Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeuti...Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.展开更多
A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. Th...A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.展开更多
A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular...A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.展开更多
In the present work, for the first time on the basis ofpoly （vinyl alcohol） （PVA）, 2- （4-dimethylaminostyryl）-l-ethylquinolinium iodide （quinaldine red （QR）） and trisodium （4E）-5-oxo- 1-（4-sulfonatophenyl...In the present work, for the first time on the basis ofpoly （vinyl alcohol） （PVA）, 2- （4-dimethylaminostyryl）-l-ethylquinolinium iodide （quinaldine red （QR）） and trisodium （4E）-5-oxo- 1-（4-sulfonatophenyl）-4-[（4-sulfonatophenyl）hydrazono]-3 pyrazolecarboxylate （tartrazine （T））, thermostable polarizing film in a wide range of spectra （λmax=394-511 nm） with polarization efficiency （PE） = 98% in absorption maximum and stretching degree （Rs） = 3.5 was developed. The basic spectral-polarization parameters （polarization efficiency and transmittance） of oriented colored PVA-films were measured and discussed. During the work it was found that oriented PVA-films are the phenomenon of anisotropy of thermal conductivity （λ｜/λ⊥）. It is a very important parameter for the development of thermostable PVA-polarizing films. For the first time quantum-chemical calculations using density functional theory （DFT） approach for structural analysis and electronic spectrum of the QR were carried out via the B3LYP/dgdzvp and TDB3LYP/dgdzvp methods. Interpretation of absorption strips in visible region of spectrum was also reported. The excitation energies, electronic transitions and oscillator strengths for the studied structures have also been calculated （B3LYP/dgdzvp）. The NBO analysis and Mulliken atomic charges of the QR were carried out.展开更多
With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were scre...With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.展开更多
Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme productio...Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.展开更多
An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was express...An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.展开更多
Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions th...Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.展开更多
Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene,...Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coll. The deduced serralysin inhibitor, LupI, shows 〈40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64mol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100℃ for 1-60 min followed by incubation at 0℃. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.展开更多
Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃a...Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.展开更多
Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available protease...Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.展开更多
The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was...The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was determined using the neutralisation test to detect PPR virus specific antibody. Vaccinated animals seroconverted and a four-fold or more rise in antibody titre were observed between pre-vaccination and post-vaccination antibodies. The vaccine elicited significant antibody response in goats through the different routes of administration (intramuscular, intranasal, intraocular, subcutaneous and orally), but was poorly transmitted between the vaccinees and in-contact animals. The sheep responded poorly to the vaccine administered through most of the routes, except for those vaccinated through intramuscular and subcutaneous routes that seroconverted significantly (≥4 fold rise). The vaccine retained a potent titre of 3.1 log10 TCID50 for more than 8 hours after reconstitution in PBS at room temperature. Based on the response of goats to oral vaccination, it is suggested that the vaccine could be administered on the field through the oral routes and has the potential to be adapted to a feed-based administration for wider application to the scattered livestock populations under the extensive system of management.展开更多
Flexible composite pipes are advantageous in ultra high strength,high modulus,pH and corrosion resistance and light weight,but there are still some hidden safety troubles because they are poorer in thermostable capaci...Flexible composite pipes are advantageous in ultra high strength,high modulus,pH and corrosion resistance and light weight,but there are still some hidden safety troubles because they are poorer in thermostable capacity.Therefore,test samples of flexible composite pipes were prepared with high-temperature polythene(PE-RT)as the neck bush and aramid fiber as the reinforcement layer.Experimental study was conducted by using HPHT vessel and differential thermal scanner for different working conditions,different temperatures,whole-pipe pressure-bearing capacity and 1000 h viability.It is shown by the environmental compatibility test that high temperature has little effect on the weight,Vicat softening temperature,mechanical properties and structures of neck bush PE-RT,but exerts an obvious effect on the tensility and wholepipe water pressure blasting of the reinforcement aramid fiber.Besides,the drop of whole-pipe pressure-bearing capacity is caused by deformation and breaking of aramid fibers when the reinforcement layer is under the force of internal pressure.Finally,disorientation and crystallization of molecular thermal motion occur with the rise of temperature,so amorphous orientation reduces,crystallinity factor and crystalline orientation factor increase gradually,thus,disorientation of macromolecular chains increases and tensile strength decreases.It is concluded that this type of flexible composite pipe can smoothly pass 1000 h viability test.And it is recommended that it be used in the situations with temperature not higher than 95℃and internal pressure not higher than 4 MPa.展开更多
A new extracellularκ-carrageenase,namely CgkP,34.0 kDa in molecular weight,was purified from Pseudoalteromonas sp.QY203.CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures r...A new extracellularκ-carrageenase,namely CgkP,34.0 kDa in molecular weight,was purified from Pseudoalteromonas sp.QY203.CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃to 50℃with the highest activity at 45℃and pH7.2.Sodium chloride increased its activity markedly,and KC1 increased its activity slightly.The divalent and trivalent metal ions including Cu^2+,Ni^2+,Zn^2+,Mn^2+,Al^3+and Fe^3+significantly inhibited its activity,while Mg^2+did not.CgkP remained 70%of original activity after being incubated at 40℃for 48h,and remained 80%of the activity after being incubated at 45℃for 1 h.It exhibited endo-κ-carrageenase activity,mainly depolymerizing theκ-carrageenan into disaccharide and tetrasaccharide.CgkP was more thermostable than most of previously reportedκ-carrageenases with a potential of being used in industry.展开更多
In order to promote the thermostability of a-diimine nickel complex by ligand backbone structure,a series of α-diimine nickel complexes with substituents on acenaphthequinone backbone were synthesized and used as cat...In order to promote the thermostability of a-diimine nickel complex by ligand backbone structure,a series of α-diimine nickel complexes with substituents on acenaphthequinone backbone were synthesized and used as catalysts for ethylene polymerization.When the hydroxyethyl phenoxyl group was introduced to the acenaphthequinone-backbone,the thermal stability and activity of the catalyst could be significantly improved.The catalytic activity of complex C2[5-(4-(2-hydroxyethyl)phenoxyl)-N,N-bis(2,6-diisopropyl)acenaphthylene-1,2-diimine]nickel(Ⅱ)dibromide with isopropyl substituents on N-aryl reached 8.2×10^6g/(molNi·h)at 70℃and 2 MPa.The activity of[5-(4-(2-hydroxyethyl)phenoxyl)-N,N-bis(2,6-dibenzhydryl-4-menthylphenyl)acenaphthylene-1,2-diimine]nickel(Ⅱ)dibromide(C3)still maintained at 6.7×10^5 g/(molNi·h)at 120℃.Compared with C3 containing bulky dibenzhydryl substituents,the activity of C2 was sensitive to the change of the polymerization pressure.However,the polyethylenes obtained from complex C3 had lower branching density.Meanwhile,the molecular weight could reach 971 kg/mol,which is almost 5 times as much as that of the polyethylene obtained from complex C2.展开更多
A thermostable superoxide dismutase （SOD） from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Seph...A thermostable superoxide dismutase （SOD） from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli （E. coli）. The prepared apo-enzyme of the purified recombinant SOD （rSOD） was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2＋ - reconstituted rSOD （Mn-rSOD） exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.展开更多
Biotechnological strategies for plastic depolymerization and recycling have emerged as transformative approaches to combat the global plastic pollution crisis,aligning with the principles of a sustainable and circular...Biotechnological strategies for plastic depolymerization and recycling have emerged as transformative approaches to combat the global plastic pollution crisis,aligning with the principles of a sustainable and circular economy.Despite advances in engineering PET hydrolases,the degradation process is frequently compromised by product inhibition and the heterogeneity of final products,thereby obstructing subsequent PET recondensation and impeding the synthesis of high-value derivatives.In this work,we utilized previously devised computational strategies to redesign a thermostable DuraMHETase,achieving an apparent melting temperature of 72℃ in complex with MHET and a 6-fold higher in total turnover number(TTN)toward MHET than the wild-type enzyme at 60℃.The fused enzyme system composed of DuraMHETase and TurboPETase demonstrated higher efficiency than other PET hydrolases and the separated dual enzyme systems.Furthermore,we identified both exo-and endo-PETase activities in DuraMHETase,whereas the endo-activity was previously unobserved at ambient temperatures.These results expand the functional scope of MHETase beyond mere intermediate hydrolysis,and may provide guidance for the development of more synergistic approaches to plastic biodepolymerization and recycling.展开更多
Cloning and expression of theα-amylase gene(AmyK2)of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring,North Sikkim,India was done in Escherichia coli BL-21(DE3).The 55.0 kDa purified recomb...Cloning and expression of theα-amylase gene(AmyK2)of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring,North Sikkim,India was done in Escherichia coli BL-21(DE3).The 55.0 kDa purified recombinant enzyme exhibited optimum activity at 70°C and pH 7.0 with significant stability in temperature and pH ranges from 30 to 90°C and 6.0-8.0,respectively.Theα-amylase is Ca^(+2)independent,and can act with reasonably high efficiency in absence of any metal.Moreover,its activity increased in the presence of Fe^(+2)ion and was inhibited by Hg^(+2)ion and EDTA.The recombinant enzyme showed a half-life of 53 min at 70°C and its Vmax and Km values were 22.22 U/mg and 5.06 mg/ml,respectively.Immobilization of the purified enzyme on low-cost coconut coir with high immobilization yield(98.27%specific activity),increased half-life(71 min),and higher thermostability with successive use up to 8 cycles with high efficacy validates the techno-economic merit of use of the immobilized biocatalyst.As a whole,cloning theα-amylase gene of thermostable bacteria into the mesophilic organism and subsequent immobilization of the enzyme will unravel its secrets within the confines of the laboratory which could expedite its commercial exploitation in future.展开更多
The selenomethionyl derivative of the thermo-stable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed, purified and crystallized. By using multiwave length anoma-lous dispersion (MAD) phasing techniques, the crystal...The selenomethionyl derivative of the thermo-stable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed, purified and crystallized. By using multiwave length anoma-lous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined. TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1-153 and 153-319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.展开更多
The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding c...The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface.Here,we report the systematic engineering of whole β-sheet regions in the core of IsPETase(a PETase from Ideonella sakaiensis)via a fluorescent high-throughput screening assay.Twenty-one beneficial substitutions were obtained and iteratively recombined.The best variant,DepoPETase β,with an increase in the melting temperatures(T_(m))of 22.9℃,exhibited superior depolymerization performance and enabled complete depolymerization of100.5 g of untreated post-consumer PET(pc-PET;0.26% W_(enzyme)/W_(PET) enzyme loading)in liter-scale bioreactor at 50℃within 4 d.Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region,a more tightly packed structure of the core sheets and the surrounding helix,and improved binding of PET to the active sites.This study not only demonstrates the importance of engineering strategy in theβ-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province(BE2021623,BK20220155)Natural Science Foundation of Jiangsu Province(BE2021623)+4 种基金National Natural Science Foundation of China(32001665,U1903205,32021005)the National Key Research and Development Program of China(2017YF0400303)the Key Scientific and Technological Research Projects in the Key Areas of the Xinjiang Production and Construction Corps(2018AB010)the Key Research and Development 303 Program of Ningxia(2020BFG02012)Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province。
文摘Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.
基金Supported by the Science and Technology Research Project of Hubei Provincial Department of Education(B2017041)
文摘A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.
基金the Science Technology Plan Foundation of Hebei Province, China (07225533)the Doctor Foundation from Agricultural University of Hebei (050031)
文摘A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.
基金supported by the National Academy of Sciences of Belarus
文摘In the present work, for the first time on the basis ofpoly （vinyl alcohol） （PVA）, 2- （4-dimethylaminostyryl）-l-ethylquinolinium iodide （quinaldine red （QR）） and trisodium （4E）-5-oxo- 1-（4-sulfonatophenyl）-4-[（4-sulfonatophenyl）hydrazono]-3 pyrazolecarboxylate （tartrazine （T））, thermostable polarizing film in a wide range of spectra （λmax=394-511 nm） with polarization efficiency （PE） = 98% in absorption maximum and stretching degree （Rs） = 3.5 was developed. The basic spectral-polarization parameters （polarization efficiency and transmittance） of oriented colored PVA-films were measured and discussed. During the work it was found that oriented PVA-films are the phenomenon of anisotropy of thermal conductivity （λ｜/λ⊥）. It is a very important parameter for the development of thermostable PVA-polarizing films. For the first time quantum-chemical calculations using density functional theory （DFT） approach for structural analysis and electronic spectrum of the QR were carried out via the B3LYP/dgdzvp and TDB3LYP/dgdzvp methods. Interpretation of absorption strips in visible region of spectrum was also reported. The excitation energies, electronic transitions and oscillator strengths for the studied structures have also been calculated （B3LYP/dgdzvp）. The NBO analysis and Mulliken atomic charges of the QR were carried out.
文摘With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
文摘Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.
基金The Natural Science Foundation of Fujian Province of China under contract No.2016J01162the Program for New Century Excellent Talents in Fujian Province University,China under contract No.B15139
文摘An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.
文摘Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.
基金Supported by the National Natural Science Foundation of China(No.41376175)the Joint Funds of the National Natural Science Foundation of China(No.U1406402-5)+3 种基金the International S&T Cooperation Program of China(No.2014DFG30890)the Qingdao Science and Technology Plan Project(No.14-2-4-11-jch)the National Science Foundation for PostDoctoral Scientists of China(No.2016M590673)the Application Foundation of Qingdao for Post-Doctoral Scientists of China(No.Q51201601)
文摘Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coll. The deduced serralysin inhibitor, LupI, shows 〈40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64mol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100℃ for 1-60 min followed by incubation at 0℃. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.
基金supported by the Natural Science Foundation of Shandong Province(No.ZR202102270334)the National Key Research and Development Program of China(No.2020YFD0901004).
文摘Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.
文摘Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.
文摘The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was determined using the neutralisation test to detect PPR virus specific antibody. Vaccinated animals seroconverted and a four-fold or more rise in antibody titre were observed between pre-vaccination and post-vaccination antibodies. The vaccine elicited significant antibody response in goats through the different routes of administration (intramuscular, intranasal, intraocular, subcutaneous and orally), but was poorly transmitted between the vaccinees and in-contact animals. The sheep responded poorly to the vaccine administered through most of the routes, except for those vaccinated through intramuscular and subcutaneous routes that seroconverted significantly (≥4 fold rise). The vaccine retained a potent titre of 3.1 log10 TCID50 for more than 8 hours after reconstitution in PBS at room temperature. Based on the response of goats to oral vaccination, it is suggested that the vaccine could be administered on the field through the oral routes and has the potential to be adapted to a feed-based administration for wider application to the scattered livestock populations under the extensive system of management.
文摘Flexible composite pipes are advantageous in ultra high strength,high modulus,pH and corrosion resistance and light weight,but there are still some hidden safety troubles because they are poorer in thermostable capacity.Therefore,test samples of flexible composite pipes were prepared with high-temperature polythene(PE-RT)as the neck bush and aramid fiber as the reinforcement layer.Experimental study was conducted by using HPHT vessel and differential thermal scanner for different working conditions,different temperatures,whole-pipe pressure-bearing capacity and 1000 h viability.It is shown by the environmental compatibility test that high temperature has little effect on the weight,Vicat softening temperature,mechanical properties and structures of neck bush PE-RT,but exerts an obvious effect on the tensility and wholepipe water pressure blasting of the reinforcement aramid fiber.Besides,the drop of whole-pipe pressure-bearing capacity is caused by deformation and breaking of aramid fibers when the reinforcement layer is under the force of internal pressure.Finally,disorientation and crystallization of molecular thermal motion occur with the rise of temperature,so amorphous orientation reduces,crystallinity factor and crystalline orientation factor increase gradually,thus,disorientation of macromolecular chains increases and tensile strength decreases.It is concluded that this type of flexible composite pipe can smoothly pass 1000 h viability test.And it is recommended that it be used in the situations with temperature not higher than 95℃and internal pressure not higher than 4 MPa.
基金supported by National Science Foundation of China(31000361 and 31070712)Program for Changjiang Scholars and Innovative Research Team in University(IRT0944)+1 种基金Special Fund for Marine Scientific Research in the Public Interest(201005024)the Fundamental Research Funds for the Central Universities(201013008)
文摘A new extracellularκ-carrageenase,namely CgkP,34.0 kDa in molecular weight,was purified from Pseudoalteromonas sp.QY203.CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃to 50℃with the highest activity at 45℃and pH7.2.Sodium chloride increased its activity markedly,and KC1 increased its activity slightly.The divalent and trivalent metal ions including Cu^2+,Ni^2+,Zn^2+,Mn^2+,Al^3+and Fe^3+significantly inhibited its activity,while Mg^2+did not.CgkP remained 70%of original activity after being incubated at 40℃for 48h,and remained 80%of the activity after being incubated at 45℃for 1 h.It exhibited endo-κ-carrageenase activity,mainly depolymerizing theκ-carrageenan into disaccharide and tetrasaccharide.CgkP was more thermostable than most of previously reportedκ-carrageenases with a potential of being used in industry.
基金the National Natural Science Foundation of China(Nos.21004017 and 21004043)the Natural Science Foundation of Hebei Provinee(No.B2015202049).
文摘In order to promote the thermostability of a-diimine nickel complex by ligand backbone structure,a series of α-diimine nickel complexes with substituents on acenaphthequinone backbone were synthesized and used as catalysts for ethylene polymerization.When the hydroxyethyl phenoxyl group was introduced to the acenaphthequinone-backbone,the thermal stability and activity of the catalyst could be significantly improved.The catalytic activity of complex C2[5-(4-(2-hydroxyethyl)phenoxyl)-N,N-bis(2,6-diisopropyl)acenaphthylene-1,2-diimine]nickel(Ⅱ)dibromide with isopropyl substituents on N-aryl reached 8.2×10^6g/(molNi·h)at 70℃and 2 MPa.The activity of[5-(4-(2-hydroxyethyl)phenoxyl)-N,N-bis(2,6-dibenzhydryl-4-menthylphenyl)acenaphthylene-1,2-diimine]nickel(Ⅱ)dibromide(C3)still maintained at 6.7×10^5 g/(molNi·h)at 120℃.Compared with C3 containing bulky dibenzhydryl substituents,the activity of C2 was sensitive to the change of the polymerization pressure.However,the polyethylenes obtained from complex C3 had lower branching density.Meanwhile,the molecular weight could reach 971 kg/mol,which is almost 5 times as much as that of the polyethylene obtained from complex C2.
基金The Natural Science Foundation of Fujian Province,China under contract Nos 2008J0067 and 2009J01033the Program for New Century Excellent Talents in Fujian Province University under contract No.NCETFJ-2007the Foundation for Innovative Research Team of Jimei University under contract No.2010A005
文摘A thermostable superoxide dismutase （SOD） from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli （E. coli）. The prepared apo-enzyme of the purified recombinant SOD （rSOD） was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2＋ - reconstituted rSOD （Mn-rSOD） exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.
文摘Biotechnological strategies for plastic depolymerization and recycling have emerged as transformative approaches to combat the global plastic pollution crisis,aligning with the principles of a sustainable and circular economy.Despite advances in engineering PET hydrolases,the degradation process is frequently compromised by product inhibition and the heterogeneity of final products,thereby obstructing subsequent PET recondensation and impeding the synthesis of high-value derivatives.In this work,we utilized previously devised computational strategies to redesign a thermostable DuraMHETase,achieving an apparent melting temperature of 72℃ in complex with MHET and a 6-fold higher in total turnover number(TTN)toward MHET than the wild-type enzyme at 60℃.The fused enzyme system composed of DuraMHETase and TurboPETase demonstrated higher efficiency than other PET hydrolases and the separated dual enzyme systems.Furthermore,we identified both exo-and endo-PETase activities in DuraMHETase,whereas the endo-activity was previously unobserved at ambient temperatures.These results expand the functional scope of MHETase beyond mere intermediate hydrolysis,and may provide guidance for the development of more synergistic approaches to plastic biodepolymerization and recycling.
基金funding from Department of Biotechnology,Government of India.
文摘Cloning and expression of theα-amylase gene(AmyK2)of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring,North Sikkim,India was done in Escherichia coli BL-21(DE3).The 55.0 kDa purified recombinant enzyme exhibited optimum activity at 70°C and pH 7.0 with significant stability in temperature and pH ranges from 30 to 90°C and 6.0-8.0,respectively.Theα-amylase is Ca^(+2)independent,and can act with reasonably high efficiency in absence of any metal.Moreover,its activity increased in the presence of Fe^(+2)ion and was inhibited by Hg^(+2)ion and EDTA.The recombinant enzyme showed a half-life of 53 min at 70°C and its Vmax and Km values were 22.22 U/mg and 5.06 mg/ml,respectively.Immobilization of the purified enzyme on low-cost coconut coir with high immobilization yield(98.27%specific activity),increased half-life(71 min),and higher thermostability with successive use up to 8 cycles with high efficacy validates the techno-economic merit of use of the immobilized biocatalyst.As a whole,cloning theα-amylase gene of thermostable bacteria into the mesophilic organism and subsequent immobilization of the enzyme will unravel its secrets within the confines of the laboratory which could expedite its commercial exploitation in future.
基金This workwas supported by the National Natural Science Foundation of China (Grant No. 30070161) the Hundreds Talents Program of the ChineseAcademy of Sciences.
文摘The selenomethionyl derivative of the thermo-stable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed, purified and crystallized. By using multiwave length anoma-lous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined. TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1-153 and 153-319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.
基金funded by the National Key Research and Development Program of China(2023YFC3903300)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIPIJCP-003,TSBICIP-KJGG-009-0203,and TSBICIP-BRFI-005)the Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00018)。
文摘The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface.Here,we report the systematic engineering of whole β-sheet regions in the core of IsPETase(a PETase from Ideonella sakaiensis)via a fluorescent high-throughput screening assay.Twenty-one beneficial substitutions were obtained and iteratively recombined.The best variant,DepoPETase β,with an increase in the melting temperatures(T_(m))of 22.9℃,exhibited superior depolymerization performance and enabled complete depolymerization of100.5 g of untreated post-consumer PET(pc-PET;0.26% W_(enzyme)/W_(PET) enzyme loading)in liter-scale bioreactor at 50℃within 4 d.Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region,a more tightly packed structure of the core sheets and the surrounding helix,and improved binding of PET to the active sites.This study not only demonstrates the importance of engineering strategy in theβ-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.
