marine drugs

Article Cloning, Expression, and Characterization of a New Glycosaminoglycan from Microbacterium sp. H14

Junhao Sun 1,2,3, Xu Han 1,2,3, Guanrui Song 1,2,3, Qianhong Gong 1,2,3,* and Wengong Yu 1,2,3,*

1 School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China; [email protected] (J.S.); [email protected] (X.H.); [email protected] (G.S.) 2 Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, Qingdao 266237, China 3 Provincial Key Laboratory of Glycoscience and Glycotechnology, Ocean University of China, 5 Yushan Road, Qingdao 266003, China * Correspondence: [email protected] (Q.G.); [email protected] (W.Y.); Tel.: +86-532-8203-2067 (Q.G.); +86-532-8203-1680 (W.Y.)  Received: 8 November 2019; Accepted: 28 November 2019; Published: 2 December 2019 

Abstract: Glycosaminoglycan (GAG) lyase is an effective tool for the structural and functional studies of glycosaminoglycans and preparation of functional oligosaccharides. A new GAG lyase from Microbacterium sp. H14 was cloned, expressed, purified, and characterized, with a molecular weight of approximately 85.9 kDa. The deduced lyase HCLaseM belonged to the polysaccharide lyase (PL) family 8. Based on the phylogenetic tree, HCLaseM could not be classified into the existing three subfamilies of this family. HCLaseM showed almost the same activity towards hyaluronan (HA), chondroitin sulfate A (CS-A), CS-B, CS-C, and CS-D, which was different from reported GAG . HCLaseM exhibited the highest activities to both HA and CS-A at its optimal temperature (35 ◦C) and pH (pH 7.0). HCLaseM was stable in the range of pH 5.0–8.0 and temperature below 30 ◦C. The enzyme activity was independent of divalent metal ions and was not obviously affected by most metal ions. HCLaseM is an endo-type enzyme yielding unsaturated disaccharides as the end products. The facilitated diffusion effect of HCLaseM is dose-dependent in animal experiments. These properties make it a candidate for further basic research and application.

Keywords: glycosaminoglycan lyase; oligosaccharide; characterization

1. Introduction Hyaluronan (HA) and chondroitin sulfate/dermatan sulfate (CS/DS) belong to the class of negatively charged linear heteropolysaccharides named glycosaminoglycans (GAGs) [1,2], which are widely distributed on the surface of cells and connective tissue of animals. degrading GAGs are associated with the regulation of various cellular processes, such as proliferation, differentiation, migration, and adhesion [3,4]. GAG-based therapeutic agents have important application prospects. According the Carbohydrate-Active enZYmes (CAZy) database, glycosaminoglycan lyases are classified into the enzymes of PL-8, PL-16, PL-29 and other families. Compared to other families, the enzymes of the PL-8 family can degrade more types of substrates. The enzymes of the PL-8 family are an important component of the GAG lyases, which can be divided into three subfamilies by phylogenetic analysis. Hyalurorate lyase (HAase), which mainly degrades HA, belongs to the first subfamily. Chondroitin ABC lyases (ChSase ABC) and chondroitin AC lyases (ChSase AC), which mainly degrade chondroitin sulfate, belong to the second and third subfamilies, respectively. Different molar masses of HA fragments produced by enzymatic degradation perform different biological functions, such as induction of endothelial cell differentiation [5], promotion

Mar. Drugs 2019, 17, 681; doi:10.3390/md17120681 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 681 2 of 15 of angiogenesis [6], stimulation of collagen production, and proliferation of fibroblasts [7], and have attracted widespread attention. CS is mainly used to treat inflammation and osteoarthritis. It is not easily absorbed and may impair therapeutic effect because of its large molecular weight. The low molecular weight chondroitin sulfate (LMWCS) produced by enzymatic degradation is more easily absorbed and exerts therapeutic effects [8]. LMWCS might prevent and treat Alzheimer's disease through the blood–brain barrier and exert neuroprotective effects [9]. Chemical degradation can also produce oligosaccharides, but may alter their structure. Enzymatic approaches are used because of their specificity, and enzymatic degradation of polysaccharide is environmentally friendly [10]. GAG lyases degrade substrates such as HA and CS, which are effective tools for preparing bioactive oligosaccharide and studying the structure–function relationship of polysaccharides. GAG lyases have broad application prospects in the field of medicine because GAGs are widely distributed in the human body, which can treat lumbar disc herniation, spinal injury, and tumors [11,12]. Hyaluronidase is widely used as a drug diffusion agent in clinic [13]. Subcutaneous administration is a safe and convenient method of administration. However, the subcutaneous space has a complex three-dimensional extracellular matrix (ECM). The main filling of ECM is hyaluronan, which hinders the diffusion of drugs for subcutaneous administration [14]. Hyaluronidase can degrade hyaluronan in the subcutaneous space, improve tissue permeability, and promote drug diffusion and absorption. The currently commercial hyaluronidase is mainly prepared by extracting bovine testicular tissue. These enzymes are low in purity and high in price, limiting their use in pharmaceutical and biochemical engineering. The marine environment contains abundant enzyme resources. The search for novel polysaccharide lyases is essential for academic research and applications. In our previous work, strain Microbacterium sp. H14 was isolated from offshore of Qingdao, which degraded HA and CS. In this study, the HA and CS lyase (HCLaseM) was cloned, expressed, purified, and characterized. Results show that HCLaseM has almost the same enzyme activity for HA and various CSs and may be an effective tool for structural analysis of HA and CS and preparation of oligosaccharides. HCLaseM has the potential to become a good drug diffusion agent.

2. Results

2.1. Cloning and Sequence Analysis of HCLaseM Gene Microbacterium sp. H14 was isolated from offshore of Qingdao, which degraded HA and CS. Only one gene (GenBank MN400984.1) encoding GAG lyase was found in Microbacterium sp. H14 by genome sequencing analysis, which consists of an open reading frame of 2415 bp encoding 804 amino acids. The theoretical molecular weight of the deduced protein is 85.9 KDa and the isoelectric point is 5.79. The results of the NCBI sequence alignment showed that HCLaseM was a new enzyme of the PL-8 family. The characterized enzymes with the highest similarity to HCLaseM are chondroitin lyase (GenBank ANC28180.1) from Arthrobacter sp. GAG (45%) [15], chondroitin lyase (AFM38168.1) from Arthrobacter sp. MAT3885 (44%), and chondroitin lyase (PDB 1RWA_A) from Arthrobacter aurescens (43%) [16,17]. These enzymes are chondroitin sulfate AC lyases and cannot degrade CS-B. Chondroitin lyase (GenBank ANC28180.1) from Arthrobacter sp. GAG and chondroitin lyase (PDB 1RWA_A) from Paenarthrobacter aurescens are exogenous enzymes. However, HCLaseM is an endo-type enzyme and degrades CS-B. HCLaseM showed extensive substrate specificity. The relatively low similarity makes HCLaseM a more interesting and unique enzyme. A phylogenetic tree was constructed for chondroitin lyase of Arthrobacter sp. GAG, HCLaseM, and other characterized enzymes of the PL-8 family from the Carbohydrate-Active enZYmes (CAZy) database (Figure1). Eleven enzymes were included in the first subfamily, four enzymes were included in the second subfamily, and three enzymes were included into the third subfamily. HCLaseM and other enzymes were not included into the existing three subfamilies. HCLaseM formed a distinct group with chondroitin lyase (PDB: 1RWA A), chondroitin lyase (AFM38168.1), and chondroitin lyase Mar. Drugs 2019, 17, 681 3 of 15 Mar. Drugs 2019 , 17 , x 3 of 14 and(ANC28180.1). chondroitin Thelyase result (ANC28180.1). show that basedThe result on the show phylogenetic that based tree, on the HCLaseM phylogenetic cannot tree, be classifiedHCLaseM into cannotthe existing be classified three subfamilies into the existing of this three family. subfamilies of this family.

FigureFigure 1. 1.PhylogeneticPhylogenetic analysis analysis of HCLaseM of HCLaseM and characterized and characterized enzymes enzymes of the PL-8 of the family. PL-8 The family. phylogeneticThe phylogenetic analysis analysis was performed was performed using the using neigh thebor neighbor joining joiningmethod method in MEGA in MEGA5.1. 5.1.

2.2.2.2. Purification Purification and and Biochemical Biochemical Characterization Characterization of ofHCLaseM HCLaseM TheThe recombinant recombinant plasmid plasmid was was successfully successfully constructe constructedd and and expressed expressed in Escherichia in Escherichia coli coliBL21BL21 (DE3).(DE3). The The recombinant recombinant protein protein was waspurified purified by Ni-S byepharose Ni-Sepharose column, column, and SDS-PAGE and SDS-PAGE showed showed that HCLaseMthat HCLaseM was successfully was successfully purified purified with an withestimated an estimated molecular molecular weight of weight 85 kDa of (Figure 85 kDa 2), (Figure which 2), waswhich consistent was consistent with the with expected the expected size. Using size. HA Using as a HAsubstrate, as a substrate, the specific the specificactivity activityof HCLaseM of HCLaseM was 278.3was 278.3U/mg, U and/mg, the and recovery the recovery was 55.9%; was 55.9%; 15.4 15.4mg of mg purified of purified HCLaseM HCLaseM was wasobtained obtained from from 1 L 1of L of bacterialbacterial culture culture (Table (Table 1).1).

Table 1. Summary of the purified HCLaseM.

Total Activity Total Protein Specific Activity Step Fold Purification Yield (%) (U) (mg) (U/mg Protein) Fermentation media 7671 299.4 25.6 1 100 Nickel column 4290 15.4 278.3 10.9 55.9 0.2% (w/v) of HA was used as substrate. One unit (U) was defined as the amount of protein needed to form 1 µmol unsaturated uronic acid/min. A millimolar absorption coefficient of 5.5 was used in the calculation.

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Figure 2. SDS-PAGE of HCLaseM. Lane M, protein marker of molecular mass; Lane 1, purified HCLaseM.

Table 1. Summary of the purified HCLaseM.

Total Total Specific Activity Fold Yield Step Activity (U) Protein (mg) (U/mg Protein) Purification (%) Fermentation media 7671 299.4 25.6 1 100 Nickel column 4290 15.4 278.3 10.9 55.9 0.2% ( w/v) of HA was used as substrate. One unit (U) was defined as the amount of protein needed Figure 2. SDS-PAGEFigureto form 2.1 µmol of SDS-PAGE HCLaseM. unsaturated of uronic HCLaseM. Lane acid/min. M, Lane A protein M,milli proteinmolar markerabsorption marker of coefficient of molecular mole ofcular 5.5 mass; was mass; Laneused in 1, Lane 1, purified purifiedthe calculation. HCLaseM. HCLaseM. Five glycosaminoglycans glycosaminoglycans were were used used as substrates as substrates to detect to detect the specificity the specificity of HCLaseM. of HCLaseM. The relative The activityrelative activity of HCLaseM of HCLaseM to CS-A, to CS-B, CS-A, CS-C, CS-B, CS-D,CS-C, andCS-D, HA and was HA 100%, was 99%,100%, 98%, 99%, 99%,98%, and99%, 102% and (Figure102% (Figure3), respectively. 3), respectively.Table The V1.Themax Summary V ofmax HCLaseMof HCLaseM of the against against purified HA HA and and HCLaseM. CS-A CS-A was was 0.0246 0.0246 ± 0.00030.0003 and ± 0.0264 ± 0.00020.0002 µM/minµM/min (Table (Table 2),2), respectively. respectively. The The Km Kofm HCLaseMof HCLaseM against against HA and HA CS-A and CS-Awas 0.419 was ± 0.419± 0.019 and0.019 0.478 and 0.478± 0.015 mg/mL,0.015 mg respectively./mL, respectively. HCLa HCLaseMseM showed showed extensive extensive substrate substrate specificity specificity and ± Total± Total Specific Activity Fold Yield Step andalmost almost the same the same enzyme enzyme activity activity towards towards HA, CS-A, HA, CS-A, CS-B, CS-B, CS-C, CS-C, and CS-D, and CS-D, which which was different was diff erentfrom fromother otherconventional conventionalActivity hyaluronidases hyaluronidases(U) andProtein chondroitinas and chondroitinase(mg)e (Table(U/mg 3). (Table Both Protein)3 the). Bothrelative the activity relativePurification (Figure activity 3) (%) Fermentation(Figureand media Vmax3) showed and Vmax thatshowed 7671 HCLaseM that HCLaseM was similarly 299.4 was similarlyactive towards active towardsHA and25.6 HA CS-A, and which CS-A, indicates which indicates that 1 it 100 thatpossessed it possessed similarly similarly affinity a ffitowardsnity towards HA and HA CS-A. and CS-A. Nickel column 4290 15.4 278.3 10.9 55.9 0.2% ( w/v) of HA was used as substrate. One unit (U) was defined as the amount of protein needed to form 1 µmol unsaturated uronic acid/min. A millimolar absorption coefficient of 5.5 was used in the calculation.

Five glycosaminoglycans were used as substrates to detect the specificity of HCLaseM. The relative activity of HCLaseM to CS-A, CS-B, CS-C, CS-D, and HA was 100%, 99%, 98%, 99%, and 102% (Figure 3), respectively. The V max of HCLaseM against HA and CS-A was 0.0246 ± 0.0003 and Figure 3. Substrate specificity specificity of HCLaseM; 0.2% ( w/v) of HA, CS-A, CS-B, CS-C, and CS-D were used 0.0264 ± 0.0002 µM/min (Table 2), respectively. The Km of HCLaseM against HA and CS-A was 0.419 as substrates. The relative activity of 100% was CS-ACS-A determined at optimal pH and temperature. ± 0.019 and 0.478 ± 0.015 mg/mL, respectively. HCLaseM showed extensive substrate specificity and almost the same enzyme activity towards HA, CS-A, CS-B, CS-C, and CS-D, which was different from other conventional hyaluronidases and chondroitinase (Table 3). Both the relative activity (Figure 3) and Vmax showed that HCLaseM was similarly active towards HA and CS-A, which indicates that it possessed similarly affinity towards HA and CS-A.

Figure 3. Substrate specificity of HCLaseM; 0.2% ( w/v) of HA, CS-A, CS-B, CS-C, and CS-D were used as substrates. The relative activity of 100% was CS-A determined at optimal pH and temperature.

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Table 2. Km and Vmax values of HCLaseM.

Substrate Km (mg/mL) Vmax (µM/min) HA 0.419 0.019 0.0246 0.0003 ± ± CS-A 0.478 0.015 0.0264 0.0002 ± ± The Km and Vmax values of HCLaseM were determined using non-linear regression.

Table 3. Comparison of substrate specificity of HCLaseM with glycosaminoglycan lyase from different microorganisms.

Substrate Enzyme Source CS-A CS-B CS-C HA Reference HCLaseM Microbacterium sp. H14 100 99 98 102 This study ChSase ABC Bacteroides Stercoris 1 100 32 40 0 [18] ChSase AC B. Stercoris 1 100 0 46 67 [18] ChSase ABC FlavobacteriumHeparinum 1 100 100 100 0 [18] ChSase AC F. Heparinum 1 100 0 110 107 [18] ChSase ABC B.Thetaiotaomicron 1 100 13–16 80–130 10–30 [18] ChSase ABC Proteus. Vulgaris 1 100 34 100 60 [18] BniHL Bacillus niacin 2 100 0 68 227 [19] HAase-B Bacillus sp. A50 2 100 0 36 256 [20] HCLase Vibrio sp. FC509 2 100 0 96 222 [21] ChSase ABC Acinetobacter sp. C26 2 100 96 95 133 [22] ChSase AC II Arthrobacter sp. CS01 2 100 0 86 255 [23] Activity on CS-A as the substrate was set at 100%. 1 Data from the [18]. 2 Data was calculated from the specific activity or relative activity in the reference.

As the temperature increased, the enzyme activity of HCLaseM gradually increased at 0–40 ◦C (Figure4A). The optimum temperature for the activity of HCLaseM towards both HA and CS-A was 40 ◦C. The HA/CS-degrading activity was stable below 30 ◦C, and remained about 50% after incubation at 40 ◦C for 60 min (Figure4B). HCLaseM showed the highest activity at pH 7.0 (Figure4C, D). HCLaseM retained about 70% of activity at pH 5.0–9.0, and retained about 90% of activity at pH 6.0–8.0 after incubation for 12 h (Figure4E,F). These properties were beneficial to the storage of the enzyme. When HCLaseM was incubated in Glycine–NaOH buffer, the residual activity of CS-A was generally slightly lower than that of HA. Compared with the HA degradation activity of HCLaseM, the CS degradation activity is less tolerant to the alkaline environment. Mar. Drugs 2019 , 17 , x 6 of 14 Mar. Drugs 2019, 17, 681 6 of 15 Mar. Drugs 2019 , 17 , x 6 of 14

FigureFigure 4. 4. Effects EEffectsffects of temperature temperature and and pH pH on on HCLaseM. HCLaseM. HCLaseM. ( A ( )A Optimal)) Optimal Optimal temperature temperature temperature of HCLaseM. of HCLaseM. ( B) The( B) The thermostabilitythermostability of of of HCLaseM. HCLaseM. ( C ( (C)C Optimal)) Optimal Optimal pH pH pH of ofHCLaseM of HCLaseM HCLaseM when when when using using usingHA HA as HA a as substrate. aas substrat a substrat (De.) Optimal ( De.) ( D) pHOptimalOptimal of HCLaseM pHpH ofof HCLaseM when using whenwhen CS-A usingusing as a CS-A substrate.CS-A as as a asubstrat (Esubstrat) Thee. pH e.( E )( stability EThe) The pH pH ofstability HCLaseMstability of HCLaseMof when HCLaseM using when HAwhen as usingausing substrate. HA HA as as ( aFa substrate.)substrate. The pH stability( F)) TheThe pH pH of stability HCLaseM stability of of HCLaseM when HCLaseM using when when CS-A using using as aCS-A substrate. CS-A as aas substrate. a Thesubstrate. relative The The relative activity relative of 100%activityactivity was of of determined100%100% was determined at optimal at at pH optimal optimal and temperature. pH pH and and temperature. temperature. Experiments Experiments Experiments were conducted were were conducted conducted three times three three and timeserrortimes barsand and representerrorerror barsbars standardrepresent deviations. standardstandard deviations. deviations.

TheThe e effectff effectect of of of various various metal metal ions, ions, ions, chelators, chelators, chelators, and and anddetergents de detergentstergents on HCLaseM on on HCLaseM HCLaseM was was examined. was examined. examined. The The activity The 2+ 2+ 2+ 3+ activityofactivity HCLaseM of of HCLaseMHCLaseM was strongly was strongly inhibited inhibitedinhibited by Hg by by, Hg Fe Hg2+ ,2+, Fe, Cu Fe2+ ,2+ Cu, andCu2+ and2+SDS, and SDS, SDS, and and slightlyand slightly slightly promoted promoted promoted by by Al by 2+ 2+ 2+ (FigureAlAl3+3+ (Figure (Figure5). Except 5). 5). ExceptExcept for Hg for Hg, Fe2+2+ ,, FeFe and2+2+ and and Cu Cu Cu 2+,2+, most ,most most metalmetal metal ionsions ions diddid did not not significantly significantly significantly affect aff affectect the the theactivity activity activity of ofHCLaseM.of HCLaseM. HCLaseM. EDTA EDTAEDTA as aas metal a metal ion ionion chelating chelatingchelating agent agent agent hardly har hardlydly reduced reduced reduced the the activitythe activity activity of of HCLaseM, HCLaseM,of HCLaseM, which which which was wasdiwasfferent different different from fromfrom previous previous reports reportsreports that that EDTAthat EDTA EDTA reduced reduc reduc enzymeeded enzyme enzyme activity activity activity [24 [24].]. [24].

Figure 5. Effects of metal ions, chelators, and detergents on HCLaseM. The activities of HCLaseM Figureagainst 5. HAEffectsEff ects and of CS-A metal were ions, measured chelators, in and 20 detergents mM Tris-HCl on HCLaseM. (pH 7.0) buffer The activities containing of 1HCLaseM mM againstconcentration HA HA and and of CS-A various CS-A were were metal measured measured ions, in chelators, 20 mM in 20 Tris-HCl and mM detergents. Tris-HCl (pH 7.0) The bu (pHff er relative 7.0) containing buffer activity 1 containing mM of 100% concentration was 1 mM concentrationofdetermined various metal in ofthe ions, various buffer chelators, without metal and ions,metal detergents. chelators,ions, chelat andTheors, relativeand detergents. detergents. activity The of relative 100% was activity determined of 100% in was the determinedbuffer without in the metal buffer ions, without chelators, metal and ions, detergents. chelators, and detergents.

Mar.Mar. Drugs Drugs 20192019,, 1717,, x 681 77 of of 14 15 Mar. Drugs 2019 , 17 , x 7 of 14 2.3. Degradation Pattern and End Production of HCLaseM 2.3. Degradation Pattern and End Production of HCLaseM 2.3. DegradationThin layer chromatography Pattern and End Production was used toof HCLaanalyzeseM the mode of degradation of HCLaseM, and the time courseThin layer of the chromatography reaction products waswas was usedused analyzed toto analyze by theusing modem odehyaluronan ofof degradation as a substrate. of HCLaseM, It was and found the thattimetime a coursecourse small ofofamount thethe reactionreaction of hyaluronan productsproducts oligosaccharide waswas analyzedanalyzed byb yw usingusingith low hyaluronanhyaluronan molecular asasweight aa substrate.substrate. was present It was at found the beginningthatthat aa smallsmall of amountamount the reaction, ofof hyaluronanhyaluronan and low oligosaccharidemolecularoligosaccharide weight withw ith oligosaccharides lowlow molecularmolecular gradually weightweight was increased presentpresent as atat the thethe reactionbeginning proceeded of the the reaction, reaction, (Figure 6A). and and The low low time molecular molecular course weight weightof the reaction oligosaccharides oligosaccharides product of gradually gradually CS-A (Figure increased 6B) was as also the the similar,reactionreaction and proceededproceeded the pattern (Figure(Figure of6 6A).A). the The The oligosaccharide time time course course of of thepro theduct reaction reaction suggests product product that of of CS-A CS-A HCLaseM (Figure (Figure may6 B)6B) was degradewas also also substratessimilar,similar, and and in the an the patternendolytic pattern of themanner. of oligosaccharide the oligosaccharide The end-product product pro was suggestsduct separated suggests that HCLaseM by that Superdex HCLaseM may peptide degrade may 10/300 substrates degrade GL gelinsubstrates anfiltration endolytic in column an manner. endolytic and The subjected manner. end-product toThe ESI–MS end-product was separatedanalysis. was The by separated Superdex main peaks by peptide Superdex of the 10 final/300 peptide products GL gel 10/300 filtration of HA GL andcolumngel filtration CS-A and were subjected column 378.10 and to m ESI–MS /subjectedz (Figure analysis. to 7A) ESI–MS and The main458.06analy peakssis. m/ zThe of(Figure themain final 7B),peaks products respectively. of the offinal HA products The and CS-A molecular of were HA weights378.10and CS-Am / arez (Figure were consistent 378.107A) and withm/ 458.06z (Figurethe unsaturatedm/z 7A)(Figure and7 B), disacch 458.06 respectively. aridesm/z (Figure of The HA molecular7B), and respectively. CS-A, weights respectively. The are consistent molecular These resultswithweights the demonstrate areunsaturated consistent that disaccharides HCLaseM with the unsaturated was of an HA endo-type and disacch CS-A, lyase,arides respectively. which of HA could These and completely CS-A, results respectively. demonstratedegrade HA These and that CS-AHCLaseMresults to demonstrate unsaturated was an endo-type thatdisaccharides HCLaseM lyase, throughwas which an endo-type coulda β-elimi completelynation lyase, mechanism.which degrade could HA completely and CS-A degrade to unsaturated HA and β disaccharidesCS-A to unsaturated through disaccharides a -elimination through mechanism. a β-elimination mechanism.

Figure 6. The reaction pattern of HCLaseM. The reactions were conducted at 40 °C using 0.2% ( w/ v)

sodiumFigure 6.6. HA The and reaction CS-A pattern in 20 mM of HCLaseM. Na 2HPO 4–NaH The reactions2PO 4 (pH were 7.0) conductedbuffer. ( A) at The 40 ◦ time°CC using course 0.2% of (w HA/v)

degradationsodiumsodium HA HA by and and HCLaseM CS-A CS-A in in 20was20 mMmM was Na Nadetermined22HPOHPO 44–NaH by 2TLC.PO 44 (pH((pHB) The 7.0) 7.0) time buffer. buff courseer. ( (AA)) of The CS-A time degradation course of HA HAby HCLaseMdegradation was by HCLaseMHCLaseM determined was by was TLC. determined Lane M, by the TLC. purified (B)) TheThe timetime monomeric coursecourse ofof sugar: CS-ACS-A degradationdegradationN-acetyl-D-(+)- byby glucosamine.HCLaseM was was determined determined by TLC. by TLC.Lane M, Lane the purified M, the monomeric purified monomeric sugar: N-acetyl- sugar:d-( +N)-glucosamine.-acetyl-D-(+)- glucosamine.

FigureFigure 7. AnalysisAnalysis of end products ofof HCLaseM.HCLaseM. ( A( A)) ESI–MS ESI–MS analysis analysis of of end end products products of of HA. HA. (B )( ESI–MSB) ESI– analysis of end products of CS-A. MSFigure analysis 7. Analysis of end ofproducts end products of CS-A. of HCLaseM. ( A) ESI–MS analysis of end products of HA. ( B) ESI– 2.4. EMSffect analysis of Hyaluronidase of end products on the of Di CS-A.ffusion of Trypan Blue 2.4. Effect of Hyaluronidase on the Diffusion of Trypan Blue The ability of hyaluronidase to promote diffusion can be determined by the increased diffusion 2.4. EffectThe ability of Hyaluronidase of hyaluronidase on the Diffusion to promote of Tr diffusionypan Blue c an be determined by the increased diffusion area of trypan blue after co-administration in the skin [25]. The diffusion experiment of trypan blue area of trypan blue after co-administration in the skin [25]. The diffusion experiment of trypan blue characterizesThe ability the of facilitated hyaluronidase diffusion to promote effect of diffusion hyaluronidase can be indetermined a short period. by the Hyaluronidase increased diffusion from characterizesarea of trypan the blue facilitated after co-administration diffusion effect ofin htheyaluronidase skin [25]. The in adiffusion short period. experiment Hyaluronidase of trypan from blue bovinecharacterizes testes (BTH) the facilitated was used diffusion as a drug effect diffusion of hyaluronidase agent in clinic. in Thea short diffusion period. area Hyaluronidase of trypan blue from in bovine testes (BTH) was used as a drug diffusion agent in clinic. The diffusion area of trypan blue in

Mar. Drugs 2019, 17, 681 8 of 15 Mar. Drugs 2019 , 17 , x 8 of 14 thebovine experimental testes (BTH) group was was used significantly as a drug di ffhigherusion tha agentn that in clinic. in the Thecontrol diffusion group area (Figure of trypan 8A, B). blue The in samethe experimental dose of BTH group and HCLaseM was significantly showed higher similar than diffusi thaton in effects. the control This group suggests (Figure that8 A,B).HCLaseM The same and BTHdose are of BTH equally and HCLaseMeffective in showed digesting similar hyaluronan diffusion i enff skinects. tissue This suggests in a short that period. HCLaseM The and potency BTH areof HCLaseMequally eff ectiveis even in slightly digesting higher hyaluronan than that in skinof BTH, tissue probably in a short because period. HCLaseM The potency has of considerable HCLaseM is activityeven slightly to degrade higher chondroitin than that of sulfate. BTH, probably The diffus becauseion area HCLaseM of trypan has blue considerable increased activity with the to increase degrade ofchondroitin HCLaseM sulfate.dose (Figure The di 8C).ffusion This area suggests of trypan that the blue facilitated increased diffusion with the effect increase of HCLaseM of HCLaseM is dose- dose dependent.(Figure8C). This suggests that the facilitated di ffusion effect of HCLaseM is dose-dependent.

FigureFigure 8. 8. DiffusionDiffusion properties properties of oftrypan trypan blue blue with with hyaluronid hyaluronidasease co-administration co-administration in dermis. in dermis. (A) Trypan(A) Trypan blue blue diffusion diffusion 60 min 60 min after after combined combined injecti injectionon with with PB, PB, BTH, BTH, and and HCLaseM. HCLaseM. (B (B) )HCLaseM HCLaseM andand BTH BTH (50 (50 U) U) was was co-injected co-injected with with trypan trypan blue blue in inmice. mice. (C) (CHCLaseM) HCLaseM (10, (10, 20 and 20 and50 U) 50 was U) wasco- injectedco-injected with with trypan trypan blue blue in mice. in mice. The The relative relative dif difusionffusion area area of of each each group group at at 0 0min min is is 100%. 100%. Error Error barsbars represent represent standard standard deviations. 3. Discussion 3. Discussion HCLaseM contains three conserved catalytic residues (N225, H275, and Y284 numbering in HCLaseM contains three conserved catalytic residues (N225, H275, and Y284 numbering in HCLaseM; Figure9), which is consistent with previous studies that Tyr residue acted as a general base HCLaseM; Figure 9), which is consistent with previous studies that Tyr residue acted as a general for the abstraction of the proton from the C5 position of the glucuronic acid, while the Asn and His base for the abstraction of the proton from the C5 position of the glucuronic acid, while the Asn and residues neutralized the charge on the glucuronic acid group [17]. Since the activity of HCLaseM is His residues neutralized the charge on the glucuronic acid group [17]. Since the activity of HCLaseM independent of divalent ions, the action of HCLaseM may utilize the catalytic mechanism of Tyr–His, is independent of divalent ions, the action of HCLaseM may utilize the catalytic mechanism of Tyr– rather than Arg/Lys acting as a Bronsted base and acid [21]. His, rather than Arg/Lys acting as a Bronsted base and acid [21]. Aromatic patch (W371, W372, F423 in Streptococcus HL) and negative patch (E468, D478, T480 in Aromatic patch (W371, W372, F423 in Streptococcus HL) and negative patch (E468, D478, T480 in Streptococcus HL) have been reported in Streptococcus HL [26,27]. These amino acids anchor the Streptococcus HL) have been reported in Streptococcus HL [26,27]. These amino acids anchor the substrate chain into a degradation site [28]. HCLaseM has several conserved amino acids associated substrate chain into a degradation site [28]. HCLaseM has several conserved amino acids associated with substrate interactions. Aromatic patches were also conserved in all enzymes (W166 and W167 with substrate interactions. Aromatic patches were also conserved in all enzymes (W166 and W167 numbering in HCLaseM; Figure9). The phenylalanine residue in the aromatic patch is not present in numbering in HCLaseM; Figure 9). The phenylalanine residue in the aromatic patch is not present in HCLaseM, which eliminates the structural conflict between HCLaseM and 4-sulfated CS. This might HCLaseM, which eliminates the structural conflict between HCLaseM and 4-sulfated CS. This might allow HCLaseM to degrade all kinds of sulfated CS. A clearly negative patch was not found in allow HCLaseM to degrade all kinds of sulfated CS. A clearly negative patch was not found in HCLaseM, which suggests that HCLaseM has a unique negative patch consisting of other amino acid HCLaseM, which suggests that HCLaseM has a unique negative patch consisting of other amino acid residues. However, the precise mechanism of action of HCLaseM's broad substrate specificity has not residues. However, the precise mechanism of action of HCLaseM's broad substrate specificity has not been determined. been determined.

Mar. Drugs 2019, 17, 681 9 of 15 Mar. Drugs 2019 , 17 , x 9 of 14

FigureFigure 9. 9.Comparison Comparison of the of partial the partial amino amino acid sequence acid sequence of HCLaseM of HC withLaseM chondroitin with chondroitin lyase (GenBank lyase ANC28180.1)(GenBank ANC28180.1) from Arthrobacter fromsp. Arthrobacter GAG (45%), sp. chondroitin GAG (45%), lyase chondroitin (AFM38168.1) lyase from (AFM38168.1)Arthrobacter fromsp. MAT3885Arthrobacter (44%), sp. and MAT3885 chondroitin (44%), lyase and (PDB chondroitin 1RWA_A) from lyasePaenarthrobacter (PDB 1RWA_A) aurescens from (43%).Paenarthrobacter Identical aminoaurescens acids (43%). are shaded Identical black. amino # Conservative acids are shaded amino black. acids. # Conservative amino acids.

TheThe enzymes enzymes of of the the PL-8 PL-8 family family can can degrade degrade many many types types of of glycosaminoglycans glycosaminoglycans and and can can be be divideddivided into into three three subfamilies subfamilies by by phylogenetic phylogenetic analysis. analysis. Hyaluronidase Hyaluronidase typically typically degrades degrades HA HA and and is relativelyis relatively less less active active against against CS. ThisCS. This type type of hyaluronidase of hyaluronidase is generally is generally the first the subfamily first subfamily of the of PL-8 the family,PL-8 family, such as such hyaluronidase as hyaluronidase from Streptococcus from Streptococcus suis [29]. suisChondroitin [29]. Chondroitin ABC lyase ABC mainly lyase degrades mainly CS-A,degrades CS-B, CS-A, and CS-C.CS-B, and The CS-C. relative The enzymatic relative enzymat activityic on activity HA is generallyon HA is generally low. This low. type This of lyase type is of generallylyase is generally the second the subfamily, second subfamily, such as chondroitin such as chondroitin ABC lyase fromABC B.lyase thetaiotaomicron from B. thetaiotaomicronwith the activity with towardsthe activity HA towards at 10–30% HA of at CS 10–30% [30]. Chondroitin of CS [30]. Chond AC lyaseroitin mainly AC lyase degrades mainly CS-A degrades and CS-C, CS-A and and has CS- certainC, and activity has certain on HA. activity This type on HA. of enzyme This type is generally of enzyme the is third generally subfamily, the thirdsuch as subfamily, chondroitin such AC as lyasechondroitin from Bacteroides AC lyase stercoris from Bacteroideswith the activitystercoris towards with the HA activity was 67%towards of CS-A HA [was18]. 67% The evolutionof CS-A [18]. of originalThe evolution chondroitinase of original into later chondroitinase hyaluronidase into plausibly later explains hyaluronidase this phenomenon. plausibly There explains are also this somephenomenon. enzymes that There are are not also included some into enzymes three subfamilies, that are no sucht included as HCLase into from threeVibrio subfamilies,sp. FC509 such [21 as]. HCLaseHCLase has from the Vibrio highest sp. activity FC509 towards [21]. HCLase HA, and has the the activity highest towards activity various towards CSs HA, was and relatively the activity high (36–73%towards of various hyaluronidase CSs was activity). relatively Most high enzymes (36–73% have of limitationshyaluronidase in degrading activity). HA Most and enzymes various CSs.have HCLaseMlimitations could in degrading degrade HA and various typesCSs. HCLase of CSs andM could showed degrade almost HA the and same various enzyme types activity of CSs towardsand showed CS-A, almost CS-B, CS-C,the same CS-D, enzyme and HA activity (100, towards 99, 98, 99, CS-A, and 102%),CS-B, CS-C, which CS-D, was diandfferent HA from(100, other99, 98, conventional99, and 102%), hyaluronidases which was anddifferent chondroitinase from other (Table conve3)ntional. Moreover, hyaluronidases both the Vmax andand chondroitinaseKm showed that(Table HCLaseM 3) . Moreover, was similarly both the active Vmax towardsand Km showed HA and that CS-A, HCLaseM and possessed was similarly similarly active affinity towards towards HA HAand and CS-A, CS-A. and A possessed phylogenetic similarly tree showed affinity that towards HCLaseM HA couldand CS-A. not be A classified phylogenetic into the tree existing showed three that subfamiliesHCLaseM ofcould the PL-8not be family, classified which into also the made existing it possible three forsubfamilies it and some of the other PL-8 unclassified family, which enzymes also tomade become it possible a new subfamily.for it and some Based other on these unclassified results, theenzymes new enzyme to become was a designatednew subfamily. as HCLaseM Based on insteadthese of results, hyaluronidase the new or chondroitinase.enzyme was designated HCLaseM enriched as HCL theaseM knowledge instead of of enzymes hyaluronidase of the PL-8 or familychondroitinase. and may have HCLaseM the potential enriched to bethe an knowledge effective tool of e fornzymes preparation of the ofPL-8 functional family and oligosaccharides may have the andpotential investigation to be an function effective relationship tool for preparation of HA and various of functional CSs. oligosaccharides and investigation functionThe optimum relationship temperature of HA and for various the activity CSs. of HCLaseM towards both HA and CS-A was 40 ◦C, whichThe was optimum similar to temperature most enzymes for ofthe the activity PL-8 familyof HCLaseM (Table4 towards). HCLaseM both keptHA and stable CS-A below was 30 40◦ °C,C. HCLaseMwhich was showed similar the to most best activityenzymes at of pH the 7.0, PL-8 which famil wasy (Table similar 4). to HCLaseM most of the kept previous stable GAGbelow lyases 30 °C. withHCLaseM an optimum showed pH the of best 6.0–8.0 activity (Table at pH4). 7.0, HCLaseM which w retainedas similar about to most 70% of of the activity previous at pH GAG 5.0–9.0, lyases andwith retained an optimum about pH 90% of of6.0–8.0 activity (Table at pH 4). 6.0–8.0.HCLaseM These retained properties about were70% of beneficial activity at to pH the 5.0–9.0, storage and of theretained enzyme. about Hyaluronidase 90% of activity activity at pH of HCLaseM6.0–8.0. These preserved properties better were than beneficial chondroitinase to the activitystorage inof anthe alkalineenzyme. environment, Hyaluronidase which activity indicates of HCLaseM that pH and preserved substrates better may than affect chondroitinase the stability of HCLaseM.activity in an alkaline environment, which indicates that pH and substrates may affect the stability of HCLaseM. Hg 2+ , Fe 2+ , and Cu 2+ strongly inhibited the activity of HCLaseM. The affinity of Hg 2+ , Fe 2+ , and Cu 2+ for SH, CO, and NH moieties of amino acids leads to structural alterations of enzyme, which inhibits enzyme activity [22]. Except Hg 2+ , Fe 2+ , and Cu 2+ , most metal ions and EDTA did not significantly affect the activity of HCLaseM. The result shows that HCLaseM was not a metal ion-

Mar. Drugs 2019, 17, 681 10 of 15

Table 4. Comparison of the properties of HCLaseM with those of glycosaminoglycan lyase from different microorganisms.

Molecular Optimal Action Enzyme Source Optimal pH References Mass (kDa) Temperature (◦C) Pattern HCLaseM Microbacterium sp. H14 85.9 7 40 Endo This study AsChnAC Arthrobacter sp. N/A 7.2 37 Exo [15] ChoA1 Arthrobacter sp. MAT3885 83.4 6.0–7.5 40 N/A[16] ChSase AC Arthrobacter aurescens N/AN/AN/A Exo [17] ChSase ABC Bacteroides stercoris 116 7 40 Exo [18] ChSase AC Bacteroides stercoris 84 5.7–6.0 45–50 Endo [18] BniHL Bacillus niacin 120 6 45 Endo [19] HAase-B Bacillus sp. A50 116 6.5 44 Endo [20] HCLase Vibrio sp. FC509 90 8 30 Endo [21] ChSase ABC Acinetobacter sp. C26 76 6 42 N/A[22] ChSase AC II Arthrobacter sp. CS01 100 6.5 37 Exo [23] cABC I Proteus vulgaris 112 8 37 Endo [31] cABC II Proteus vulgaris 105 8 37 Exo [32] ChonABC Bacteroides thetaiotaomicron 115 7.6 37 Exo [33] ChSase AC Flavobacterium heparinum 74 6.8 40 Endo [34] HylB Streptococcus zooepidemicus N/A 6 37 N/A[35] HAase Arthrobacter globiformis 74 6 42 N/A[36] ChSase ABC Sphingomonas paucimobilis 82 6.5 40 N/A[37] N/A: There are no clear data in the reference.

Hg2+, Fe2+, and Cu2+ strongly inhibited the activity of HCLaseM. The affinity of Hg2+, Fe2+, and Cu2+ for SH, CO, and NH moieties of amino acids leads to structural alterations of enzyme, which inhibits enzyme activity [22]. Except Hg2+, Fe2+, and Cu2+, most metal ions and EDTA did not significantly affect the activity of HCLaseM. The result shows that HCLaseM was not a metal ion-dependent enzyme and was resistant to many metal ions. This property is contributed to application of HCLaseM in complex environment. Studies have shown that HA oligosaccharides have important application prospects in the field of medicine [2], such as applications in promotion of angiogenesis [7], immunomodulation, and anti-tumor [38]. Chondroitin sulfate oligosaccharides have antioxidant effects and exert neuroprotective effects to prevent and treat Alzheimer's disease [9]. HCLaseM showed an endogenous mode of action and degraded the substrates to disaccharides. HCLaseM has certain application prospects in the structural and functional studies of glycosaminoglycans and preparation of functional oligosaccharides. One of the most important fields of application of hyaluronidase is as a drug diffusion agent. The same dose of HCLaseM and commercially available BTH produced similar diffusion-promoting effects. It showed that in a relatively short period of time, the diffusion of trypan blue had nothing to do with the type of enzymes. The diffusion-promoting effect of HCLaseM is dose-dependent, which is beneficial to explore the curative effect of combination therapy with hyaluronidase and drugs. Compared with BTH, HCLaseM is easier to purify and has higher purity. HCLaseM has the potential to become a good drug diffusion agent. Immune problems still need to be considered, and we consider building long-acting HCLaseM to improve application value. Mar. Drugs 2019, 17, 681 11 of 15

4. Materials and Methods

4.1. Material and Animals Microbacterium sp. H14 was isolated from offshore of Qingdao and was preserved in our lab (School of Medicine and Pharmacy, Ocean University of China). Genomic sequencing was performed by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). PrimeSTAR HS DNA polymerases, restriction endonuclease, T4-DNA Kit, and other genetic engineering enzymes were purchased from Takara Inc. Hyaluronidase from bovine testes (BTH) and HA was purchased from Sigma. CS-A, CS-B, CS-C, and CS-D were obtained by Bomei Biotechnology Co. Ltd. (Hefei, China). Superdex peptide 10/300 GL was purchased from GE Healthcare. E. coli DH5α and BL21 (DE3) were grown in Luria-Bertani (LB). Female Kunming mice weighing 18–22 g were purchased from Qingdao Daren Fucheng Co. Ltd. (Qingdao, China). All animal experiments for this research conformed to the rules of international moral principles and the Guidelines for the Care and Use of Laboratory Animals [39]. All animal procedures adhered to compliance according to Animal Ethics Committee of School of Medicine and Pharmacy, Ocean University of China (Qingdao, China; Approval Date: 5 March 2018).

4.2. Cloning and Sequence Analysis of GAG Lyase Gene According to the genomic sequence of Microbacterium sp. H14, primers HCLaseM-F (GGAATTCC ATATGTTCACCCCCTCTCGCC) and HCLaseM-R (CCGCTCGAGGCGGTGCAGCGAGAACTCC) containing Nde I and Xho I restriction sites were designed and synthesized. The target gene was amplified using the genomic DNA of Microbacterium sp. H14 as a template and ligated into plasmid pET-28a. The recombinant plasmid was sequenced in Beijing Ruibo Xingke Biotechnology Co. Ltd. (Beijing, China). The similarity search of HCLaseM was performed using the BLASTp server of NCBI. The theoretical molecular mass and isoelectric point of the protein was estimated using the Compute pI/Mw tool on the ExPASy server. Phylogenetic analysis was performed using MEGA tools.

4.3. Expression and Purification of Recombinant HCLaseM The recombinant plasmid was transformed to E. coli BL21 (DE3) and cultured in LB with kanamycin (30 µg/mL) at 37 ◦C. The recombinant strains were induced with 0.02 mM IPTG to express recombinant HCLaseM at 18 ◦C for 24 h when the OD600 reached 0.6. Cells were harvested by centrifugation and disrupted by high pressure cracker. The supernatant was obtained by centrifugation and the target protein was purified using an Ni-Sepharose column, and the molecular weight and purity of the target protein were examined by SDS-PAGE. Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China).

4.4. Assay of HCLaseM Activity First, 0.1 mL of HCLaseM was added to 0.9 mL of 0.2% (w/v) HA/CS substrate (20 mM PB buffer, pH 7.0), and the enzymatic reaction was carried out at 40 ◦C for 10 min. HCLaseM forms a double bond in the non-reducing end of the sugar ring by β-elimination mechanism, which can be detected by a change in absorbance at 232 nm. One unit (U) was defined as the amount of protein needed to form 1 µmol of 4, 5-unsaturated uronic acid/min. Millimolar absorption coefficients for HA CS-A, CS-B, CS-C, and CS-D were 5.5, 5.1, 5.1, 5.5, and 5.1 respectively [40].

4.5. Substrate Specificity of HCLaseM HA, CS-A, CS-B, CS-C, and CS-D (0.2% w/v) were used as substrates to study the substrate specificity of HCLaseM. Mar. Drugs 2019, 17, 681 12 of 15

4.6. Effects of Temperature, pH, Metal Ions, Chelators, and Detergents on HCLaseM To determine the optimal reaction temperature for HCLaseM activity, HA and CS-A were digested at a range of 0–60 ◦C, respectively. After incubation at 0–60 ◦C for 1 h, the residual activity was measured to detect the thermostability. To detect the optimum pH, 0.2% (w/v) HA substrate was dissolved in different buffers (50 mM Na2HPO4–NaH2PO4 (pH 6.0–8.0), Tris-HCl (pH 7.05–8.95), NaH2PO4–citric acid (pH 3.0–8.0), and glycine–NaOH (pH 8.6–10.6)) with pH 3.0 to 10.0. To determine acidity stability, residual activity was measured after the enzyme was incubated in different buffers at 4 ◦C for 12 h. Enzyme activity was investigated in the presence of different metal ions, chelators and detergents to detect their effects on HCLaseM.

4.7. Enzymatic Reaction Kinetics of HCLaseM Towards HA and CS-A

Km and Vmax values of HCLaseM were obtained using Lineweaver–Burk plots. Optimal reaction conditions were used in experiments designed to calculate reaction rate at each substrate concentration to determine Km and Vmax values of HCLaseM.

4.8. Degradation Pattern of HCLaseM HA and CS-A were used as substrates to judge the degradation pattern of HCLaseM. First, 1 mL (0.5 U) of enzymes was added to 9 mL of 0.2% (w/v) HA/CS substrate (20 mM PB buffer, pH 7.0), and incubated at 40 ◦C. Aliquots of the reaction products were removed for time course experiments and separated on a TLC aluminum silica gel plate developed with n-butanol/glacial acetic acid/water (2:1:1, by vol.), and the reaction products were visualized by heating the TLC plate after spraying with a diphenylamine–aniline–phosphate reagent.

4.9. End Products of HCLaseM First, 2 U of enzymes was added to 2 mL of 0.2% (w/v) HA and CS-A substrate solution and incubated at 40 ◦C for 24 h to obtain end products. The final products were separated by a mobile phase (0.2 M ammonium bicarbonate) at a flow rate of 0.2 mL/min using Superdex peptide 10/300 GL gel filtration column, and then analyzed by negative ion electrospray ionization mass spectroscopy (ESI–MS). The analysis was set in the negative ion mode and the mass acquisition range was set at 200–1000.

4.10. Effect of Hyaluronidase on the Diffusion of Trypan Blue Female Kunming mice were selected as the research animals and trypan blue was used as a visual tracer to simulate a combination drug. Hyaluronidase activity was determined according to published recommendations [41]. To investigate the effects of HCLaseM and BTH on the diffusion of trypan blue, mice were randomly divided into three groups with ten cases each group. After mice were thoroughly anesthetized, the median villi of the back were removed and the epidermis was exposed. Trypan blue and test article (50 U BTH, 50 U HCLaseM, PB as negative control) were injected subcutaneously in the median back, with a final volume of 50 µL. The diffusion area at different time points (0, 5, 10, 15, 20, 30, 40, 50 and 60 min) was measured with a caliper after the injection. The trypan blue diffusion area at each time point was calculated according to the area calculation formula (Area = D D π/4). 1 × 2 × D1 and D2 represent the lateral diameter and the longitudinal diameter, respectively. To investigate the effect of different concentrations of HCLaseM on the diffusion of trypan blue, mice were randomly divided into four groups, ten cases each group. Trypan blue and different concentrations of HCLaseM (10, 20 and 50 U, PB as negative control) were injected. Subsequent steps are as described above.

5. Conclusions We cloned, expressed, and characterized a new GAG lyase HCLaseM from Microbacterium sp. H14. The stability under a wide range of pH, metal ion-resisted property, and substrate specificity Mar. Drugs 2019, 17, 681 13 of 15 of HCLaseM make it a novel enzyme for further basic research and application. The enzyme shows almost the same enzyme activity towards HA, CS-A, CS-B, CS-C, and CS-D, which is different from the reported enzymes and makes HCLaseM a more unique enzyme. HCLaseM may be an effective tool for structural analysis of HA and CS and preparation of oligosaccharides. HCLaseM has the potential to be a good drug diffusion agent.

Author Contributions: W.Y. initiated the project. Q.G. conceived and designed the experiments. J.S., X.H., and G.S. performed the experiments and analyzed the data. J.S. and Q.G. wrote the paper. Funding: This work was supported by the Key R&D Program of Shandong Province (2018GHY115047), the National Science and Technology Major Project for Significant New Drugs Development (2018ZX09735004) and the Qingdao Scientific and Technological Innovation center for Marine Biomedicine Development Grant (2017-CXZX01-3-8). Conflicts of Interest: The authors declare no conflicts of interest.

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