Hyaluronic acid degradation during initial steps of downstream processing

Schiraldi, Chiara; Andreozzi, Lucía; Marzaioli, Iolanda; Vinciguerra, S.; D’Avino, Alberto; Volpe, Felice; Panariello, Andrea; Rosa, Mario De

doi:10.3109/10242420903408344

Cited by 11 publications

(6 citation statements)

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“…HA productivity titers in E. coli strains Rosetta2 (DE3) plysS and Rosetta-gami B (DE3)pLysS were higher than those previously reported for the same media [18,34]. The improved productivity is probably attributed to the solid T7 promoter and the buffering of the TB medium, which could maintain the neutral pH crucial to HA production [56]. However, lysozyme has been previously suggested to increase HA productivity of Lactococcus lactis when applied 24 h after induction [19].…”

Section: Discussionmentioning

confidence: 57%

“…Although TB medium resulted in a relatively high HA titer in B. megaterium, HA production was not observed until 16 h from the onset of growth. The enhancing effects of glucose and sucrose on HA productivity have been previously reported in several studies [16,[54][55][56]. The solid buffering capacity of MOPSO seems to play a significant role in optimizing the culture conditions by maintaining a neutral pH throughout the entire growth period, eventually leading to improved HA production [44,57,58].…”

Section: Discussionmentioning

confidence: 86%

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The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production

et al. 2022

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(1) Background: Hyaluronic acid (HA) is a polyanionic mucopolysaccharide extensively used in biomedical and cosmetic industries due to its unique rheological properties. Recombinant HA production using other microbial platforms has received increasing interest to avoid potential toxin contamination associated with its production by streptococcal fermentation. In this study, the Gram-negative strains Escherichia coli (pLysY/Iq), E. coli Rosetta2, E. coli Rosetta (DE3) pLysS, E. coli Rosetta2 (DE3), E. coli Rosetta gammiB(DE3)pLysS, and the Gram-positive Bacillus megaterium (MS941) were investigated as new platforms for the heterologous production of HA. (2) Results: The HA biosynthesis gene hasA, cloned from Streptococcus equi subsp. zoopedemicus, was ligated into plasmid pMM1522 (MoBiTec), resulting in pMM1522 hasA, which was introduced into E. coli Rosetta-2(DE3) and B. megaterium (MS941). The initial HA titer by the two hosts in the LB medium was 5 mg/L and 50 mg/L, respectively. Streptococcal hasABC and hasABCDE genes were ligated into plasmid pPT7 (MoBiTec) and different E. coli host strains were then transformed with the resulting plasmids pPT7hasABC and pPT7hasABCDE. For E. coli Rosetta-gamiB(DE3)pLysS transformed with pPT7hasABC, HA production was 500 ± 11.4 mg/L in terrific broth (TB) medium. Productivity was slightly higher (585 ± 2.9 mg/L) when the same host was transformed with pPT7 carrying the entire HA operon. We also transformed B. megaterium (MS941) protoplasts carrying T7-RNAP with pPT7hasABC and pPT7hasABCDE. In comparison, the former plasmid resulted in HA titers of 2116.7 ± 44 and 1988.3 ± 19.6 mg/L in LB media supplemented with 5% sucrose and A5 medium + MOPSO, respectively; the latter plasmid boosted the titer final concentration further to reach 2476.7 ± 14.5 mg/L and 2350 ± 28.8 mg/L in the two media, respectively. The molecular mass of representative HA samples ranged from 105 − 106 Daltons (Da), and the polydispersity index (PDI) was <2. Fourier transform infrared spectroscopy (FTIR) spectra of the HA product were identical to those obtained for commercially available standard polymers. Finally, scanning electron microscopic examination revealed the presence of extensive HA capsules in E. coli Rosetta-gamiB(DE3)pLysS, while no HA capsules were produced by B. megaterium. (3) Conclusions: Our results suggested that Gram-positive bacteria are probably superior host strains for recombinant HA production over their Gram-negative counters. The titers and the molecular weight (MW) of HA produced by B. megaterium were significantly higher than those obtained by different E. coli host strains used in this study.

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Section: Discussionmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 86%

The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production

et al. 2022

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“…However, the authors still point out that reducing ethanol precipitation steps would be more advantageous since the handling of flammable material and the utilization of ultracentrifuges represent relatively high risks and costs. Few examples of processes that use only filtration technology have been reported 12, 20…”

Section: Resultsmentioning

confidence: 99%

Application of a 22L scale membrane bioreactor and cross‐flow ultrafiltration to obtain purified chondroitin

Schiraldi

Alfano

Cimini

et al. 2012

Biotechnology Progress

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Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed-batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale-up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨︁KTA cross-flow instrument, was translated to a UNIFLUX-10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C-3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled-up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild-type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity.

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“…The mutant strain (hyaluronidase-free and haemolytic negative) S. equi subsp. zooepidemicus BA06 strain was obtained as previously described (Schiraldi et al 2010 ). Plasmid pNZ8148 was obtained from Nizo (Netherlands).…”

Section: Methodsmentioning

confidence: 99%

“…In the present work the E. coli K4 genes, kfoC and kfoA , were expressed in a previously obtained non hemolytic, hyaluronidase negative S. equi subsp. zooepidemicus BA06 host (Schiraldi et al 2010 ) to implement its UDP-biosynthesis pathway and polymer assembly machinery for the production of chondroitin, thereby generating a cell factory for the obtainment of both biopolymers in a single fermentation event.…”

Section: Introductionmentioning

confidence: 99%

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

et al. 2017

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Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitin-like polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0364-7) contains supplementary material, which is available to authorized users.

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Hyaluronic acid degradation during initial steps of downstream processing

Cited by 11 publications

References 19 publications

The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production

The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production

Application of a 22L scale membrane bioreactor and cross‐flow ultrafiltration to obtain purified chondroitin

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

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