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Extraction of Hyaluronic Acid from Aloe barbadensis (Aloe Vera)

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Hyaluronic acid have a high moisture preservation and biocompatibility characteristic, thus allowing various usage of this substance in pharmaceutical, medicinal, and skin care products. Present manufacturing process of hyaluronic acid requires extraction of animal cells or through other methods utilizing bacteria. The aim of this research is to investigate an alternative source of hyaluronic acid extraction using plant based which is aloe vera (A. Barbadensis). Three main parts of aloe vera (rind, mesophyll and gel) underwent several steps of extraction process and the result was compared to the sample of hyaluronic acid from goat brain. The evaluation including comparison of total carbohydrates, reducing sugars and degradation using heat treatment. The results of extraction were analyzed using UV-Spectrophotometer at 230 nm and compare to the extraction result of goat brain to ensure the presence of hyaluronic acid. It was found out that the rind part of aloe vera have the highest potential of high content of hyaluronic acid.
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Journal Home: https://journal.sgu.ac.id/jffn 2020:1(2), pp.95-102
95
EXTRACTION OF HYALURONIC ACID
FROM ALOE BARBADENSIS (ALOE VERA)
Runita Rizkiyanti Putri
Tutun Nugraha
Stephanie Christy
Department of Chemical Engineering, Faculty of Life Sciences,
International University Liaison Indonesia, 15345
ABSTRACT
Hyaluronic acid have a high moisture preservation and biocompatibility characteristic, thus
allowing various usage of this substance in pharmaceutical, medicinal, and skin care products.
Present manufacturing process of hyaluronic acid requires extraction of animal cells or through
other methods utilizing bacteria. The aim of this research is to investigate an alternative source of
hyaluronic acid extraction using plant based which is Aloe barbadensis (aloe vera). Three main
parts of aloe vera (rind, mesophyll and gel) underwent several steps of extraction process and the
result was compared to the sample of hyaluronic acid from goat brain. The evaluation including
comparison of total carbohydrates, reducing sugars and degradation using heat treatment. The
results of extraction were analyzed using UV-Spectrophotometer at 230 nm and compare to the
extraction result of goat brain to ensure the presence of hyaluronic acid. It was found out that the
rind part of aloe vera have the highest potential of high content of hyaluronic acid.
Keywords: Aloe vera; extraction; hyaluronic acid.
ABSTRAK
Asam hialuronat memiliki kemampuan untuk mempertahankan kelembapan serta
biokompatibilitas yang tinggi, hal ini menjadi alasan asam hialuronat banyak digunakan dalam
produk farmasi baik yang berhubungan dengan obat maupun perawatan kulit. Pada proses
pembuatan asam hialuronat, ektraksi dari sel hewan masih merupakan sumber utama disamping
penggunaan metode alternatif menggunakan beberapa jenis bakteria. Tujuan dari penelitian ini
adalah untuk menginvestigasi sumber alternatif pengekstrasian asam hialuronat menggunakan
bahan dasar tumbuhan yaitu Aloe barbadensis (liday buaya). Terdapat tiga bagian dari lidah buaya
yang melewati beberapa tahap ekstraksi (kulit, mesofil, dan jel), hasil dari ekstraksi kemudian
dibandingkan dengan sampel asam hialuronat dari otak kambing. Evaluasi mencakup perbandingan
karbohidrat total, penurunan kadar gula, dan degradasi molekul menggunakan panas. Analisis
terakhir menggunakan UV-Spektrofotometer di panjang gelombang 230 nm dan dibandingkan
dengan hasil ekstraksi dari otak kambing untuk memastikan keberadaan asam hyaluronat. Hasil
analisis menunjukan bahwa kulit lidah buaya memiliki potensi mengandung asam hialuronat yang
cukup tinggi.
Kata kunci: Asam hialuronat; ekstraksi; lidah buaya.
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
2020:1(2), pp.95-102 J. Functional Food & Nutraceutical
96
INTRODUCTION
Hyaluronic acid (HA) is a biological occurring
polymer which has substantial biological functions
in almost every organism (Necas, et.al., 2008). In
humans, HA can be found in skin, vitreous of the
eye, umbilical cord, and synovial fluid, but it is
also present in body’s tissues such as skeletal
tissues, heart valves, lungs, brain, and many others
(Meyer K., Palmer, J.W., 1934). Hyaluronic acid
was located predominantly within extracellular and
peri cellular matrix, although correspondingly
existed on the intracellular cell (Balazs, et al.,
1986).
Resources to gain hyaluronic acid were commonly
taken from various animal tissues such as human
umbilical cords, rooster combs, bovine vitreous
humor, and bovine synovial fluid (Liu, et. Al.,
2011). At present day, even though production
through animal-based tissues still remain unshaken
to be the major pathway for large HA production,
another possibility of production systems have been
demanded because of some disadvantages of the
existing process. Due to the grinding procedure and
several repetition of using acid and organic
solvents, both practical and mechanical issues will
always happened in animal extraction in terms of
cost and safety (Widner, et. al., 2005).
Another issue is that HA from animal tissues may
remain connected to a HA-specific binding cellular
proteins of hyaluronidase (Fraser, et al., 1997).
Hyaluronidase is undesirable since it may trigger
the risk to prohibit an immune response.
Furthermore, transmitter of infectious diseases in
form of nucleic acids, prions, and viruses may well
increases within extraction procedure (Shiedlin, et
al., 2004). Lastly, the procedure are expensive and
require a long period of time, labor, and advanced
facilities to accommodate processes involved from
animal extraction until purification of HA (Shlini, et
al., 2017). Hence, it is preferred to generate
hyaluronic acid via an animal cell-free system that
could reduce contagion of undesirable contaminant
and expense of manufacturing (Widner, et al.,
2005) and (Yu & Stephanopoulos, 2008).
Therefore, this research was arranged to find
another pathway of extracting hyaluronic acid from
a plant source, which according to (Shlini, et al.,
2017) has proved to be successfully done from
sweet potato and tapioca (Sana, et al., 2017).
Moreover, aloe vera (A. barbadensis) was chosen
due to its popularity to the public and considerably
easy to be harvested in Indonesia.
In this research A. barbadensis is chosen as the
potential source of HA due to similarities with HA
in compositions and biological activities. Both aloe
vera and hyaluronic acid proven to promotes
wound healing (including dermatology
applications), anti-inflammatory and therapeutic
benefits. Moreover, A. barbadensis and hyaluronic
acid have been used for dermatology purposes due
to their abilities to retain water. There are three
major parts of A. barbadensis used in this research,
those are: rind, mesophyll, and gel. Rind is the
external surface waxy cuticle which performs as a
wall in a contradiction to moisture loss. Rind
covers several levels of structures, with slight
beneath from the waxy cuticle remains an area
where the aloe related bacteria live (Sushruta, et
al., 2013). Mesophyll is a liquid yellow-brownish
part of aloe vera which holds the xylem and
phloem vascular bundles. Mesophyll has the
biggest concentration of anthraquinones and
chromones of the whole aloe vera. Last part of aloe
vera is the gel which located inside the inner
parenchyma part of aloe vera. It consist of two
components: juice of the gel and fibrous pulp
enriched with cellulose.
Commercial manufacturing of hyaluronic acid is
built on either animal-based extraction or
genetically modified strains of bacterial
fermentation. Both of these pathways are
commonly applied and proved to manufacture
hyaluronic acid products with molecular weights
above 10 kDa that was suitable for medicine and
dermatology usage (Liu, et al., 2011). Biological
properties of hyaluronic acid are connected with its
molecular weight, hence there is a great interest in
HA degradation and evaluation of the biological
behavior of HA fragments. Mechanisms of the HA
cleavage into its smaller fragments involve
enzymatic, free radical, thermal, ultrasonic, and
chemical methods such as acid and alkaline
hydrolysis (Soltes, et al., 2007).
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
J. Functional Food & Nutraceutical 2020:1(2), pp.95-102
97
MATERIALS AND METHOD
Materials
All aloe vera (A. barbadensis) and fresh goat brain
were purchased from a market in Tangerang,
Indonesia. The chemicals used for this research
were acetone (Amresco), chloroform (Merck),
methanol (FULLTIME), sodium acetate (CV. Bina
Sejahtera), L-cysteine (Merck), acetic acid (Merck),
37% hydrochloric acid (Sigma Aldrich),
ethylenediaminetetraacetic acid/EDTA (Disolvin),
distilled water, sodium chloride (HiMedia
Laboratories), absolute ethanol (FULLTIME),
sulfuric acid (J.T Baker), ice cubes, sea salt,
sodium carbonate (Merck), anthrone (Merck),
sodium hydroxide (Merck), potassium sodium
tartrate tetrahydrate (PUDAK Scientific),
dinitrosalicylic acid/DNS (Sigma Aldrich).
Equipment
M254A BEL Engineering Weighing balance, water
filtration system (Hydro Water Solution PT. Hydro
Water Technology), hotplate stirrer (WiseStir
MSH-20D), MColorpHast pH-indicator strips,
centrifuge (Type 80-2 China), refrigerator
(Electrolux), autoclave HG 50 Hirayama, Phillips
food processor/grinder, PG Instruments T60 UV-
Visible Spectrophotometer, and VWR V-1200
Visible Spectrophotometer.
Extraction Process
The extraction methodology is based on the studies
being performed by (Shlini, et al., 2017) with sweet
potato (Ipomoea batatas) and (Sana, et al., 2017)
with tapioca (Manihot esculenta). In this research,
aloe vera (A. barbadensis) will be taken as the plant
source and goat brain as sample of pure hyaluronic
acid. The samples were washed thoroughly, parts
of aloe vera were separated by knife and each of
the four samples were homogenously crushed. 50 g
of each sample was submerged in 50 mL of
acetone and stirred for an hour. Chloroform and
methanol with ratio 2:1 was used to incubate 100
mL sample for 24 hours at 25C. Followed by
digestion buffer (100mM sodium acetate pH 5.0,
5.0mM cysteine and 5.0mM disodium EDTA) that
arranged in a ratio 2 mL of buffer to 100mg of
tissue. The sample was hydrated inside the
digestion buffer for 44 hours at 5C before
centrifuge at 3200rpm for 30 minutes. The solvents
was removed and the solid filtrate was splashed by
3 mL of 2.0M sodium chloride and followed by
absolute ethanol. Absolute ethanol was inserted in
ratio of 2:1 and kept for 24 hours at -16C. The
next procedure was centrifugation at 3200 rpm for
30 minutes. Sequentially, the supernatant was taken
away and the solid filtrate was washed with 80%
ethanol. Second centrifugation was done as
previous one before supernatant was discarded and
the solid filtrate dried for 24 hours at 25C. The
final solid was re-suspended in 5 mL of distilled
water and stored inside a test tube.
Total Carbohydrate Analysis using Anthrone’s
Method (Hodge, et al., 1962)
0.1 g of sample was boiled for 3 hours with 5 mL
of 2.5N-HCl, then cooled to room temperature with
ice and salt. The sample was neutralized by adding
solid sodium carbonate until the effervescence
ends. The sample was made up to the volume of
100 mL and centrifuged at 3200 rpm for 15
minutes. The supernatant was collected to prepare
1mL aliquots for analysis. The sample was added by
4mL of fresh anthrone reagent (dissolve 0.2 g of
anthrone in 100 mL of ice cold H2SO4) and heated
for 8 minutes in a boiling water. The sample was
rapidly cooled with ice and sea salt and observed at
absorbance of 630 nm in a visible
spectrophotometer.
Reducing Sugar Analysis using DNS Method
(Garriga, et al., 2017)
DNS reagent was prepared by making two
mixtures; Solution A (1 g of DNS was dissolved in
20 mL of NaOH 2M) and Solution B (30 g of
potassium sodium tartrate tetrahydrate was dissolved
in 50 mL of distilled water). Solution A was added
into Solution B, heated, and mixed on a hot plate at
300C and 370 rpm. This new solution was
completed to the volume of 100 mL with distilled
water and stored in amber bottle at refrigerator
(4C). This solution was named as DNS reagent. 1
mL of each sample was placed into a test tube and
added by 1 mL of DNS reagent. The test tube was
heated in a boiling water for 5 minutes and cooled
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
2020:1(2), pp.95-102 J. Functional Food & Nutraceutical
98
by ice and sea salt to room temperature. The
sample was added by 8 mL of distilled water and
read at 540 nm in a visible spectrophotometer.
Fragmentation of Hyaluronic Acid (Lowry and
Beavers, 1994) and (Botner, et al., 1988)
Degradation of the pre-assumed HA sample and
goat brain sample were done through thermal
degradation. 10 mL of each sample was taken into
small bottle and inserted into the autoclave for 4
hours at 128C. Sequentially, the sample was
observed using UV-spectrophotometer in 230 nm
wavelength.
RESULTS AND DISCUSSION
Total Carbohydrate Measurement
Hyaluronic acid is a carbohydrate compound, more
specifically a repeated glycosaminoglycan (GAG)
which formed of ß4-glucuronic acid and ß3-N-
acetylglucosamine (Meyer K, 1934). Hyaluronic
acid occurred in a high molecular weight due to the
repetition of glucuronic acid and N-
acetylglucosamine that able to goes up to a
thousand repetition even further as can be seen
from figure 1.
Figure 1. Structure of hyaluronic acid monomer (Cowman & Matsuoka, 2005)
Anthrone’s method was used to measure total
carbohydrate content from three different part of
aloe vera samples (rind, mesophyll and gel) to be
compared to total carbohydrate content of
hyaluronic acid from natural source, in this case
goat brain. This method used as the initial stage to
identify hyaluronic acid.
As can be seen from Figure 2, all of aloe vera’s
parts (rind, mesophyll, and gel) were proved to
show some value of absorbance at 630 nm, which
showed that aloe vera does contains carbohydrate.
Goat brain as the hyaluronic acid source showed
highest peak with the value of absorbance of 0.034
followed by rind with absorbance of 0.023. From
three parts of aloe vera (rind, mesophyll and gel),
rind part showed highest and closest absorbance to
hyaluronic source from goat brain, but to be certain
further analysis through reducing sugar needs to be
done.
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
J. Functional Food & Nutraceutical 2020:1(2), pp.95-102
99
Figure 2. Graph showing comparison of total carbohydrate of aloe parts with goat brain
Reducing Sugar Measurement.
Anthrone method only cover the general picture of
finding carbohydrate, hence another method is used
to observe more specific compositions of
carbohydrate which downgrade the structure from
polysaccharides into smaller fragments of
carbohydrates; reducing sugar. Moreover,
hyaluronic acid chemical structure is particularly
included a form of reducing sugar: ß-D-glucose
(Gunawardena, 2015), which made the essential on
doing DNS is highly proposed.
Figure 3. ß-D-glucose (Gunawardena, 2015)
DNS method was done as a complement procedure
from anthrone’s result to specifically qualify any
reducing sugars inside the sample. Total
carbohydrate analysis through anthrone’s methods
already showed that rind and goat brain has highest
and closest absorbance compared to other part of
aloe vera. Figure 4 below showed that all parts of
A. barbadensis have shown value of absorbance
which suggested contains reducing sugar. It should
be highlighted that both in anthrone and DNS
method, rind part of aloe vera showed the highest
absorbance 0.333 in comparison to mesophyll and
gel. In addition, rind have the closest absorbance to
goat brain (0.288) that contain high concentration
of hyaluronic acid in both anthrone and DNS
method thus conforming that rind have a very high
chance to contain hyaluronic acid. Based on these
findings, rind was chosen to undergo further
analysis step which is thermal degradation.
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
2020:1(2), pp.95-102 J. Functional Food & Nutraceutical
100
Figure 4. Graph showing comparison of reducing sugar of aloe parts with goat brain
Thermal Degradation
Hyaluronic acid is naturally occurred in a high
molecular weight, but since there are various
applications which came from different sizes of
molecular weight of hyaluronan, it prompted a HA
cleavage method to be performed. There were
numerous ways to decrease the molecular weight of
hyaluronic acid into smaller fragments which
engage with enzyme, free radical, heat, ultrasound,
and chemicals. Unfortunately, most of those
methods will produce unwanted toxic impurities
and demand a high cost. Thermal degradation of
hyaluronic acid proved to be successfully done by
(Botner, et al., 1988) at 128C in an autoclave.
Based on total carbohydrate and reducing sugar
measurement, rind has the highest chance of
containing hyaluronic acid, hence thermal
degradation analysis was done to conforming the
presence of hyaluronic acid in rind compared to
goat brain. Hyaluronic acid was proved to be
existed on the wavelength of 230 nm based on
several studies being done by (Shlini, et al., 2017)
and (Sana, et al., 2017). Therefore, the rind sample
and goat brain were gone through UV-
spectrophotometer before and after thermal
degradation to showed the existence of hyaluronic
acid.
Figure 5. Absorbance of rind before and after
thermal degradation
Figure 6. Absorbance of goat brain before and
after thermal degradation
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
J. Functional Food & Nutraceutical 2020:1(2), pp.95-102
101
Hyaluronic acid is naturally occurred in a high
molecular weight, but since there are various
applications which came from different sizes of
molecular weight of hyaluronan, it prompted a HA
cleavage method to be performed. As can be seen
from Figure 5 and Figure 6, three repetitions of
both samples showed a decrease of absorbance
with very similar value, hence showed degradation
process using heat treatment to be successful and
hyaluronic acid component from both samples was
successfully fragmented as the end product. One
law that affirm molecular weight of the end product
after degradation will decreased is the law of
conservation of mass. The law stated that mass is
neither created nor destroyed in chemical reactions
(Sterner, R and Hood, J., 2011). Since thermal
degradation was not a chemical reaction, it only
shrinks the structure molecules which produced a
less bulky compound with smaller weight of mass.
Another supportive evidence to show the declining
of its molecular weight is the smaller value of the
concentration after degradation procedure. If the
chemical structure of HA were cut during thermal
degradation, it ends with less bulky chemical
compounds which leads to smaller value of
concentration. The concentration of the sample was
declined after degradation as can be seen in the
decreased of absorbance value. This can be
explained through the Lambert Beer’s Law,
expressed through:
A = c l (Equation. 1)
Whereas A is absorbance, is molar absorption
coefficient, c is molar concentration and l is optical
path length passed by the UV light. Since the value
of absorbance after thermal degradation was lower
compared from before degradation process, it
concluded that concentration after degradation was
also dropped due to proportionally equivalent value
of absorbance and concentration according to the
Equation. 1. It can be clearly seen that the drop of
concentrations was constant through three
repetitions of sampling using UV-
spectrophotometer which referring back to Figure.
5 and Figure. 6.
This result also supported by the fact that rind is
highly composed by one of the hyaluronic acid
structures; carboxyl group which are richly present
in form of oxalic acid inside rind. Moreover, rind
has anti-inflammatory property due to chromones
which someway equaled with hyaluronic acid’s
anti-inflammation property. Chromones also have
skin protection effects which matched with one of
hyaluronic acid’s benefits for skin; protection of
water loss to the skin. Lastly, on just below the
waxy cuticle of rind, there is an area where aloe
correlated bacteria live. Gram-positive microbes
(including Group A and group C Streptococci)
which able to produce hyaluronic acid through
bacterial pathway, were only found on the surface
of aloe vera (A. barbadensis), whereas coccobacilli
(streptococcus morbillorium, enterococcus
faecium, and other Gram-negative rods) are
observed only in gel part.
CONCLUSION
Anthrone method showed that all parts of aloe vera
containing carbohydrate with rind has the highest
absorbance, just below the absorbance of goat
brain. This result was confirmed by DNS method
which showed that again rind has the highest
absorbance just like the goat brain. Furthermore,
thermal degradation process was done to degrade
high molecular weight HA into small molecular
weight HA. The result of thermal degradation can
be seen through UV-Spectrophotometer which
showed constant and very similar decrease of
absorbance on both rind and goat brain sample,
thus showed that rind is containing hyaluronic acid.
For further studies, isolation and purification of
hyaluronic acid and quantification of its
concentration, ion exchange chromatography is
preferred due to anionic nature of hyaluronic acid.
The elution obtained by ion exchange
chromatography can be further purified using gel
permeation chromatography and for determination
of precise structure of HA, NMR (Nuclear
Magnetic Resonance) followed by FT-IR can be
used in future research.
EXTRACTION OF HYALURONIC ACID FROM
ALOE BARBADENSIS (ALOE VERA)
Putri, R.R., Nugraha, T., Christy S.
2020:1(2), pp.95-102 J. Functional Food & Nutraceutical
102
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... Pengaplikasian wound healing sheet ekstrak daun Aloe vera dan albumin telur ayam kampung dapat mempercepat penyembuhan luka insisi gingiva serta mengefisienkan waktu tindakan karena mudah diaplikasikan daripadi prosedur suturing. 25,26 Tindakan needleless ini dapat mengurangi kecemasan dan meningkatkan kenyamanan pasien sebab efek inflamasi yang disebabkan oleh jarum suturing bisa dikurangi. 3 Penelitian ini masih menggunakan teknik konvensional untuk mengaplikasikan sediaan wound healing sheet dan albumin telur ayam kampung pada luka insisi gingiva, diperlukan inovasi atau teknologi baru untuk menciptakan sediaan yang lebih praktis dan lebih menempel ketika diaplikasikan. ...
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Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals. Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.
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Since its identification 60 years ago as a ubiquitous component of the body of mammals, hyaluronic acid has been widely studied, primarily in the fields of medicine and biology. On the other hand, our research has dealt with hyaluronic acid as a chemical intermediate in the synthesis of novel lubricious coatings, and in this connection data were needed on stability of aqueous solutions of the polymer over a range of temperatures from 25-100 degrees C. The investigation reported here provides that information, obtained by exposing samples in sealed ampules in baths at controlled temperatures and determining the resulting change in viscosity of the solutions. Data of this kind have not previously been reported on sodium hyaluronate freed from the proteins and other organics normally associated with the polymer in its natural environment.
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Sodium hyaluronate (HA) is widely distributed in extracellular matrixes and can play a role in orchestrating cell function. Consequently, many investigators have looked at the effect of exogenous HA on cell behavior in vitro. HA can be isolated from several sources (e.g., bacterial, rooster comb, umbilical cord) and therefore can possess diverse impurities. This current study compares the measured impurities and the differences in biological activity between HA preparations from these sources. It was demonstrated that nucleic acid and protein content was highest in human umbilical cord and bovine vitreous HA and was low in bacterial and rooster comb HA. Macrophages exposed to human umbilical cord HA produced significantly higher amounts of TNF-alpha relative to control or bacterial-derived HA. These results indicate that the source of HA should be considered due to differences in the amounts and types of contaminants that could lead to widely different behaviors in vitro and in vivo.
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The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.
Article
Engineering of hyaluronic acid (HA) biosynthetic pathway in recombinant Escherichia coli as production host is reported in this work. A hyaluronic acid synthase (HAS) gene, sphasA, from Sreptococcus pyogenes with the start codon gtg to atg mutant, was expressed in recombinant E. coli with or without the genes ugd, galF and glmU, which are analogs of hasB, hasC and hasD from Streptococcus, respectively, encoding UDP-glucose 6-dehygrogenase, Glucose-1-P uridyltransferase, and N-acetyl glucosamine uridyltransferase enzymes in the HA biosynthetic pathway. The single, double and triple organized artificial operons of sphasA, ugd, galF and glmU were designed and constructed using the inducible plasmid backbone of pMBAD. Only the triple expression recombinant, Top10/pMBAD-spABC, generated a relatively high titer of HA (approximately 48 mg/l at 48 h), indicating that both of the enzymes encoded by ugd and galF are essential for HA biosynthesis. A new gene of ssehasA with identical protein sequence of seHAS from Streptococcus equisimilis, was artificially synthesized after substituting all of the rare codons in the natural sehasA. The HA titer at 24 h flask culture increased to approximately 190 mg/l in sseAB and 160 mg/l in sseABC, respectively. Sorbitol could be used as another carbon source for HA accumulation, and the metabolic pathway for HA synthesis in a recombinant E. coli was presented. The concentration of Mg(2+) cofactor of HA synthase was optimized and a cell growth inhibition phenomenon was observed during HA accumulation. Molecular weight (MW) measurements revealed that the mean MW of HA produced from the recombinant E. coli under different conditions ranges from approximately 3.5x10(5) to 1.9x10(6)Da, indicating that the recombinant E. coli can be used as a potential host candidate for industrial production of HA.
Limiting viscosity number and weight average molecular weight of hyaluronate samples produced by heat degradation
  • H Botner
  • T Waaler
  • O Wik
Botner, H., Waaler, T., Wik, O. 1988. Limiting viscosity number and weight average molecular weight of hyaluronate samples produced by heat degradation, International Journal Biology Macromolecules, 10, pp.27-291.
Experimental approaches to hyaluronan structure
  • M Cowman
  • S Matsuoka
Cowman, M. & Matsuoka, S., 2005. Experimental approaches to hyaluronan structure.