Cloning, expression, and characterization of a novel heparinase I from <i>Bacteroides eggerthii</i>

Liu, Caiyun; Su, Wen-Bin; Guo, Li-Bin; Zhang, Ye-Wang

doi:10.1080/10826068.2019.1709977

Cited by 17 publications

(14 citation statements)

References 40 publications

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“…60 min at 30 and 37˚C, respectively) [14]. These thermostability results exhibited that the heparinase I from B. cellulosilyticus has practical applications in the clinic and industry.…”

Section: Plos Onementioning

confidence: 68%

“…According to the previous report [14], the enzyme assay was performed by an ultraviolet spectrophotometer at 232 nm. The enzymatic reaction was carried out at 30˚C in pH 7.5 Tris-HCl buffers (containing 200 mM NaCl, 10 mM CaCl 2 , and 20 mM Tris-HCl).…”

Section: Enzyme Assaymentioning

confidence: 99%

“…Escherichia coli was usually chosen as the host which is one of the most successful and economical systems of expression. The Hep-Is from F. heparinum [12], B. thetaiotaomicron [13], and Bacteroides eggerthii [14] have been successfully expressed in E. coli. However, the recombinant enzymes still suffer the problems of an inclusion body and low activity recovery that lead to a low yield [15,16].…”

Section: Introductionmentioning

confidence: 99%

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A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

et al. 2020

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As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca 2+ , Mg 2+ , and Mn 2+ , while severely inhibited by Zn 2+ , Co 2+ , and EDTA. The specific activity of the enzyme was 738.3 U�mg-1 which is the highest activity ever reported. The K m and V max were calculated as 0.17 mg�mL-1 and 740.58 U�mg-1 , respectively. Besides, the half-life of 300 min at 30˚C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.

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“…60 min at 30 and 37˚C, respectively) [14]. These thermostability results exhibited that the heparinase I from B. cellulosilyticus has practical applications in the clinic and industry.…”

Section: Plos Onementioning

confidence: 68%

Section: Enzyme Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

et al. 2020

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“…The enzyme assay for ChSaseAC was performed with an ultraviolet spectrophotometer at 232 nm, according to our previous report ( Liu et al, 2020 ; Guo et al, 2021 ). The enzymatic reaction mixture consisted of 890 μl of 20 mM, pH 7.5 Tris–HCl buffer containing 10 mM CaCl 2 , 100 μl of 5 mg/ml CS-A, and 10 μl of enzyme solution (∼10 μg).…”

Section: Methodsmentioning

confidence: 99%

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization

Zhou

Guo

Wei

et al. 2021

Front. Bioeng. Biotechnol.

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Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

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“…B. eggerthii 1_2_48FAA is predicted to encode 39 PULs, and consequently a plethora of corresponding putative CAZymes 14 . Only a handful of enzymes from B. eggerthii have been characterized to date, including the heparinase Hep I 26 , the endo -xylanase Be Xyn5A 27 , and the “multicatalytic” arabinofuranosidase-feruloyl esterase Be GH43/FAE 28 . Multicatalytic enzymes contain multiple connected catalytic domains with discrete functions, and while only few have been characterized so far, multicatalytic enzymes often display synergistic activities between these domains.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

Kmezik

Krska

Mazurkewich

et al. 2021

Sci Rep

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Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

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Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii

Cited by 17 publications

References 40 publications

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization

Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

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