technology, Faculty of Chemistry, Institute
of Biotechnology, Warszawa, Poland; 2Academy of Cosmetics and
Health Care, Warszawa, Poland; 3The H. Niewodniczanski Institute of
Nuclear Physics, Polish Academy of Sciences, Kraków, Poland; 4Medical
University of Warsaw, Warszawa, Poland;
e-mail: Tomasz Kobiela <[email protected]>
The knowledge how surfactants and hydrolyzed proteins
influence epidermal kerationcytes elasticity is sparse. During past decades atomic force microscopy (AFM) was
widely used in the study of the structure and function of
a variety of biological systems in a liquid environment [1].
A great advantage of AFM is its ability to measure the
sample stiffness attributed to the state of the cytoskeleton
structure of living cells [2]. In the present study the effects
of sodium lauryl sulphate (SLS) and hydrolyzed collagen
(HK) of molecular weight 9kDa were examined with respect to human HaCaT keratinocytes viability and elasticity. In order to assess the impact of SLS and HK on cells
viability we applied the microtitration (MTT) viability test.
The viability of keratonicytes treated with SLS and HK of
various molar ratio was determined. The increase of SLS
concentration resulted in decrease of cells proliferation. It
was found that HK can inhibit this process. The strongest
effect was observed for the SLS:HK molar ratio 2:1. AFM
measurements of the keratinocytes stiffness were carried
out immediately after exposure of cells to the SLS and
HK respectively. After the treatment with SLS a dramatic
decrease in the cell stiffness was observed. On the other
hand the keratinocytes treated with SLS:HK molar ratio 2:1
showed the Young’s modulus values obtained for control
measurements.
References:
1. Matzke R et al. (2001) Nat Cell Biol 3: 607–610.
2. Lekka M et al. (2006) Chem Biol 13: 505–512.
Abstracts
54
O3.3
O3.4
Skin bioengineering in the field of cosmetic
efficacy testing
Cell cultures in assessment of safety
and efficiency of cosmetic products
Katarzyna Pytkowska, Jacek Arct
Karolina Leleń, Sławomir Majewski
Academy of Cosmetics and Health Care in Warsaw, Warsaw, Poland
Academy of Cosmetics and Health Care in Warsaw, Warsaw, Poland
e-mail: Katarzyna Pytkowska <[email protected]>
e-mail: Karolina Leleń <[email protected]>
The term “skin bioengineering” was introduced for the application of engineering tools in dermatology and cosmetology in late 1970’s. In the European cosmetic industry
appropriate product claim substantiation is required by law
from many years. This fact caused a mushroom growth of
non-invasive, instrumental, in vivo efficacy testing methods
at the end of the 20th century. The other direction of fast
skin bioengineering development is creating of in vitro epidermis and/or skin models suitable for safety assessment
of cosmetics and cosmetic raw materials.
Nowadays the majority of cosmetic efficacy testing methods are based on the skin bioengineering technologies.
The data obtained give precise, objective information concerning influence of the cosmetic on the skin condition
like barrier properties, level of moisturisation, mechanical
properties, relief and colour, bioavailability of cosmetic actives etc. Historically speaking the methods were designed
for supporting simple claims like “the cream moisturize the
skin” or “the cream decrease the number of fine wrinkles”.
At present these methodologies are applied commercially
also for more detailed claims and direct comparing the efficacy of different products, the third way of application
is an diagnostic support in cosmetology and dermatology.
Many valuable skin bioengineering tools like Fourier transform infrared spectroscopy (FTIR) and laser confocal
microscopy are still used for strictly scientific and experimental purposes, but some like optical digital imaging, ultrasound imaging and laser profilometry are used in clinical
practice and commercial efficacy testing. The most popular
simple tools, still developing, are techniques based on stratum corneum impedance and conductance measurements
(indirect assessment of skin water content in vivo) and skinsurface-level temperature and humidity gradient registering
sensors (transepidermal water loss in vivo measurement).
Safety assessment of cosmetics use and their efficiency is
very important issue both for the cosmetic manufacturers
and consumers. The cosmetic manufacturing companies
make efforts to fulfill all the legal norms and regulations in
force for this section of market. Based on the act, the cosmetic used in accordance with its indication and instruction
cannot endanger human health.
Cell cultures, mostly immortalized keratinocyte and fibroblast cell lines or epidermis equivalents are often employed
to assess cosmetic ingredient action as well as to confirm
marketing declarations of manufacturers. The present
study focuses on the opportunity to use cell culture-based
approaches for safety and efficiacy assesment of cosmetics
action as well as discusses opportunities and limitations of
these methods and their reference to in vivo conditions. The
study shows results of research on effect of the surface
active substances (basic ingredients in majority of cosmetic
products) on elementary biological functions of keratinocytes.
Eurobiotech 2010
Posters
P3.2
P3.1
An Antarctic alkalistable subtilisin-like
serine protease from marine bacterium
Pseudoalteromonas sp., strain 93
Polymer membranes for ophthalmic
surgery applications
Maria Grolik1, Krzysztof Szczubiałka1, Maria Nowakowska1,
Bogumił Wowra2, Dariusz Dobrowolski2, Edward Wylęgała2
1Faculty of Chemistry, Jagiellonian University, Cracow, Poland;
2Department of Ophthalmology, District Railway Hospital, Katowice,
Poland
e-mail: Maria Grolik <[email protected]>
Natural polymers have found widespread biomedical applications including drug and gene delivery, biosensors, and
biomaterials as they have numerous advantages, such as
biocompatibility, biodegradability, and anti-bacterial properties. They are safe for human use.
The aim of this work was to synthesize and to determine
the properties of a polymeric material for ophthalmic surgery applications. The cytotoxicity of the hydrogel scaffold
in the form of a membrane, that we have obtained from
chitosan (CH_UM) cross-linked with genipin, a naturally
occurring and nontoxic agent, was investigated.
A biodegradable chitosan membrane containing collagen,
seeded with keratinocyte stem cells was prepared as a novel
corneal epithelium substitute.
The result reveals that the CH_UM – genipin - collagen
scaffolds treated with 5% w/v of genipin had the highest porosity and swelling ratio (at the pH characteristic of
blood = 7.4). The optical microscope technique was used
to visualize the microstructure of scaffolds. Atomic Force
Microscopy (AFM) measurements revealed the topography
of the surfaces of membranes.
55
Ewa Gromek, Marianna Turkiewicz
Institute of Technical Biochemistry, Technical University of Lodz, Lodz,
Poland
e-mail: Ewa Gromek <[email protected]>
The Antarctic strains of microorganisms have been
screened for interesting bioactivities, particularly for coldadapted enzymes, called psychrozymes. These true psychrophilic biocatalysts are not only optimally active at low
temperature (15–20°C lower than their mesophilic counterparts) but also display the relatively high activity (even up
to 60% of the maximal one), and high catalytic efficiency
at temperatures characteristic of Antarctic habitats (–2÷
+4°C). These enzymes are interesting from both scientific
(structural background of cold-adaptation) and application
standpoints (possibility of lower temperatures of technological processes).
The strain of Pseudoalteromonas sp. no 93 from the pure
culture collection at the Institute of Technical Biochemistry of the Technical University of Lodz, isolated from
alimentary tract of Antarctic krill Euphausia superba Dana,
secretes extracellularly an alkalistable subtilisin-like serine
protease. The purified enzyme has a molecular mass of approximately 45 kDa and is stabilized by Ca2+ ions. Optimal
activity is exhibited at 45°C and pH 10.0 against natural
and synthetic substrates and is retained 12–30% maximum
activity in the temperature range between 0 and 20°C. The
protease is stable up to 55°C for 60 minutes, and at pH
11.5 it displays 70% maximum activity. The enzyme exhibits maximal activity in 0.5 M solution of NaCl and KCl, and
it is able to catalyze the hydrolysis of proteins in presence
of 2% solution of SDS and 10 M urea solution (50% and
88% of maximal activity, respectively). The protease had a
Km of 2.49 mM for N-SuccAAPFpNA at 30°C and the
value of activation energy in N-SuccAAPFpNA hydrolysis
was found to be 40.7 kJ×mol–1 in the temperature range
of 15–45°C.
The properties of the Antarctic serine protease from marine bacterium Pseudoalteromonas sp., strain 93, such as relatively high activity at low temperatures, alkaline optimal
pH, alkalistability and halotolerance demonstrate its high
potential for applied research.
Abstracts
56
P3.3
P3.4
Potential use of Melocanna baccifera
(Bamboo) raw and activated charcoals for
removal of Cd(II) from aqueous solutions
The microscopic filamentous fungus
Gliocephalotrichum simplex IM 2358 is
able to produce silver nanoparticles
H. Lalhruaitluanga, M. N. V. Prasad
M. Wawrzyniuk1, S. Różalska1, W. Kozłowski2,
Z. Klusek2, J. Długoński1
1University of Hyderabad, Department of Plant Sciences, Hyderabad,
India
e-mail: Lalhruaitluanga H <[email protected]>
Bamboo is the most abundant vegetation in the state of
Mizoram, which constitutes 14% of the total India’s bamboo area. Melocanna baccifera is the most abundant and economically important non-timber product in the state of
Mizoram, contributing about 95% of the growing stock of
bamboo. The community of the region uses this potential
resource in many ways, charcoal production is one of them.
Charcoal is mainly used to keep them warm during winter
in this community. Activated charcoal is prepared from M.
baccifera raw charcoal by chemical pretreatment. The motivation of the present study is to investigate further application of this bamboo charcoal in the field of environmental problem, so that this plant would be more valuable
for the community. For this, batch mode experiments were
conducted for pH (2.0–6.0), adsorbent dose (0.1–0.5 g),
contact time (15-360 min) and metal ion concentrations
(5.0–30.0 mg/L). SEM and FTIR analysis was also incorporated. Redlich-Peterson, Dubinin-Radushkevich, Freundlich and Langmuir isotherms were used to study adsorption isotherm. Pseudo-first-order, Pseudo-second-order,
Intra-particle diffusion and External mass transfer model
were used to study the kinetic of adsorption. From these
studies, the charcoal biomass could be used as adsorbents
for efficient treatment of Cd(II) from aqueous solution.
1Department of Industrial Microbiology and Biotechnology, University
of Lodz, Lodz, Poland; 2Division of Physics and Technology of
Nanometric Structures, University of Lodz, Lodz, Poland
e-mail: Magdalena Wawrzyniuk <[email protected]>
Due to the growing number of applications of metal nanoparticles, their synthesis becomes an interesting field in the
biotechnology research. Developing new, feasible, environmental-friendly methods seems to be an attractive area of
the study. Biological methods for nanoparticles production,
based on the biosynthesis, do not involve the use of toxic
reagents or formation of hazardous by-products, and are
relatively cost-effective. In this study, we report a possibility
of application of the microscopic filamentous fungus Gliocephalotrichum simplex IM 2358 [1] isolated from contaminated soil for silver nanoparticles manufacture. Mycelia-free
water extracts were used as a reducing agent. Additionally,
a biotic control (a fungal culture without silver ions) and
abiotic control (samples with silver ions but without the
fungus) were applied. After 72 h of silver ions treatment
with fungal filtrate, probes were tested on UV-Vis spectroscopy to confirm the presence of nanoparticles. A typical
peak for silver nanoparticles absorption (420 nm) was observed. Moreover, XPS and AFM analysis confirmed that
silver ions incubated with fungal extracts were successfully
reduced to metal nanoparticles. The XPS spectrum shows
a significant increase of carbon-characteristic peak and
further analysis indicate a significant amount of carbonoxide bounds which are present in nanoparticles coating.
The obtained results showed that the investigated fungus
Gliocephalotrichum simplex IM 2358 can be applied for silver
nanoparticles manufacture. References:
Rózalska S, Szewczyk R, Długoński J (2010) J Hazard Mater 180: 323–331.