Huang JW

References (11)

Title : Complete decomposition of poly(ethylene terephthalate) by crude PET hydrolytic enzyme produced in Pichia pastoris - Chen_2024_Chem.Eng.J_481_148418
Author(s) : Chen CC , Li X , Min J , Zeng Z , Ning Z , He H , Long X , Niu D , Peng R , Liu X , Yang Y , Huang JW , Guo RT
Ref : Chemical Engineering Journal , 481 :148418 , 2024
Abstract : Using enzymes to decompose poly(ethylene terephthalate) (PET) is an attractive strategy to the sustainable utilization of PET, and an effective production platform of PET degrading enzyme is a prerequisite to achieve this goal. Here, we exploited the industrial yeast strain Pichia pastoris to produce a potent PET hydrolase termed FAST-PETase, whose performance was further elevated by removing two N-linked glycosylations through molecular engineering. The expression of the yielded variant, FAST-PETase-212/277, was elevated by antibiotics selection and chaperon co-expression to exceed 3 g/L in a 30-L fermenter. Notably, the crude fermentation product can be directly applied to decompose PET without purification. More than 95 % postconsumer PET can be achieved by 0.5 mgenzyme gPET-1 in 24 h in a 10-L reaction system in a reactor. These results demonstrate the economic viability of producing PET hydrolytic enzyme with modern fermentation facilities for large scale PET decomposition.
ESTHER : Chen_2024_Chem.Eng.J_481_148418
PubMedSearch : Chen_2024_Chem.Eng.J_481_148418
Gene_locus related to this paper: idesa-peth

Title : Complete bio-degradation of poly(butylene adipate-co-terephthalate) via engineered cutinases - Yang_2023_Nat.Commun_14_1645
Author(s) : Yang Y , Min J , Xue T , Jiang P , Liu X , Peng R , Huang JW , Qu Y , Li X , Ma N , Tsai FC , Dai L , Zhang Q , Liu Y , Chen CC , Guo RT
Ref : Nat Commun , 14 :1645 , 2023
Abstract : Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.
ESTHER : Yang_2023_Nat.Commun_14_1645
PubMedSearch : Yang_2023_Nat.Commun_14_1645
PubMedID: 36964144
Gene_locus related to this paper: idesa-peth , thefu-q6a0i4

Title : Remodeling the polymer-binding cavity to improve the efficacy of PBAT-degrading enzyme - Yang_2023_J.Hazard.Mater_464_132965
Author(s) : Yang Y , Cheng S , Zheng Y , Xue T , Huang JW , Zhang L , Guo RT , Chen CC
Ref : J Hazard Mater , 464 :132965 , 2023
Abstract : Poly(butylene adipate-co-terephthalate) (PBAT) is among the most widely applied synthetic polyesters that are utilized in the packaging and agricultural industries, but the accumulation of PBAT wastes has posed a great burden to ecosystems. Using renewable enzymes to decompose PBAT is an eco-friendly solution to tackle this problem. Recently, we demonstrated that cutinase is the most effective PBAT-degrading enzyme and that an engineered cutinase termed TfCut-DM could completely decompose PBAT film to terephthalate (TPA). Here, we report crystal structures of a variant of leaf compost cutinase in complex with soluble fragments of PBAT, including BTa and TaBTa. In the TaBTa complex, one TPA moiety was located at a polymer-binding site distal to the catalytic center that has never been experimentally validated. Intriguingly, the composition of the distal TPA-binding site shows higher diversity relative to the one proximal to the catalytic center in various cutinases. We thus modified the distal TPA-binding site of TfCut-DM and obtained variants that exhibit higher activity. Notably, the time needed to completely degrade the PBAT film to TPA was shortened to within 24 h by TfCut-DM Q132Y (5813 mol per mol protein). Taken together, the structural information regarding the substrate-binding behavior of PBAT-degrading enzymes could be useful guidance for direct enzyme engineering.
ESTHER : Yang_2023_J.Hazard.Mater_464_132965
PubMedSearch : Yang_2023_J.Hazard.Mater_464_132965
PubMedID: 37979420
Gene_locus related to this paper: 9bact-g9by57

Title : Substrate-Binding Mode of a Thermophilic PET Hydrolase and Engineering the Enzyme to Enhance the Hydrolytic Efficacy - Zeng_2022_ACS.Catal_12_3033
Author(s) : Zeng W , Li X , Yang Y , Min J , Huang JW , Liu W , Niu D , Yang X , Han X , Zhang L , Dai L , Chen CC , Guo RT
Ref : ACS Catal , 12 :3033 , 2022
Abstract : Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amounts of PET wastes that have accumulated in the environment have become a serious threat to the ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analogue, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme-substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temperature approaching 99 C and elevated PET hydrolytic activity were obtained. Finally, crystallographic analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegradation platforms.
ESTHER : Zeng_2022_ACS.Catal_12_3033
PubMedSearch : Zeng_2022_ACS.Catal_12_3033
Gene_locus related to this paper: 9bact-g9by57

Title : General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis - Chen_2021_Nat.Catal_4_425
Author(s) : Chen CC , Han X , Li X , Jiang P , Niu D , Ma L , Liu W , Li S , Qu Y , Hu H , Min J , Yang Y , Zhang L , Zeng W , Huang JW , Dai L , Guo RT , Chen, CC
Ref : Nature Catalysis , 4 :425 , 2021
Abstract : Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and a major contributor to plastic pollution. IsPETase, from the PET-assimilating bacterium Ideonella sakaiensis, is a unique PET-hydrolytic enzyme that shares high sequence identity to canonical cutinases, but shows substrate preference towards PET and exhibits higher PET-hydrolytic activity at ambient temperature. Structural analyses suggest that IsPETase harbours a substrate-binding residue, W185, with a wobbling conformation and a highly flexible W185-locating beta6-beta7 loop. Here, we show that these features result from the presence of S214 and I218 in IsPETase, whose equivalents are strictly His and Phe, respectively, in all other homologous enzymes. We found that mutating His/Phe residues to Ser/Ile could enhance the PET-hydrolytic activity of several IsPETase-like enzymes. In conclusion, the Ser/Ile mutations should provide an important strategy to improve the activity of potential PET-hydrolytic enzymes with properties that may be useful for various applications.
ESTHER : Chen_2021_Nat.Catal_4_425
PubMedSearch : Chen_2021_Nat.Catal_4_425
Gene_locus related to this paper: 9burk-a0a1f4jxw8 , idesa-peth

Title : Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei - Dai_2021_J.Biotechnol_334_47
Author(s) : Dai L , Qu Y , Huang JW , Hu Y , Hu H , Li S , Chen CC , Guo RT
Ref : J Biotechnol , 334 :47 , 2021
Abstract : The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETase(S121E/D186H/R280A) (IsPETase(EHA)) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETase(EHA) showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETase(EHA)_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 degC and 40 degC, respectively. The catalytic activity of IsPETase(EHA)_CBM was increased by 86 % when the protein concentration was increased from 2.5 microg/mL to 20microg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.
ESTHER : Dai_2021_J.Biotechnol_334_47
PubMedSearch : Dai_2021_J.Biotechnol_334_47
PubMedID: 34044062
Gene_locus related to this paper: idesa-peth

Title : Structure of a gut microbial diltiazem-metabolizing enzyme suggests possible substrate binding mode - Zhou_2020_Biochem.Biophys.Res.Commun__
Author(s) : Zhou S , Ko TP , Huang JW , Liu W , Zheng Y , Wu S , Wang Q , Xie Z , Liu Z , Chen CC , Guo RT
Ref : Biochemical & Biophysical Research Communications , : , 2020
Abstract : When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a beta-barrel subdomain and an alpha-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The beta-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident.
ESTHER : Zhou_2020_Biochem.Biophys.Res.Commun__
PubMedSearch : Zhou_2020_Biochem.Biophys.Res.Commun__
PubMedID: 32423809

Title : Crystal Structure of a Mycoestrogen-Detoxifying Lactonase from Rhinocladiella mackenziei: Molecular Insight into ZHD Substrate Selectivity - Zheng_2018_ACS.Catal_8_4294
Author(s) : Zheng YY , Liu WT , Chen CC , Hu XY , Liu WD , Ko TP , Tang XK , Wei HL , Huang JW , Guo RT
Ref : ACS Catal , 8 :4294 , 2018
Abstract : Development of potent biocatalysts for enzymatic detoxification of estrogenic mycotoxin zearalenone (ZEN) and its more toxic derivative alpha-zearalenol (alpha-ZOL) is of great interest. Here, we report the crystal structures of a ZEN-hydrolyzing enzyme from Rhinocladiella mackenziei (RmZHD), including substrate complexes. A molecular mechanism for the distinct activity of RmZHD in hydrolyzing the structurally similar ZEN and alpha-ZOL is then proposed. In addition, structure-based engineering to modify the substrate-binding pocket and improve the RmZHD activity toward alpha-ZOL is presented. These results expand our scope in understanding the catalytic mechanism of ZHD-family enzymes and are of vital importance in further industrial applications.
ESTHER : Zheng_2018_ACS.Catal_8_4294
PubMedSearch : Zheng_2018_ACS.Catal_8_4294
Gene_locus related to this paper: 9euro-a0a0d2ilk1

Title : Structural insight into catalytic mechanism of PET hydrolase - Han_2017_Nat.Commun_8_2106
Author(s) : Han X , Liu W , Huang JW , Ma J , Zheng Y , Ko TP , Xu L , Cheng YS , Chen CC , Guo RT
Ref : Nat Commun , 8 :2106 , 2017
Abstract : PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.
ESTHER : Han_2017_Nat.Commun_8_2106
PubMedSearch : Han_2017_Nat.Commun_8_2106
PubMedID: 29235460
Gene_locus related to this paper: idesa-peth

Title : Enhanced alpha-Zearalenol Hydrolyzing Activity of a Mycoestrogen-Detoxifying Lactonase by Structure-Based Engineering - Xu_2016_ACS.Catal_6_7657
Author(s) : Xu Z , Liu W , Chen CC , Li Q , Huang JW , Ko TP , Liu G , Peng W , Cheng YS , Chen Y , Jin J , Li H , Zheng Y , Guo RT
Ref : ACS Catal , 6 :7657 , 2016
Abstract : The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate, but ZOL, especially the -form, shows higher estrogenic toxicity than ZEN. To enhance alpha-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against alpha-ZOL, with an 2.7-fold reduction in substrate affinity but a 5.2-fold higher turnover rate. We then determined two V153H/ZOL complex structures. Here, the alpha-ZOL lactone ring is hydrogen-bonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity toward the more toxic alpha-ZOL, with great potential in further industrial applications.
ESTHER : Xu_2016_ACS.Catal_6_7657
PubMedSearch : Xu_2016_ACS.Catal_6_7657
Gene_locus related to this paper: biooc-ZHD101

Title : Crystal structure and substrate-binding mode of the mycoestrogen-detoxifying lactonase ZHD from Clonostachys rosea - Peng_2014_RSC.Adv_4_62321
Author(s) : Peng W , Ko TP , Yang YY , Zheng YY , Chen CC , Zhu Z , Huang CH , Zeng YF , Huang JW , Wang AHJ , Liu JR , Guo RT
Ref : RSC Advances , 4 :62321 , 2014
Abstract : The mycotoxin zearalenone has been contaminating maize and other grains. It can be hydrolyzed and inactivated by the lactonase ZHD, which belongs to the alpha/beta-hydrolase family. Besides the catalytic core domain, the enzyme comprises an alpha-helical cap domain. Zearalenone differs from other quorum-sensing lactones in its chemical structure. As revealed by the complex structure, the substrate binds into a deep pocket between the core and cap domains, adjacent to the catalytic triad Ser102-His242-Glu126. The enzyme-substrate interactions include three direct hydrogen bonds and several nonpolar contacts. In particular, the Trp183 side chain is engaged in both hydrogen bonding and T-stacking interactions with the benzoate ring. The central role of Trp183 in substrate binding was verified by the mutants W183A, W183H and W183F. Several mutants were also produced to investigate the roles of nearby amino-acid residues. Interestingly, mutants that destabilize the dimer had adverse functional effects on ZHD.
ESTHER : Peng_2014_RSC.Adv_4_62321
PubMedSearch : Peng_2014_RSC.Adv_4_62321
Gene_locus related to this paper: biooc-ZHD101