Xiaotang Wang

A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases.

β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism.

Identification of a laccase Glac15 from Ganoderma lucidum 77002 and its application in bioethanol production

BackgroundLaccases have potential applications in detoxification of lignocellulosic biomass after thermochemical pretreatment and production of value-added products or biofuels from renewable biomass. However, their application in large-scale industrial and environmental processes has been severely thwarted by the high cost of commercial laccases. Therefore, it is necessary to identify new laccases with lower cost but higher activity to detoxify lignocellulosic hydrolysates and better efficiency to produce biofuels such as bioethanol. Laccases from Ganoderma lucidum represent proper candidates in processing of lignocellulosic biomass.ResultsG. lucidum 77002 produces three laccase isoenzymes with a total laccase activity of 141.1 U/mL within 6xa0days when using wheat bran and peanut powder as energy sources in liquid culture medium. A new isoenzyme named Glac15 was identified, purified, and characterized. Glac15 possesses an optimum pH of 4.5 to 5.0 and a temperature range of 45°C to 55°C for the substrates tested. It was stable at pH values ranging from 5.0 to 7.0 and temperatures lower than 55°C, with more than 80% activity retained after incubation for 2xa0h. When used in bioethanol production process, 0.05 U/mL Glac15 removed 84% of the phenolic compounds in prehydrolysate, and the yeast biomass reached 11.81 (optimal density at 600xa0nm (OD600)), compared to no growth in the untreated one. Addition of Glac15 before cellulase hydrolysis had no significant effect on glucose recovery. However, ethanol yield were improved in samples treated with laccases compared to that in control samples. The final ethanol concentration of 9.74, 10.05, 10.11, and 10.81xa0g/L were obtained from samples containing only solid content, solid content treated with Glac15, solid content containing 50% prehydrolysate, and solid content containing 50% prehydrolysate treated with Glac15, respectively.ConclusionsThe G. lucidum laccase Glac15 has potentials in bioethanol production industry.

Covalent Immobilization of Penicillin G Acylase onto Fe3O4@Chitosan Magnetic Nanoparticles.

Penicillin G acylase (PGA) was immobilized on magnetic Fe3O4@chitosan nanoparticles through the Schiff base reaction. The immobilization conditions were optimized as follows: enzyme/support 8.8 mg/g, pH 6.0, time 40 min, and temperature 25°C. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 6.2 mg/g-support were obtained. Broader working pH and higher thermostability were achieved by the immobilization. In addition, the immobilized PGA retained 75% initial activity after ten cycles. Kinetic parameters Vmax and Km of the free and immobilized PGAs were determined as 0.91 mmol/min and 0.53 mmol/min, and 0.68 mM and 1.19 mM, respectively. Synthesis of amoxicillin with the immobilized PGA was carried out in 40% ethylene glycol at 25°C and a conversion of 72% was obtained. These results showed that the immobilization of PGA onto magnetic chitosan nanoparticles is an efficient and simple way for preparation of stable PGA.

Improve ethanol tolerance of β-glucosidase Bgl1A by semi-rational engineering for the hydrolysis of soybean isoflavone glycosides.

A β-glucosidase Bgl1A variant (A24S/F297Y) with improved ethanol tolerance was obtained by semi-rational engineering. At 30-40°C, IC50 values (the amount required for inhibiting 50% enzyme activity) of the variant for ethanol were 17-30% (v/v), 1.4- to 2.4-fold of Bgl1A. When incubating in 15% (v/v) ethanol at 30°C, the half-life of A24S/F297Y was 13min; whereas Bgl1A lost all enzyme activity within 5min. A24S/F297Y was more stable at pH 7.5 than at pH 6.5, and more than 50% of the original activity remained after incubation at 30°C for 10h. At 35°C and pH 7.5, the half-life of A24S/F297Y was 80min, 4.3 times longer than that of Bgl1A. When converting isoflavone glycosides to aglycones using A24S/F297Y as catalyst, the hydrolysis rates were 99% for daidzin and 98% for genistin. The concentrations of daidzein and genistein rapidly increased by 7.02mM and 4.35mM within 10min, respectively. These results showed that A24S/F297Y was a promising candidate for the enzymatic hydrolysis of soybean isoflavone glycosides.

Characterization of a novel β-glucosidase from Gongronella sp. W5 and its application in the hydrolysis of soybean isoflavone glycosides.

A novel β-glucosidase named BglW5 from Gongronella sp. was isolated, purified, and characterized for the first time. Under solid state fermentation, the yield of BglW5 was 49.9 U/g fermented medium. BglW5 was stable over a wide pH range of 3.0-8.5 and retained more than 50% of its maximal activity after incubation at 25 °C for 96 h. The half-lives of BglW5 were 20 h at 60 °C, and 1 h at 70 °C. The activity of BglW5 was stimulated by xylose and fructose at concentrations up to 500 mM, with maximal stimulatory effect of 1.6-fold and 2.2-fold, respectively. BglW5 converted isoflavone glycosides to aglycones, with a hydrolysis rate of 96.2% for daidzin and 96.7% for genistin. The productivities were 1.5 mmol L(-1) h(-1) for daidzein and 1.23 mmol L(-1) h(-1) for genistein, respectively. These features suggest that BglW5 has great application potential in the hydrolysis of soybean isoflavone glycosides.

A cold-adapted and glucose-stimulated type II α-glucosidase from a deep-sea bacterium Pseudoalteromonas sp. K8

ObjectivesTo express and characterize a putative α-glucosidase, Pagl, from Pseudoalteromonas sp. K8 obtained via genome mining approach.ResultsPagl was expressed and purified to homogeneity, with a molecular mass of 60xa0kDa. It was optimally active at pH 8.5 and 30xa0°C, and showed cold-adapted activity. Pagl exhibited specific activity towards substrates with α-1,4-linkage, with the highest specific activity of 19.4xa0U/mg for maltose, followed by pNPαG and maltodextrins, suggesting that Pagl belongs to the type II α-glucosidase. Interestingly, the activity of Pagl is significantly enhanced (2.7 times) in the presence of 200xa0mM glucose.ConclusionThe unique catalytic properties of Pagl make it an attractive candidate for several industrial applications.

Pseudoruegeria marinistellae sp. nov., isolated from an unidentified starfish in Sanya, China

A taxonomic study was carried out on a Gram-stain negative, rod-shaped, non-flagellated, and facultatively anaerobic bacterial strain designated as strain SF-16T, which was isolated from an unidentified starfish in Sanya, China. Strain SF-16T was found to be 5.0–7.0xa0μm long, and oxidase and catalase positive. Cell growth was observed at pH 6.0–8.5 (optimum, 7.0–8.0), temperatures of 10–41xa0°C (optimum, 25–30xa0°C), and salinities of 0–12xa0% (optimum, 3.0–6.0xa0%). The predominant fatty acids (>20xa0%) were found to be C18:1ω7c and/or C18:1ω6c (summed feature 8). Ubiquinone 10 was identified as the predominant quinone for strain SF-16T. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, two unidentified lipids, and three unidentified phospholipids. The DNA G+C content of the genomic DNA of strain SF-16T was determined to be 63.0xa0mol%. Phylogenetic analysis based on the 16S rRNA gene showed that strain SF-16T belongs to the genus Pseudoruegeria and is closely related to Pseudoruegeria sabulilitoris GJMS-35T (98.42xa0% similarity). The ANI value between strain SF-16T and P. sabulilitoris GJMS-35T was found to be 74.98xa0%, and DNA–DNA hybridization value was 21.1xa0±xa02.3xa0% in silico and 57xa0% in vitro. Based on the low level of the genetic relatedness, phylogenetic and phenotypic data, a novel species Pseudoruegeria marinistellae sp. nov. is proposed. The type strain is SF-16T (=MCCC 1K01155Txa0=xa0KCTC 42910T).

PspAG97A: A Halophilic α-Glucoside Hydrolase with Wide Substrate Specificity from Glycoside Hydrolase Family 97

A novel α-glucoside hydrolase (named PspAG97A) from glycoside hydrolase family 97 (GH97) was cloned from the deep-sea bacterium Pseudoalteromonas sp. K8, which was screened from the sediment of Kongsfjorden. Sequence analysis showed that PspAG97A belonged to GH97, and shared 41% sequence identity with the characterized α-glucosidase BtGH97a. PspAG97A possessed three key catalytically related glutamate residues. Mutation of the glutamate residues indicated that PspAG97A belonged to the inverting subfamily of GH97. PspAG97A showed significant reversibility against changes in salt concentration. It exhibited halophilic ability and improved thermostability in NaCl solution, with maximal activity at 1.0 M NaCl/KCl, and retained more than 80% activity at NaCl concentrations ranging from 0.8 to 2.0 M for over 50 h. Furthermore, PspAG97A hydrolyzed not only α-1,4-glucosidic linkage, but also α-1,6- and α-1,2-glucosidic linkages. Interestingly, PspAG97A possessed high catalytic efficiency for long-chain substrates with α-1,6-linkage. These characteristics are clearly different from other known α-glucoside hydrolases in GH97, implying that PspAG97A is a unique α-glucoside hydrolase of GH97.

Structures of PspAG97A α-glucoside hydrolase reveal a novel mechanism for chloride induced activation

Here we report the first crystal structure of a secretory α-glucoside hydrolase isolated from Pseudoalteromonas sp. K8, PspAG97A, which belongs to glycoside hydrolase family 97 and exhibits halophilic property. PspAG97A lacks an acidic surface, that is considered essential for protein stability at high salinity. Interestingly, PspAG97A unusually contains a chloride ion coordinated by the guanidinium group of Arg171 and the main chain amide groups of Tyr172 and Glu173 at the active site. The structures of PspAG97A complexed with acarbose and panose demonstrate that residues Glu173, Arg171 and Asn170 for subsite +1 decide the substrate specificity of the enzyme for the α-1,6-glucosidic linkage. Structural alterations observed in the R171K variant and enzyme kinetic experiments focusing on chloride assisted activation suggest that the active site chloride serves to properly orient Glu173, Arg171 and Asn170 to facilitate substrate recognition. Furthermore, the chloride assists the binding of Glu173 to the conserved calcium ion and plays an essential role in properly positioning the base catalyst Glu456. In sum, our results provide valuable insight into the structural basis of protein halophilicity.

Lutimaribacter marinistellae sp. nov., isolated from a starfish

A taxonomic study was carried out on a Gram-staining-negative bacterium, strain SF-12T, isolated from an unidentified starfish living in Sanya, PR China. Cells of SF-12T were non-spore-forming rods, 0.5-0.8 µm wide, 2.2-2.5 µm long and motile by means of flagella. SF-12T was facultatively anaerobic, heterotrophic, oxidase- and catalase-positive. Growth of SF-12T occurred at 15-38u2009°C (optimum, 30u2009°C), at pH 6.5-8.5 (optimum, pH 7.0), and in the presence of 2.0-7.0u2009% (w/v) NaCl (optimum, 3.0-4.0u2009%). The predominant fatty acids of SF-12T were C18u2009:u20091ω7c and/or C18u2009:u20091ω6c. Ubiquinone 10 was the sole respiratory quinone of SF-12T. The major polar lipids of SF-12T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unknown aminolipids, and seven unknown phospholipids. The DNA G+C content was 61 mol%. SF-12T showed the highest 16S rRNA gene sequence similarity to Lutimaribacter pacificus W11-2BT (96.06u2009%), followed by Cribrihabitans neustonicus CC-AMHB-3T (96.02u2009%), Lutimaribacter saemankumensis SMK-117T (96.0u2009%), Cribrihabitans marinus CZ-AM5T (95.92u2009%), Lutimaribacter litoralis KU5D5T (95.92u2009%) and other species of the family Rhodobacteraceae(<95.9u2009%). However, phylogenetic trees based on 16S rRNA gene sequences showed that SF-12T formed a lineage with members of the genus Lutimaribacter in the trees. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, SF-12T is considered to represent a novel species of the genus Lutimaribacter, for which the name Lutimaribacter marinistellae sp. nov. is proposed. The type strain is SF-12T (=MCCC 1K01154T=KCTC 42911T).