Accepted Manuscript Title: Enzymatic production of specifically distributed hyaluronan oligosaccharides Author: Panhong Yuan Mengxian Lv Peng Jin Miao Wang Guocheng Du Jian Chen Zhen Kang PII: DOI: Reference:
S0144-8617(15)00394-X http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.068 CARP 9899
To appear in: Received date: Revised date: Accepted date:
7-1-2015 24-4-2015 25-4-2015
Please cite this article as: Yuan, P., Lv, M., Jin, P., Wang, M., Du, G., Chen, J., and Kang, Z.,Enzymatic production of specifically distributed hyaluronan oligosaccharides, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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Pure aqueous solution system was established for hyaluronan depolymerization.
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Hyaluronidase concentration and hydrolysis time determined molecular mass
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Target hyaluronan fragments with specific molecular mass could be effectively
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distribution.
produced.
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The lowest polydispersity index of the hyaluronan oligosaccharide was 1.16.
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The hyaluronan tetrasccharide HA4 was successfully isolated with a high purity.
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Large-scale production of hyaluronan oligosaccharides was achieved (40 g/L).
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Enzymatic production of specifically distributed hyaluronan
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oligosaccharides
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Panhong Yuana,b,c, Mengxian Lve, Peng Jina,b,c, Miao Wange, Guocheng Du a,b,d, Jian Chen b,c,
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Zhen Kang a,b,c*
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a
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Wuxi 214122, China
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b
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Jiangsu 214122, China.
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c
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d
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Jiangnan University, Wuxi 214122, China
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e
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Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi,
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School of Biotechnology, Jiangnan University, Wuxi 214122, China
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The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education,
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
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The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University,
*
Corresponding author,
Zhen Kang, Phone: +86-510-85918307 Fax: +86-510-85918309, E-mail: [email protected]
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Abstract
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High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous
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solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit
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HA and hydrolysis time played important roles in molecular mass distribution. By modulating
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the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA
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oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA
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oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000 Da and end
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hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size
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exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis,
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and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed
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low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore,
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large-scale production of defined HA oligosaccharides with narrow molecular mass
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distribution will significantly promote progress in related research and its potential
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applications.
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Keywords: Hyaluronan; Recombinant leech hyaluronidase; Molecular mass distribution; Oligosaccharides; Polydispersity; Molecular mass distribution; Large-scale enzymatic production
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1. Introduction Hyaluronan (HA), an anionic linear polymer composed of repeated disaccharide
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N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcUA) units linked through β
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(1,3) and β (1,4) glycosidic bonds, is widely distributed in the extracellular matrix of almost
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all the animal tissues and is present in significant quantities in the skin, brain, and central
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nervous system (Liu, Sun, Heggeness, Yeh & Luo, 2004; Vigetti et al., 2014). High molecular
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mass HA has been demonstrated to have important physiological roles in living organisms
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owing to its associated unique viscoelastic and rheological properties, and has been
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extensively used in cosmetics and pharmaceutics (such as tissue augmentation, ocular surgery,
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osteoarthritis, and drug delivery) (Stern, Asari & Sugahara, 2006). Nevertheless, interestingly,
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it has been discovered and confirmed that the function of HA fragments is strongly dependent
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on its chain length despite its exceedingly simple primary structure (Chang et al., 2012;
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Holubova et al., 2014; Kogan, Soltes, Stern & Gemeiner, 2007). Recently, several studies
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have demonstrated that HA oligosaccharides with different molecular sizes are involved in angiogenesis, wound healing, cell differentiation and proliferation, apoptosis, and tumor cell migration (Aya & Stern, 2014; Stern, Asari & Sugahara, 2006; Toole, Ghatak & Misra, 2008). It has been reported that low-molecular-mass (70,000-120,000 Da) HA fragments are best
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suitable for the delivery of proteins and peptides with applications in chronic wound healing
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and contribute to the development of novel HA conjugates (Ferguson, Roberts, Moseley,
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Griffiths & Thomas, 2011). In contrast, HA oligosaccharides (molecular mass < 10,000 Da)
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can selectively kill many types of cancer cells via disruption of the receptor-HA interaction 5 Page 4 of 28
(Toole, Ghatak & Misra, 2008; Zeng, Toole, Kinney, Kuo & Stamenkovic, 1998). In addition,
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when compared with high-molecular-mass HA, the HA oligosaccharides can be easily
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absorbed by the body and serve as precursors for the synthesis of higher molecular mass HA
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molecules (Boltje, Buskas & Boons, 2009). Consequently, efficient production of HA
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oligosaccharides with narrow size distribution will be significant for its related in-depth study
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and potential broad applications in cosmetics, medicine, and food industries.
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Currently, many physical (ultrasonication, microwave irradiation, and heating) and
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chemical (acidic and alkaline hydrolysis, oxidant hydrolysis) methods have been developed
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and applied for depolymerizing HA polymers into lower molecular mass HA oligosaccharides
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(Bezáková et al., 2008; Drimalova, Velebny, Sasinkova, Hromadkova & Ebringerova, 2005;
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Melander & Tømmeraas, 2010; Stern, Kogan, Jedrzejas & Soltes, 2007). However, in most
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cases, the harsh degradation conditions give rise to a mixture of oligosaccharides and
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monosaccharides with high polydispersity, which complicate subsequent purification
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processes and decrease the transformation efficiency with respect to specific weight-average
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molar mass (Mw) HA oligosaccharides (Ferguson, Roberts, Moseley, Griffiths & Thomas, 2011; Holubova et al., 2014). Although many approaches for de novo chemical synthesis of HA oligosaccharides have been developed, the complex and time-consuming processes as well as rare and expensive substrates (uridine diphosphate (UDP) sugars) limit their practical
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applications for large-scale production of HA oligosaccharides (Boltje, Buskas & Boons,
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2009; Huang & Huang, 2007). In contrast, enzymatic production of narrow-spectrum HA
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oligosaccharides with hyaluronidase (HAase) under mild operation conditions is a promising
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and attractive approach. However, application of commercial bovine testicular hyaluronidase 6 Page 5 of 28
(BTH) extracted from bovine testes for enzymatic production of specific or narrow-spectrum
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HA oligosaccharides has some disadvantages, such as risk of viral contamination, broad-size
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distributed HA fragments, low-yield and complex manipulation (Chen, et al., 2009;
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Deschrevel, Tranchepain & Vincent, 2008; Kakizaki, Ibori, Kojima, Yamaguchi & Endo,
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2010), which have limited its industrial applications (El-Safory, Fazary & Lee, 2010). Thus,
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large-scale production of recombinant HAase robust microbial factories and development of
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enzymatic process for the preparation of specific HA oligosaccharides would be attractive.
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More recently, the first leech HAase-encoding gene was cloned and characterized. By
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combining protein engineering and high-density culture, the production of recombinant
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HAase was improved to 8.42×105 U/ml in the secretory expression system of the yeast Pichia
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pastoris, which paved way for its application (Jin, Kang, Zhang, Du & Chen, 2014).
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Additionally, the HA oligosaccharides produced from leech HAase contained glucuronic acid
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at the reducing end, which was quite different from that of BTH (Jin, Kang, Zhang, Du &
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Chen, 2014; Linker, Meyer & Hoffman, 1960). In the present study, to expedite enzymatic
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preparation of HA oligosaccharides, the whole hydrolysis process and products in pure water solution without the addition of any metal ions were systematically investigated and characterized. Impressively, by controlling the hydrolysis time, high-level enzymatic production of the designed HA oligosaccharides with low polydispersity (approximately 1.16)
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was achieved.
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2. Materials and methods
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2.1. Chemicals and reagents
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HA (1.21×106 Da) sample (fermented with Streptococcus zooepidemics) of high purity 7 Page 6 of 28
was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Standard dextranum
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samples with standard molecular mass distribution (180, 2700, 9750, 13,050, 36,800, 135,350,
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and 2,000,000 Da) were purchased (National Institute for Food and Drug Control). HAase
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from the yeast P. pastoris secretory expression system with a specificity value of 1.20× 106
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U/mg was applied for HA hydrolysis. Specifically, the specific activity was defined as units of
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enzyme per mg of protein. BTH and 8-Aminonaphthalene-1, 3, 6-trisulfonic acid disodium
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salt (ANTS) was purchased (Sigma-Aldrich). Dinitrosalicylic acid (DNS) reagent contained
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1% DNS, 2% NaOH, and 20% sodium potassium tartrate. The matrix solution for the
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matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)
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was prepared by dissolving 20 mg of 2,5-dihydroxy-benzoic acid (DHB) and 58.5 mg of
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sodium chloride in 1 mL of 10% ethanol.
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2.2. Preparation of HA oligosaccharides
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Before preparation of HA oligosaccharides, the recombinant leech HAase activity was
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determined by measuring the amount of reducing sugar using the DNS colorimetric method
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(Ghose, 1987; Linker, Hoffman & Meyer, 1957; Silveira, Aguiar, Siika-aho & Ramos, 2014; Yuki & Fishman, 1963). The absorbance of each reaction mixture was detected at 540 nm after an appropriate dilution for calculating the HAase activity. In the preparation of molar-mass-defined HA fragments, the amount of HAase could be controlled. The
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constant-temperature fermentation reaction was conducted in a 3-L fermentor (LiFlus GM
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BioTRON, Korea) containing 2 L of the medium, maintaining the temperature at 45°C using
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a temperature sensor. In addition, an agitator, operating at 500 rpm, was mounted to the
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bottom of the fermentation tank, which ensured uniform mixing of HA and water. 8 Page 7 of 28
Subsequently, HAase was added and the reaction proceeded smoothly without the addition of
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acid, alkali, and anti-foaming agent to achieve product of high purity. According to the size of
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HA fragments required, the reaction was terminated at a suitable kinetic time point. The
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samples were collected and filtered to remove insoluble particle. Furthermore, HA was
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hydrolyzed using BTH at the same conditions.
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2.3. High performance liquid chromatography-size exclusion chromatography-refractive
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index detector analysis
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To determine the molecular masses and polydispersity of the HA hydrolysate, high
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performance liquid chromatography-size exclusion chromatography-refractive index detector
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(HPLC-SEC-RI) analysis was employed (Tranchepain et al., 2006). The Agilent 1260
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HPLC-SEC-RI device was composed of a G1310A pump, a G1329B injector, an
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UltrahydrogelTM linear column (7.8×300 mm; Shimadzu, Kyoto, USA), size exclusion
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chromatograph using Sephadex G-100 as the sieving material, and a G1362A RI detector. The
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mobile phase was sodium nitrate (0.1 mol/L). A total of 40 µL of the reaction mixture were
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injected with 0.05% sodium azide and each sample was eluted under a constant flow rate (0.9 mL/min) for 25 min. The temperature of the column was controlled at 40°C and the flow cell temperature was controlled at 35°C. The signals measured by the RI detector were analyzed using the GPC software, which calculated the number-average molar mass (Mn), Mw, and
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polydispersity index (Ip, Ip = Mw/Mn). The standard curve plotting the HA fragment Mw as a
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function of the elution volume was simulated using the GPC software (Agilent Technologies).
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Then, the Mw of each hydrolysate was calculated based on the standard curve by the GPC
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profile. In conclusion, HPLC-SEC-RI is highly suitable to study the kinetics of the HA 9 Page 8 of 28
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hydrolysis reaction catalyzed using recombinant leech HAase in case it has been calibrated
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using standard dextranum samples in advance (Tranchepain et al., 2006).
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2.4. MALDI-TOF MS analysis of HA tetrasaccharides and HA hexasaccharides MALDI-TOF MS is a powerful method to quickly and accurately determine the masses
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of saccharides. In the present study, the MALDI-TOF-MS experiments were conducted on
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TofSpecE mass spectrometer equipped with N2 laser at a wavelength of 337 nm. The machine
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was run in negative-ion reflector mode using 20-kV acceleration voltage (Mahoney, Aplin,
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Calabro, Hascall & Day, 2001). Spectra external calibration was conducted before measuring
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the samples (Tranchepain et al., 2006). A total of 0.5 μL of the hydrolysate beads mixed with
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0.5 μL of DHB solution were spotted on the iron target of the spectrometer and crystallized
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under vacuum conditions. Raw data were analyzed using Flex Analysis version 3.3 software,
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and no smoothing was performed on the spectra. Hydrolysate masses were calculated using
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the Prowl program in monoisotopic mode. Each sample solution was performed under the
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same operating conditions.
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2.5. Preparation and characterization of the HA tetrasaccharides HA oligosaccharides were isolated from the hydrolysates by anion exchange
chromatography according to the previous description (Tawada, et al., 2002). The purified HA4 was dissolved in distilled water with concentration of 1.0 g/L. ESI-MS analysis was
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carried out on a MALDI SYNAPT Q-TOF MS with scanning over the m/z range 50-2200 in
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10 s/scan. 5 μL sample solution was introduced into the ion source of the mass spectrometer
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through an autosampler of Agilent 1260 series HPLC system for analysis.
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2.6. Polyacrylamide gel electrophoresis analysis of molar-mass-defined HAFs 10 Page 9 of 28
The molar-mass-defined fragments were frozen and lyophilized in a vacuum
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concentrator. For the derivatization of molar-mass-defined HA fragments using ANTS, 2
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nmol of each fragments were employed. All the samples were derived by adding 5 μL of
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ANTS reagent solution for 15 min at room temperature and then 5 μL of sodium
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cyanoborohydride solution for 16 h at 37°C (Tawada et al., 2002). After incubation, each
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sample was centrifuged at 10,000 × g for 10 min at 4°C. 10% polyacrylamide gel be used to
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obtain high resolution separation of molar-mass-defined HA fragments. For electrophoresis,
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the comb was removed from the gel, and the gel was moved to an electrophoresis unit. Each
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sample of 5 µL was run on a polyacrylamide gel (Bhilocha et al., 2011) on a vertical gel
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apparatus at 4°C first 18mA/gel and then at 7mA/gel for 3h in Tris-borate-EDTA (TBE) buffer.
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After electrophoresis, the gel was illuminated with UV light (365 nm) and photographed.
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2.7. Analysis of molar-mass-defined HAFs by capillary zone electrophoresis
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The samples were lyophilized in a vacuum concentrator. The molar-mass-defined HAFs
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were dissolved in 200 µL of tenfold diluted capillary zone electrophoresis (CZE) operating
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buffer and centrifuged for 15 min at 11,000 × g. The supernatant was used for CZE analysis. CZE analysis was performed on a P/ACETM MDQ Capillary Electrophoresis System equipped with elastic quarts capillary vessel column (65 cm×75 µm) and 32 Karat Software. The operating buffer contained 50 mM disodium hydrogen phosphate and 20 mM sodium
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tetraborate (pH 9) and the sample buffer was prepared by diluting the operating buffer tenfold
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with Milli-Q water (Hofinger, Hoechstetter, Oettl, Bernhardt & Buschauer, 2008). The rinse
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pressure was 20 psi, inject pressure was 2 psi, and duration was 4 s. The analysis was
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performed at a separate voltage of 15 kV, capillary temperature of 20°C, and detector 11 Page 10 of 28
wavelength of 200 nm (Grimshaw et al., 1996). Between each analysis, the capillary was
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operated with Milli-Q water (1 min), 50 mM sodium hydroxide (1 min), Milli-Q water (1
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min), and finally operating buffer (5 min).
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3. Results and discussion
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3.1. Stability evaluation of the recombinant HAase in aqueous solution
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To facilitate and simplify the subsequent purification processes, a pure aqueous solution
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without the addition of any metal ions was chosen and utilized for HA hydrolysis.
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Accordingly, the stability of the recombinant HAase in this system was first investigated. Our
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previous results (Jin, Kang, Zhang, Du & Chen, 2014) revealed that the system should be
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incubated at 45°C with a near-neutral optimum pH of 6.5. In the present study, the samples
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were collected at regular intervals and analyzed. Interestingly, an obvious increase in the
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HAase activity was observed in the initial stage, which was consistent with the experiments
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conducted using the buffer systems. As shown in Fig. 1, after incubation for 24 h, no
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significant decrease in the HAase activity was detected, suggesting its desirable stability in pure aqueous solution. Moreover, the results also indicated that application of this pure aqueous solution system for the enzymatic production of HA oligosaccharides is practicable.
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Fig.1. Evaluation of the stability of the recombinant leech HAase. The remaining enzymatic
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activity was determined at 45°C and rotation speed of 500 rpm in a 3-L fermentor.
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3.2. Analysis of the hydrolysis process
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To rapidly estimate the practical feasibility of the hydrolysis process, high concentration
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of high-molecular-mass hyaluronan (40 g/L) was added as substrate into a 3-L fermenter for
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analysis. In particular, four different HAase concentrations (1.00×103, 1.25×104, 2.00×104,
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and 4.00×104 U/ml) were applied. As shown in Fig. 2, the Mw of the HA fragments sharply decreased in the initial stage. Moreover, it was obvious that higher degradation rates were achieved with increased concentration of HAase. After 2 h, the Mw of the HA fragments generated with different concentrations of HAase (as indicated earlier) decreased to 36,000,
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16,000, 8500, and 5000 Da, respectively. However, the decrease in Mw was very slow beyond
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that time although the theoretical end products were HA hexasaccharides (HA6) and HA
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tetrasaccharides (HA4) (Jin, Kang, Zhang, Du & Chen, 2014; Linker, Meyer & Hoffman,
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1960; Stern & Jedrzejas, 2006), suggesting that the hydrolysis rate rapidly decreased with the 13 Page 12 of 28
accumulation of shorter HA fragments, which is consistent with the previous reports
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conducted with BTH (Deschrevel, Tranchepain & Vincent, 2008). In fact, no further obvious
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decrease in the Mw was detected and the final Mw of the generated HA oligosaccharides at 24
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h was approximate 20,000, 10,000, 4000, and 3000 Da, respectively. In fact, this is the first
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HA depolymerization curve reported because partial digestion of HA with BTH always gives
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rise to a wide variety of fragments with high polydispersity (Kakizaki, Ibori, Kojima,
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Yamaguchi & Endo, 2010; Tawada, et al., 2002; Tranchepain et al., 2006). Conversely, the
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results obtained here indicated that the partial digestion products of the recombinant leech
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HAase should have a low polydispersity value, which was confirmed by further analysis
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results (Table 1).
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Fig.2. Time course of HA hydrolysis in the fermentation tank. The high-molecular-mass HA
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was digested under a rotation speed of 500 rpm and temperature of 45°C. (A) HAase,
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1.00×103 U/mL; (B) HAase, 1.25×104 U/mL; (C) HAase, 2.00×104 U/mL; (D) HAase, 14 Page 13 of 28
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4.00×104 U/mL.
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3.3. Influence of HA concentration and hydrolysis time on polydispersity
The biofunction of HA molecules are closely related to its Mw. However, in most cases,
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the HA polysaccharide fragments produced using traditional methods are widely distributed
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with high polydispersity (Stern, Kogan, Jedrzejas & Soltes, 2007). As a result, the production
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of HA fragments with low polydispersity is attractive. In this regard, the effects of HA
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concentration and hydrolysis time were further investigated. As shown in Table 1, it could be
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obviously observed that although hydrolysates at any hydrolysis time point have different Mw
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values, higher polydispersity was achieved from higher concentrations of HA. Meanwhile, it
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could also be noted that the polydispersity index of all the hydrolysates with different
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concentrations of HA (20, 40 and 60 g/L) declined with prolonged hydrolysis. These results
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indicated that comparatively low concentrations of HA and long hydrolysis time could give
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rise to HA oligosaccharides with low polydispersity, whereas BTH with high
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polydispersity (Tawada, et al., 2002). Table 1 Characteristics of hydrolysates from different concentrations of high-molecular-mass HA.
HAase (U/mL)
HA (g/L)
0.5 h
1h
1.5 h
2h
3h
Mw
Ip
Mw
Ip
Mw
Ip
Mw
Ip
Mw
Ip
1.5×104
20
10,000
1.86
7000
1.6
5600
1.46
4900
1.33
4000
1.20
1.5×104
40
20,000
2.43
13,000
2.06
10,000
1.88
8400
1.73
6700
1.54
1.5×104
60
32,000
2.70
18,000
2.24
16,200
2.14
14,100
2.00
10,000
1.90
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Mw = Weight-average molar mass, Ip = Polydispersity index
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3.4. Enzymatic production of defined HA oligosaccharides 15 Page 14 of 28
Based on the above-mentioned results, partial degradation for the production of desirable
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HA fragments with specific Mw was investigated. The HA oligosaccharides with Mw values of
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30,000, 10,000, and 4000 Da were individually prepared by controllable degradation of 40
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g/L high-molecular-size HA with different concentrations of HAase and at various hydrolysis
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time. As expected, the defined HA oligosaccharides were successfully and efficiently
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produced. More interestingly, it could be noted that all the products catalyzed by
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comparatively lower HAase and longer hydrolysis time exhibited lower polydispersity (Table
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2). For instance, although the HA oligosaccharides (40 g/L) produced using 1.25×104 and
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2.00×104 U/mL HAases showed only one peak at the same time, the molecular mass
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distribution graph of the HA oligosaccharides obtained from 1.25×104 U/mL HAase was more
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concentrated (Fig. 3). Impressively, the polydispersity index of the HA fragments with Mw of
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4000 Da was only 1.16, suggesting an extremely narrow size distribution. Previously,
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although many attempts have been made using BTH, it was impractical to prepare specific
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molecular-mass HA fragments (Kakizaki, Ibori, Kojima, Yamaguchi & Endo, 2010; Mahoney,
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Aplin, Calabro, Hascall & Day, 2001). In comparison, the recombinant leech HAase is more valuable and acceptable for the enzymatic production of the desirable HA oligosaccharides. In addition, several efficient isolation and purification methods for obtaining pure and size-uniform HA oligosaccharides have been established (Kakehi, Kinoshita & Yasueda,
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2003),
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oligosaccharides.
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Table 2 Polydispersity of specifically distributed HA oligosaccharides produced with
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different concentrations of HAase.
which
will
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large-scale
production
of
size-specific
HA
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HAase (U/ml)
Hydrolysis time (h)
3
1.00×10 1.25×10 4 1.25×10 4 2.00×10 4 2.00×10 4 4.00×10 4
30,000 10,000 4000 Mw = Weight-average molar mass
1.69 2.07 1.68 1.86 1.16 1.19
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3 0.5 15 1.3 24 4.5
Polydispersity index
ip t
Mw (Da)
Fig.3. HPLC-SEC profile of the HA oligosaccharide samples with Mw of 10,000 Da. (A) The
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elution peak of the samples produced using 1.25×104 U/mL HAase; (B) Molecular mass
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distribution graph of the samples hydrolyzed using 1.25×104 U/mL HAase; (C) The elution peak of the samples produced using 2.00×104 U/mL HAase; (D) Molecular mass distribution graph of the samples hydrolyzed using 2.00×104 U/mL HAase.
3.5. Enzymatic production of the end products HA6 and HA4
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In recent years, HA6 and HA4 have attracted much attention because of its potential
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applications in medicine (Torigoe et al., 2011; Wakao et al., 2011). However, their large-scale
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production has always been a big challenge owing to the inability of traditional degradation
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methods (Stern, Kogan, Jedrzejas & Soltes, 2007). Although HA6 and HA4 could be 17 Page 16 of 28
generated with BTH, the proportion was still low (Jin, Kang, Zhang, Du & Chen, 2014;
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Tawada, et al., 2002), which eventually decreased the conversion rate. Consequently, in the
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present study, large-scale enzymatic production of HA6 and HA4 was investigated. In
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particular, 6.00×104 U/mL HAase was added to the hydrolysis system containing 40 g/L HA.
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MALDI-TOF MS spectrometry was applied for analyzing the degradation products. At 0.5 h,
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trace amounts of HA oligomers HA10, HA8, HA6, and HA4 were generated (Fig. 4). When
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the incubation period was extended to 5.5 h, HA10 was completely converted to HA6 and
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HA4. At 6.5 h, the peak corresponding to HA8 disappeared and only two ion peaks
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corresponding to HA6 and HA4 were detected, indicating that all the higher HA oligomers
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were exhaustively digested to the end products HA6 and HA4. Moreover, it has been
317
demonstrated that the reducing end of HA oligomers from leech HAase was glucuronic acid
318
but not N-acetyl-D-glucosamine (Jin, Kang, Zhang, Du & Chen, 2014; Yuki & Fishman,
319
1963), indicating its potential different biological function. Thus, HA4 was further isolated
320
from the hydrolysate with the anion exchange chromatography. Specifically, Electrospray
322 323
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Ionization Mass Spectrometry (ESI-MS) was used with the negative ion mode to analyze the separated products. As shown in Fig. 5, all the multistage mass spectrometry data confirmed a high purity of the isolated HA4.
18 Page 17 of 28
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Fig.4. MALDI-TOF MS profile of the hydrolysates catalyzed by 6.00×104 U/mL HAase. In
326
particular, the mass-to-charge ratio of 797, 1176, 1555, and 1934 showed [HA4-2H+Na]-,
327
[HA6-2H+Na]-, [HA8-2H+Na]-, and [HA10-2H+Na]-, respectively.
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Description: C:\Users\yph\Desktop\
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Fig.5. Negative-ion electrospray mass spectra of separated product: (A) HA4 mass spectra with
331
corresponding structure of 775.1[M-H]-; (B) The precursor ion 775.1[M-H]- is bombarded to
332
generate fragment ions 396.0[M-H]- and 599.1[M-H]- in MS2 mass spectra.
333
3.6. Characterization of polydispersity of the hydrolysis products
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To gain more detailed insights, the produced HA oligosaccharides were further
335
characterized by powerful polyacrylamide gel electrophoresis method developed recently
336
(Bhilocha et al., 2011). Accordingly, 10% polyacrylamide gel in TBE buffer was employed
337
for separating the intermediate HA products with Mw of 4000, 10,000, and 30,000 Da, the end
338
products containing HA6 and HA4, and the isolated HA4. The hydrolysis products with Mw
339
of 30,000 Da that produced by BTH were also comparatively investigated at the same time.
340
As shown in Fig. 6, it could be obviously discovered that the hydrolysate with smaller Mw,
341
especially the isolated HA4 (Lane 1) showed much sharper band, suggesting its low
342
polydispersity value. In comparison, it could be found that the HA oligomers from BTH
344
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trailed a longer band (Lane 6) than that with the same Mw from the recombinant leech HAase (Lane 5), suggesting a broader distribution of the Mw values of the BTH hydrolysates.
345
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Fig.6. Polyacrylamide gel electrophoresis analysis of the molecular mass distribution of the
347
prepared oligosaccharide samples: Lane 1, the isolated product of HA4; Lane 2, end
348
hydrolysates containing HA6 and HA4; Lane 3, samples with Mw of 4000 Da; Lane 4,
349
samples with Mw of 10,000 Da; Lane 5, samples with Mw of 30,000 Da; Lane 6, samples with
350
Mw of 30,000 Da from bovine testicular hyaluronidase hydrolysis.
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Furthermore, the HA oligomers with Mw of 30,000 Da were separated and characterized
352
using CZE, which has high resolution power particularly in analyzing complex carbohydrate
353
mixtures such as the negatively charged molar-mass-defined HA fragments (Hong, Sudor,
354
Stefansson & Novotny, 1998; Hofinger, Hoechstetter, Oettl, Bernhardt & Buschauer, 2008).
355
Obviously, the HA oligomers hydrolyzed by the recombinant leech HAase showed only one
356
main peak (Fig. 7). In contrast, several peaks were observed with the HA oligomers from
357
BTH. The results further confirmed the concentrated molecular mass distribution of the
358
hydrolysates from the recombinant leech HAase, which might be ascribed to the preferential
359
digestion of longer HA chains by the recombinant leech HAase through a nonprocessive
361 362
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endolytic mode (Lepperdinger, Mullegger & Kreil, 2001). Thus, it can be concluded that enzymatic production of HA oligosaccharides with specific molecular mass distribution using recombinant leech HAase was efficient and applicable.
21 Page 20 of 28
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Fig.7. CZE profile of the prepared HA oligosaccharide samples. (A) Samples with Mw of
365
30,000 Da from bovine testicular hyaluronidase hydrolysis; (B) Samples with Mw of 30,000
366
Da from leech hyaluronidase hydrolysis.
367
Conclusion
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HAase concentration per unit HA played an important role in hydrolysis rate and
369
molecular mass distribution. Longer hydrolysis time produced molar-mass-defined HA
371 372 373 374
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fragments with lower polydispersity. By modulating HAase concentrations and hydrolysis time, any molar-mass-defined HA oligomers could be efficiently produced. All the obtained molar-mass-defined
HA
oligosaccharides
were
extremely
concentrated
with
low
polydispersity. Thus, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will promote progress in related research and potential
375
applications in areas, including chemical synthesis, medical therapy, cosmetics, and health
376
food.
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Conflict of interest 22 Page 21 of 28
The authors declare that there is no conflict of interest.
379
Acknowledgments
380
This work was financially supported by a grant from the Key Technologies R & D Program of
381
Jiangsu Province, China (BE2014607); National High Technology Research and
382
Development Program of China (863 Program, 2012AA022005); Program for Changjiang
383
Scholars and Innovative Research Team in University (no. IRT1135); National Natural
384
Science Foundation of China (31200020); and 111 Project.
385
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Stern, R., & Jedrzejas, M. J. (2006). Hyaluronidases: Their genomics, structures, and mechanisms of action. Chemical Reviews, 106, 818-839. Stern, R., Kogan, G., Jedrzejas, M. J., & Soltes, L. (2007). The many ways to cleave hyaluronan. Biotechnology Advances, 25, 537-557. 26 Page 25 of 28
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C., & Vincent, J. C. (2006). A complete set of hyaluronan fragments obtained from
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Zeng, C. X., Toole, B. P., Kinney, S. D., Kuo, J. W., & Stamenkovic, I. (1998). Inhibition of
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S0144-8617(15)00394-X http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.068 CARP 9899
To appear in: Received date: Revised date: Accepted date:
7-1-2015 24-4-2015 25-4-2015
Please cite this article as: Yuan, P., Lv, M., Jin, P., Wang, M., Du, G., Chen, J., and Kang, Z.,Enzymatic production of specifically distributed hyaluronan oligosaccharides, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
2
Pure aqueous solution system was established for hyaluronan depolymerization.
3
Hyaluronidase concentration and hydrolysis time determined molecular mass
6
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Target hyaluronan fragments with specific molecular mass could be effectively
cr
5
distribution.
produced.
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4
The lowest polydispersity index of the hyaluronan oligosaccharide was 1.16.
8
The hyaluronan tetrasccharide HA4 was successfully isolated with a high purity.
9
Large-scale production of hyaluronan oligosaccharides was achieved (40 g/L).
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1 Page 1 of 28
Enzymatic production of specifically distributed hyaluronan
13
oligosaccharides
14
Panhong Yuana,b,c, Mengxian Lve, Peng Jina,b,c, Miao Wange, Guocheng Du a,b,d, Jian Chen b,c,
15
Zhen Kang a,b,c*
ip t
12
cr
16 17
a
18
Wuxi 214122, China
19
b
20
Jiangsu 214122, China.
21
c
22
d
23
Jiangnan University, Wuxi 214122, China
24
e
26 27 28 29
us
an
Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi,
M
School of Biotechnology, Jiangnan University, Wuxi 214122, China
te
d
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education,
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
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The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University,
*
Corresponding author,
Zhen Kang, Phone: +86-510-85918307 Fax: +86-510-85918309, E-mail: [email protected]
30 31 32 33
3 Page 2 of 28
Abstract
35
High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous
36
solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit
37
HA and hydrolysis time played important roles in molecular mass distribution. By modulating
38
the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA
39
oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA
40
oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000 Da and end
41
hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size
42
exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis,
43
and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed
44
low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore,
45
large-scale production of defined HA oligosaccharides with narrow molecular mass
46
distribution will significantly promote progress in related research and its potential
47
applications.
49 50 51
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Keywords: Hyaluronan; Recombinant leech hyaluronidase; Molecular mass distribution; Oligosaccharides; Polydispersity; Molecular mass distribution; Large-scale enzymatic production
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4 Page 3 of 28
56 57
1. Introduction Hyaluronan (HA), an anionic linear polymer composed of repeated disaccharide
59
N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcUA) units linked through β
60
(1,3) and β (1,4) glycosidic bonds, is widely distributed in the extracellular matrix of almost
61
all the animal tissues and is present in significant quantities in the skin, brain, and central
62
nervous system (Liu, Sun, Heggeness, Yeh & Luo, 2004; Vigetti et al., 2014). High molecular
63
mass HA has been demonstrated to have important physiological roles in living organisms
64
owing to its associated unique viscoelastic and rheological properties, and has been
65
extensively used in cosmetics and pharmaceutics (such as tissue augmentation, ocular surgery,
66
osteoarthritis, and drug delivery) (Stern, Asari & Sugahara, 2006). Nevertheless, interestingly,
67
it has been discovered and confirmed that the function of HA fragments is strongly dependent
68
on its chain length despite its exceedingly simple primary structure (Chang et al., 2012;
69
Holubova et al., 2014; Kogan, Soltes, Stern & Gemeiner, 2007). Recently, several studies
71 72 73
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have demonstrated that HA oligosaccharides with different molecular sizes are involved in angiogenesis, wound healing, cell differentiation and proliferation, apoptosis, and tumor cell migration (Aya & Stern, 2014; Stern, Asari & Sugahara, 2006; Toole, Ghatak & Misra, 2008). It has been reported that low-molecular-mass (70,000-120,000 Da) HA fragments are best
74
suitable for the delivery of proteins and peptides with applications in chronic wound healing
75
and contribute to the development of novel HA conjugates (Ferguson, Roberts, Moseley,
76
Griffiths & Thomas, 2011). In contrast, HA oligosaccharides (molecular mass < 10,000 Da)
77
can selectively kill many types of cancer cells via disruption of the receptor-HA interaction 5 Page 4 of 28
(Toole, Ghatak & Misra, 2008; Zeng, Toole, Kinney, Kuo & Stamenkovic, 1998). In addition,
79
when compared with high-molecular-mass HA, the HA oligosaccharides can be easily
80
absorbed by the body and serve as precursors for the synthesis of higher molecular mass HA
81
molecules (Boltje, Buskas & Boons, 2009). Consequently, efficient production of HA
82
oligosaccharides with narrow size distribution will be significant for its related in-depth study
83
and potential broad applications in cosmetics, medicine, and food industries.
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Currently, many physical (ultrasonication, microwave irradiation, and heating) and
85
chemical (acidic and alkaline hydrolysis, oxidant hydrolysis) methods have been developed
86
and applied for depolymerizing HA polymers into lower molecular mass HA oligosaccharides
87
(Bezáková et al., 2008; Drimalova, Velebny, Sasinkova, Hromadkova & Ebringerova, 2005;
88
Melander & Tømmeraas, 2010; Stern, Kogan, Jedrzejas & Soltes, 2007). However, in most
89
cases, the harsh degradation conditions give rise to a mixture of oligosaccharides and
90
monosaccharides with high polydispersity, which complicate subsequent purification
91
processes and decrease the transformation efficiency with respect to specific weight-average
93 94 95
M
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molar mass (Mw) HA oligosaccharides (Ferguson, Roberts, Moseley, Griffiths & Thomas, 2011; Holubova et al., 2014). Although many approaches for de novo chemical synthesis of HA oligosaccharides have been developed, the complex and time-consuming processes as well as rare and expensive substrates (uridine diphosphate (UDP) sugars) limit their practical
96
applications for large-scale production of HA oligosaccharides (Boltje, Buskas & Boons,
97
2009; Huang & Huang, 2007). In contrast, enzymatic production of narrow-spectrum HA
98
oligosaccharides with hyaluronidase (HAase) under mild operation conditions is a promising
99
and attractive approach. However, application of commercial bovine testicular hyaluronidase 6 Page 5 of 28
(BTH) extracted from bovine testes for enzymatic production of specific or narrow-spectrum
101
HA oligosaccharides has some disadvantages, such as risk of viral contamination, broad-size
102
distributed HA fragments, low-yield and complex manipulation (Chen, et al., 2009;
103
Deschrevel, Tranchepain & Vincent, 2008; Kakizaki, Ibori, Kojima, Yamaguchi & Endo,
104
2010), which have limited its industrial applications (El-Safory, Fazary & Lee, 2010). Thus,
105
large-scale production of recombinant HAase robust microbial factories and development of
106
enzymatic process for the preparation of specific HA oligosaccharides would be attractive.
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More recently, the first leech HAase-encoding gene was cloned and characterized. By
108
combining protein engineering and high-density culture, the production of recombinant
109
HAase was improved to 8.42×105 U/ml in the secretory expression system of the yeast Pichia
110
pastoris, which paved way for its application (Jin, Kang, Zhang, Du & Chen, 2014).
111
Additionally, the HA oligosaccharides produced from leech HAase contained glucuronic acid
112
at the reducing end, which was quite different from that of BTH (Jin, Kang, Zhang, Du &
113
Chen, 2014; Linker, Meyer & Hoffman, 1960). In the present study, to expedite enzymatic
115 116 117
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preparation of HA oligosaccharides, the whole hydrolysis process and products in pure water solution without the addition of any metal ions were systematically investigated and characterized. Impressively, by controlling the hydrolysis time, high-level enzymatic production of the designed HA oligosaccharides with low polydispersity (approximately 1.16)
118
was achieved.
119
2. Materials and methods
120
2.1. Chemicals and reagents
121
HA (1.21×106 Da) sample (fermented with Streptococcus zooepidemics) of high purity 7 Page 6 of 28
was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Standard dextranum
123
samples with standard molecular mass distribution (180, 2700, 9750, 13,050, 36,800, 135,350,
124
and 2,000,000 Da) were purchased (National Institute for Food and Drug Control). HAase
125
from the yeast P. pastoris secretory expression system with a specificity value of 1.20× 106
126
U/mg was applied for HA hydrolysis. Specifically, the specific activity was defined as units of
127
enzyme per mg of protein. BTH and 8-Aminonaphthalene-1, 3, 6-trisulfonic acid disodium
128
salt (ANTS) was purchased (Sigma-Aldrich). Dinitrosalicylic acid (DNS) reagent contained
129
1% DNS, 2% NaOH, and 20% sodium potassium tartrate. The matrix solution for the
130
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)
131
was prepared by dissolving 20 mg of 2,5-dihydroxy-benzoic acid (DHB) and 58.5 mg of
132
sodium chloride in 1 mL of 10% ethanol.
133
2.2. Preparation of HA oligosaccharides
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Before preparation of HA oligosaccharides, the recombinant leech HAase activity was
135
determined by measuring the amount of reducing sugar using the DNS colorimetric method
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(Ghose, 1987; Linker, Hoffman & Meyer, 1957; Silveira, Aguiar, Siika-aho & Ramos, 2014; Yuki & Fishman, 1963). The absorbance of each reaction mixture was detected at 540 nm after an appropriate dilution for calculating the HAase activity. In the preparation of molar-mass-defined HA fragments, the amount of HAase could be controlled. The
140
constant-temperature fermentation reaction was conducted in a 3-L fermentor (LiFlus GM
141
BioTRON, Korea) containing 2 L of the medium, maintaining the temperature at 45°C using
142
a temperature sensor. In addition, an agitator, operating at 500 rpm, was mounted to the
143
bottom of the fermentation tank, which ensured uniform mixing of HA and water. 8 Page 7 of 28
Subsequently, HAase was added and the reaction proceeded smoothly without the addition of
145
acid, alkali, and anti-foaming agent to achieve product of high purity. According to the size of
146
HA fragments required, the reaction was terminated at a suitable kinetic time point. The
147
samples were collected and filtered to remove insoluble particle. Furthermore, HA was
148
hydrolyzed using BTH at the same conditions.
149
2.3. High performance liquid chromatography-size exclusion chromatography-refractive
150
index detector analysis
us
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144
To determine the molecular masses and polydispersity of the HA hydrolysate, high
152
performance liquid chromatography-size exclusion chromatography-refractive index detector
153
(HPLC-SEC-RI) analysis was employed (Tranchepain et al., 2006). The Agilent 1260
154
HPLC-SEC-RI device was composed of a G1310A pump, a G1329B injector, an
155
UltrahydrogelTM linear column (7.8×300 mm; Shimadzu, Kyoto, USA), size exclusion
156
chromatograph using Sephadex G-100 as the sieving material, and a G1362A RI detector. The
157
mobile phase was sodium nitrate (0.1 mol/L). A total of 40 µL of the reaction mixture were
159 160 161
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158
an
151
injected with 0.05% sodium azide and each sample was eluted under a constant flow rate (0.9 mL/min) for 25 min. The temperature of the column was controlled at 40°C and the flow cell temperature was controlled at 35°C. The signals measured by the RI detector were analyzed using the GPC software, which calculated the number-average molar mass (Mn), Mw, and
162
polydispersity index (Ip, Ip = Mw/Mn). The standard curve plotting the HA fragment Mw as a
163
function of the elution volume was simulated using the GPC software (Agilent Technologies).
164
Then, the Mw of each hydrolysate was calculated based on the standard curve by the GPC
165
profile. In conclusion, HPLC-SEC-RI is highly suitable to study the kinetics of the HA 9 Page 8 of 28
166
hydrolysis reaction catalyzed using recombinant leech HAase in case it has been calibrated
167
using standard dextranum samples in advance (Tranchepain et al., 2006).
168
2.4. MALDI-TOF MS analysis of HA tetrasaccharides and HA hexasaccharides MALDI-TOF MS is a powerful method to quickly and accurately determine the masses
170
of saccharides. In the present study, the MALDI-TOF-MS experiments were conducted on
171
TofSpecE mass spectrometer equipped with N2 laser at a wavelength of 337 nm. The machine
172
was run in negative-ion reflector mode using 20-kV acceleration voltage (Mahoney, Aplin,
173
Calabro, Hascall & Day, 2001). Spectra external calibration was conducted before measuring
174
the samples (Tranchepain et al., 2006). A total of 0.5 μL of the hydrolysate beads mixed with
175
0.5 μL of DHB solution were spotted on the iron target of the spectrometer and crystallized
176
under vacuum conditions. Raw data were analyzed using Flex Analysis version 3.3 software,
177
and no smoothing was performed on the spectra. Hydrolysate masses were calculated using
178
the Prowl program in monoisotopic mode. Each sample solution was performed under the
179
same operating conditions.
181 182 183
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2.5. Preparation and characterization of the HA tetrasaccharides HA oligosaccharides were isolated from the hydrolysates by anion exchange
chromatography according to the previous description (Tawada, et al., 2002). The purified HA4 was dissolved in distilled water with concentration of 1.0 g/L. ESI-MS analysis was
184
carried out on a MALDI SYNAPT Q-TOF MS with scanning over the m/z range 50-2200 in
185
10 s/scan. 5 μL sample solution was introduced into the ion source of the mass spectrometer
186
through an autosampler of Agilent 1260 series HPLC system for analysis.
187
2.6. Polyacrylamide gel electrophoresis analysis of molar-mass-defined HAFs 10 Page 9 of 28
The molar-mass-defined fragments were frozen and lyophilized in a vacuum
189
concentrator. For the derivatization of molar-mass-defined HA fragments using ANTS, 2
190
nmol of each fragments were employed. All the samples were derived by adding 5 μL of
191
ANTS reagent solution for 15 min at room temperature and then 5 μL of sodium
192
cyanoborohydride solution for 16 h at 37°C (Tawada et al., 2002). After incubation, each
193
sample was centrifuged at 10,000 × g for 10 min at 4°C. 10% polyacrylamide gel be used to
194
obtain high resolution separation of molar-mass-defined HA fragments. For electrophoresis,
195
the comb was removed from the gel, and the gel was moved to an electrophoresis unit. Each
196
sample of 5 µL was run on a polyacrylamide gel (Bhilocha et al., 2011) on a vertical gel
197
apparatus at 4°C first 18mA/gel and then at 7mA/gel for 3h in Tris-borate-EDTA (TBE) buffer.
198
After electrophoresis, the gel was illuminated with UV light (365 nm) and photographed.
199
2.7. Analysis of molar-mass-defined HAFs by capillary zone electrophoresis
te
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The samples were lyophilized in a vacuum concentrator. The molar-mass-defined HAFs
201
were dissolved in 200 µL of tenfold diluted capillary zone electrophoresis (CZE) operating
202 203 204 205
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200
buffer and centrifuged for 15 min at 11,000 × g. The supernatant was used for CZE analysis. CZE analysis was performed on a P/ACETM MDQ Capillary Electrophoresis System equipped with elastic quarts capillary vessel column (65 cm×75 µm) and 32 Karat Software. The operating buffer contained 50 mM disodium hydrogen phosphate and 20 mM sodium
206
tetraborate (pH 9) and the sample buffer was prepared by diluting the operating buffer tenfold
207
with Milli-Q water (Hofinger, Hoechstetter, Oettl, Bernhardt & Buschauer, 2008). The rinse
208
pressure was 20 psi, inject pressure was 2 psi, and duration was 4 s. The analysis was
209
performed at a separate voltage of 15 kV, capillary temperature of 20°C, and detector 11 Page 10 of 28
wavelength of 200 nm (Grimshaw et al., 1996). Between each analysis, the capillary was
211
operated with Milli-Q water (1 min), 50 mM sodium hydroxide (1 min), Milli-Q water (1
212
min), and finally operating buffer (5 min).
213
3. Results and discussion
214
3.1. Stability evaluation of the recombinant HAase in aqueous solution
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To facilitate and simplify the subsequent purification processes, a pure aqueous solution
216
without the addition of any metal ions was chosen and utilized for HA hydrolysis.
217
Accordingly, the stability of the recombinant HAase in this system was first investigated. Our
218
previous results (Jin, Kang, Zhang, Du & Chen, 2014) revealed that the system should be
219
incubated at 45°C with a near-neutral optimum pH of 6.5. In the present study, the samples
220
were collected at regular intervals and analyzed. Interestingly, an obvious increase in the
221
HAase activity was observed in the initial stage, which was consistent with the experiments
222
conducted using the buffer systems. As shown in Fig. 1, after incubation for 24 h, no
224 225
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223
an
215
significant decrease in the HAase activity was detected, suggesting its desirable stability in pure aqueous solution. Moreover, the results also indicated that application of this pure aqueous solution system for the enzymatic production of HA oligosaccharides is practicable.
12 Page 11 of 28
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226
Fig.1. Evaluation of the stability of the recombinant leech HAase. The remaining enzymatic
228
activity was determined at 45°C and rotation speed of 500 rpm in a 3-L fermentor.
229
3.2. Analysis of the hydrolysis process
M
an
227
To rapidly estimate the practical feasibility of the hydrolysis process, high concentration
231
of high-molecular-mass hyaluronan (40 g/L) was added as substrate into a 3-L fermenter for
232
analysis. In particular, four different HAase concentrations (1.00×103, 1.25×104, 2.00×104,
234 235 236
te
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233
d
230
and 4.00×104 U/ml) were applied. As shown in Fig. 2, the Mw of the HA fragments sharply decreased in the initial stage. Moreover, it was obvious that higher degradation rates were achieved with increased concentration of HAase. After 2 h, the Mw of the HA fragments generated with different concentrations of HAase (as indicated earlier) decreased to 36,000,
237
16,000, 8500, and 5000 Da, respectively. However, the decrease in Mw was very slow beyond
238
that time although the theoretical end products were HA hexasaccharides (HA6) and HA
239
tetrasaccharides (HA4) (Jin, Kang, Zhang, Du & Chen, 2014; Linker, Meyer & Hoffman,
240
1960; Stern & Jedrzejas, 2006), suggesting that the hydrolysis rate rapidly decreased with the 13 Page 12 of 28
accumulation of shorter HA fragments, which is consistent with the previous reports
242
conducted with BTH (Deschrevel, Tranchepain & Vincent, 2008). In fact, no further obvious
243
decrease in the Mw was detected and the final Mw of the generated HA oligosaccharides at 24
244
h was approximate 20,000, 10,000, 4000, and 3000 Da, respectively. In fact, this is the first
245
HA depolymerization curve reported because partial digestion of HA with BTH always gives
246
rise to a wide variety of fragments with high polydispersity (Kakizaki, Ibori, Kojima,
247
Yamaguchi & Endo, 2010; Tawada, et al., 2002; Tranchepain et al., 2006). Conversely, the
248
results obtained here indicated that the partial digestion products of the recombinant leech
249
HAase should have a low polydispersity value, which was confirmed by further analysis
250
results (Table 1).
Ac ce p
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241
251 252
Fig.2. Time course of HA hydrolysis in the fermentation tank. The high-molecular-mass HA
253
was digested under a rotation speed of 500 rpm and temperature of 45°C. (A) HAase,
254
1.00×103 U/mL; (B) HAase, 1.25×104 U/mL; (C) HAase, 2.00×104 U/mL; (D) HAase, 14 Page 13 of 28
255
4.00×104 U/mL.
256
3.3. Influence of HA concentration and hydrolysis time on polydispersity
The biofunction of HA molecules are closely related to its Mw. However, in most cases,
258
the HA polysaccharide fragments produced using traditional methods are widely distributed
259
with high polydispersity (Stern, Kogan, Jedrzejas & Soltes, 2007). As a result, the production
260
of HA fragments with low polydispersity is attractive. In this regard, the effects of HA
261
concentration and hydrolysis time were further investigated. As shown in Table 1, it could be
262
obviously observed that although hydrolysates at any hydrolysis time point have different Mw
263
values, higher polydispersity was achieved from higher concentrations of HA. Meanwhile, it
264
could also be noted that the polydispersity index of all the hydrolysates with different
265
concentrations of HA (20, 40 and 60 g/L) declined with prolonged hydrolysis. These results
266
indicated that comparatively low concentrations of HA and long hydrolysis time could give
267
rise to HA oligosaccharides with low polydispersity, whereas BTH with high
269 270
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ip t
257
polydispersity (Tawada, et al., 2002). Table 1 Characteristics of hydrolysates from different concentrations of high-molecular-mass HA.
HAase (U/mL)
HA (g/L)
0.5 h
1h
1.5 h
2h
3h
Mw
Ip
Mw
Ip
Mw
Ip
Mw
Ip
Mw
Ip
1.5×104
20
10,000
1.86
7000
1.6
5600
1.46
4900
1.33
4000
1.20
1.5×104
40
20,000
2.43
13,000
2.06
10,000
1.88
8400
1.73
6700
1.54
1.5×104
60
32,000
2.70
18,000
2.24
16,200
2.14
14,100
2.00
10,000
1.90
271
Mw = Weight-average molar mass, Ip = Polydispersity index
272
3.4. Enzymatic production of defined HA oligosaccharides 15 Page 14 of 28
Based on the above-mentioned results, partial degradation for the production of desirable
274
HA fragments with specific Mw was investigated. The HA oligosaccharides with Mw values of
275
30,000, 10,000, and 4000 Da were individually prepared by controllable degradation of 40
276
g/L high-molecular-size HA with different concentrations of HAase and at various hydrolysis
277
time. As expected, the defined HA oligosaccharides were successfully and efficiently
278
produced. More interestingly, it could be noted that all the products catalyzed by
279
comparatively lower HAase and longer hydrolysis time exhibited lower polydispersity (Table
280
2). For instance, although the HA oligosaccharides (40 g/L) produced using 1.25×104 and
281
2.00×104 U/mL HAases showed only one peak at the same time, the molecular mass
282
distribution graph of the HA oligosaccharides obtained from 1.25×104 U/mL HAase was more
283
concentrated (Fig. 3). Impressively, the polydispersity index of the HA fragments with Mw of
284
4000 Da was only 1.16, suggesting an extremely narrow size distribution. Previously,
285
although many attempts have been made using BTH, it was impractical to prepare specific
286
molecular-mass HA fragments (Kakizaki, Ibori, Kojima, Yamaguchi & Endo, 2010; Mahoney,
288 289 290
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287
ip t
273
Aplin, Calabro, Hascall & Day, 2001). In comparison, the recombinant leech HAase is more valuable and acceptable for the enzymatic production of the desirable HA oligosaccharides. In addition, several efficient isolation and purification methods for obtaining pure and size-uniform HA oligosaccharides have been established (Kakehi, Kinoshita & Yasueda,
291
2003),
292
oligosaccharides.
293
Table 2 Polydispersity of specifically distributed HA oligosaccharides produced with
294
different concentrations of HAase.
which
will
further
promote
large-scale
production
of
size-specific
HA
16 Page 15 of 28
HAase (U/ml)
Hydrolysis time (h)
3
1.00×10 1.25×10 4 1.25×10 4 2.00×10 4 2.00×10 4 4.00×10 4
30,000 10,000 4000 Mw = Weight-average molar mass
1.69 2.07 1.68 1.86 1.16 1.19
296
d
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cr
295
3 0.5 15 1.3 24 4.5
Polydispersity index
ip t
Mw (Da)
Fig.3. HPLC-SEC profile of the HA oligosaccharide samples with Mw of 10,000 Da. (A) The
298
elution peak of the samples produced using 1.25×104 U/mL HAase; (B) Molecular mass
300 301
302
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299
te
297
distribution graph of the samples hydrolyzed using 1.25×104 U/mL HAase; (C) The elution peak of the samples produced using 2.00×104 U/mL HAase; (D) Molecular mass distribution graph of the samples hydrolyzed using 2.00×104 U/mL HAase.
3.5. Enzymatic production of the end products HA6 and HA4
303
In recent years, HA6 and HA4 have attracted much attention because of its potential
304
applications in medicine (Torigoe et al., 2011; Wakao et al., 2011). However, their large-scale
305
production has always been a big challenge owing to the inability of traditional degradation
306
methods (Stern, Kogan, Jedrzejas & Soltes, 2007). Although HA6 and HA4 could be 17 Page 16 of 28
generated with BTH, the proportion was still low (Jin, Kang, Zhang, Du & Chen, 2014;
308
Tawada, et al., 2002), which eventually decreased the conversion rate. Consequently, in the
309
present study, large-scale enzymatic production of HA6 and HA4 was investigated. In
310
particular, 6.00×104 U/mL HAase was added to the hydrolysis system containing 40 g/L HA.
311
MALDI-TOF MS spectrometry was applied for analyzing the degradation products. At 0.5 h,
312
trace amounts of HA oligomers HA10, HA8, HA6, and HA4 were generated (Fig. 4). When
313
the incubation period was extended to 5.5 h, HA10 was completely converted to HA6 and
314
HA4. At 6.5 h, the peak corresponding to HA8 disappeared and only two ion peaks
315
corresponding to HA6 and HA4 were detected, indicating that all the higher HA oligomers
316
were exhaustively digested to the end products HA6 and HA4. Moreover, it has been
317
demonstrated that the reducing end of HA oligomers from leech HAase was glucuronic acid
318
but not N-acetyl-D-glucosamine (Jin, Kang, Zhang, Du & Chen, 2014; Yuki & Fishman,
319
1963), indicating its potential different biological function. Thus, HA4 was further isolated
320
from the hydrolysate with the anion exchange chromatography. Specifically, Electrospray
322 323
cr
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d
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Ac ce p
321
ip t
307
Ionization Mass Spectrometry (ESI-MS) was used with the negative ion mode to analyze the separated products. As shown in Fig. 5, all the multistage mass spectrometry data confirmed a high purity of the isolated HA4.
18 Page 17 of 28
ip t cr us an
324
Fig.4. MALDI-TOF MS profile of the hydrolysates catalyzed by 6.00×104 U/mL HAase. In
326
particular, the mass-to-charge ratio of 797, 1176, 1555, and 1934 showed [HA4-2H+Na]-,
327
[HA6-2H+Na]-, [HA8-2H+Na]-, and [HA10-2H+Na]-, respectively.
d te
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M
325
未标题-2.jpg
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Description: C:\Users\yph\Desktop\
329
19 Page 18 of 28
Fig.5. Negative-ion electrospray mass spectra of separated product: (A) HA4 mass spectra with
331
corresponding structure of 775.1[M-H]-; (B) The precursor ion 775.1[M-H]- is bombarded to
332
generate fragment ions 396.0[M-H]- and 599.1[M-H]- in MS2 mass spectra.
333
3.6. Characterization of polydispersity of the hydrolysis products
cr
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330
To gain more detailed insights, the produced HA oligosaccharides were further
335
characterized by powerful polyacrylamide gel electrophoresis method developed recently
336
(Bhilocha et al., 2011). Accordingly, 10% polyacrylamide gel in TBE buffer was employed
337
for separating the intermediate HA products with Mw of 4000, 10,000, and 30,000 Da, the end
338
products containing HA6 and HA4, and the isolated HA4. The hydrolysis products with Mw
339
of 30,000 Da that produced by BTH were also comparatively investigated at the same time.
340
As shown in Fig. 6, it could be obviously discovered that the hydrolysate with smaller Mw,
341
especially the isolated HA4 (Lane 1) showed much sharper band, suggesting its low
342
polydispersity value. In comparison, it could be found that the HA oligomers from BTH
344
an
M
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343
us
334
trailed a longer band (Lane 6) than that with the same Mw from the recombinant leech HAase (Lane 5), suggesting a broader distribution of the Mw values of the BTH hydrolysates.
345
20 Page 19 of 28
Fig.6. Polyacrylamide gel electrophoresis analysis of the molecular mass distribution of the
347
prepared oligosaccharide samples: Lane 1, the isolated product of HA4; Lane 2, end
348
hydrolysates containing HA6 and HA4; Lane 3, samples with Mw of 4000 Da; Lane 4,
349
samples with Mw of 10,000 Da; Lane 5, samples with Mw of 30,000 Da; Lane 6, samples with
350
Mw of 30,000 Da from bovine testicular hyaluronidase hydrolysis.
cr
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346
Furthermore, the HA oligomers with Mw of 30,000 Da were separated and characterized
352
using CZE, which has high resolution power particularly in analyzing complex carbohydrate
353
mixtures such as the negatively charged molar-mass-defined HA fragments (Hong, Sudor,
354
Stefansson & Novotny, 1998; Hofinger, Hoechstetter, Oettl, Bernhardt & Buschauer, 2008).
355
Obviously, the HA oligomers hydrolyzed by the recombinant leech HAase showed only one
356
main peak (Fig. 7). In contrast, several peaks were observed with the HA oligomers from
357
BTH. The results further confirmed the concentrated molecular mass distribution of the
358
hydrolysates from the recombinant leech HAase, which might be ascribed to the preferential
359
digestion of longer HA chains by the recombinant leech HAase through a nonprocessive
361 362
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M
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Ac ce p
360
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351
endolytic mode (Lepperdinger, Mullegger & Kreil, 2001). Thus, it can be concluded that enzymatic production of HA oligosaccharides with specific molecular mass distribution using recombinant leech HAase was efficient and applicable.
21 Page 20 of 28
ip t cr us
363
Fig.7. CZE profile of the prepared HA oligosaccharide samples. (A) Samples with Mw of
365
30,000 Da from bovine testicular hyaluronidase hydrolysis; (B) Samples with Mw of 30,000
366
Da from leech hyaluronidase hydrolysis.
367
Conclusion
d
M
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364
HAase concentration per unit HA played an important role in hydrolysis rate and
369
molecular mass distribution. Longer hydrolysis time produced molar-mass-defined HA
371 372 373 374
Ac ce p
370
te
368
fragments with lower polydispersity. By modulating HAase concentrations and hydrolysis time, any molar-mass-defined HA oligomers could be efficiently produced. All the obtained molar-mass-defined
HA
oligosaccharides
were
extremely
concentrated
with
low
polydispersity. Thus, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will promote progress in related research and potential
375
applications in areas, including chemical synthesis, medical therapy, cosmetics, and health
376
food.
377
Conflict of interest 22 Page 21 of 28
The authors declare that there is no conflict of interest.
379
Acknowledgments
380
This work was financially supported by a grant from the Key Technologies R & D Program of
381
Jiangsu Province, China (BE2014607); National High Technology Research and
382
Development Program of China (863 Program, 2012AA022005); Program for Changjiang
383
Scholars and Innovative Research Team in University (no. IRT1135); National Natural
384
Science Foundation of China (31200020); and 111 Project.
385
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Bhilocha, S., Amin, R., Pandya, M., Yuan, H., Tank, M., LoBello, J., Shytuhina, A., Wang, W. L., Wisniewski, H. G., de la Motte, C., & Cowman, M. K. (2011). Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5-to 500-kDa hyaluronan. Analytical Biochemistry, 417, 41-49. Boltje, T. J., Buskas, T., & Boons, G. J. (2009). Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. Nature Chemistry, 1, 611-622. Chang, C. C., Hsieh, M. S., Liao, S. T., Chen, Y. H., Cheng, C. W., Huang, P. T., Lin, Y. F., & 23 Page 22 of 28
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