Cloning, Heterologous Expression, and Characterization of Novel Protease- Resistant α-Galactosidase from New Sphingomonas Strain

( Jun Pei Zhou ),( Yan Yan Dong ),( Jun Jun Li ),( Rui Zhang ),( Xianghua Tang ),( Yuelin Mu ),( Bo Xu ),( Qian Wu ),( Zun Xi Huang ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.11

The α-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ≤97.2% with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 α-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas α-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas α-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-α-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and 60oC and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant α-galactosidases showing thermolability at 50oC or 60oC and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at 60oC) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas α-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

Molecular and Biochemical Characterization of a Novel Intracellular Low-Temperature-Active Xylanase

( Jun Pei Zhou,),( Yan Yan Dong ),( Xiang Hua Tang ),( Jun Jun Li ),( Bo Xu ),( Qian Wu ),( Ya Jie Gao ),( Lu Pan ),( Zun Xi Huang ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.4

A 990 bp full-length gene (xynAHJ2) encoding a 329- residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperatureactive enzyme (exhibiting optimum activity at 35 o C, 62% at 20 o C, and 38% at 10 o C; thermolability at ≥45 o C). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to Ni 2+ , Ca 2+ , or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperatureactive xylanase.

Production and Characterization of Ethanol- and Protease-Tolerant and Xylooligosaccharides-Producing Endoxylanase from Humicola sp Ly01

( Jun Pei Zhou ),( Qian Wu ),( Rui Zhang ),( Yu Ying Yang ),( Xiang Hua Tang ),( Jun Jun Li ),( Jun Mei Ding ),( Yan Yan Dong ),( Zun Xi Huang ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.6

This paper reports the production and characterization of crude xylanase from the newly isolated Humicola sp. Ly01. The highest (41.8 U/ml) production of the crude xylanase was obtained under the optimized conditions (w/v): 0.5% wheat bran, 0.2% KH2PO4, and 0.5% peptone; initial pH 7.0; incubation time 72 h; 30℃; and 150 rpm. A considerable amount of the crude xylanase was induced using hulless barley bran or soybean meal as the carbon source, but a small amount of the enzyme was produced when supplementary urea was used as the nitrogen source to wheat bran. The crude xylanase showed apparent optimal cellulase-free xylanase activity at 60℃ and pH 6.0, more than 71.8% of the maximum xylanase activity in 3.0-30.0% (v/v) ethanol and more than 82.3% of the initial xylanase activity after incubation in 3.0-30.0% (v/v) ethanol at 30℃ for 2 h. The crude xylanase was moderately resistant to both acid and neutral protease digestion, and released 7.9 and 10.9 μmol/ml reducing sugar from xylan in the simulated gastric and intestinal fluids, respectively. The xylooligosaccharides were the main products of the hydrolysis of xylan by the crude xylanase. These properties suggested the potential of the crude enzyme for being applied in the animal feed industry, xylooligosaccharides production, and high-alcohol conditions such as ethanol production and brewing.

Experimental study on the compression of concrete filled steel tubular latticed columns with variable cross section

Yan Yang,Jun Zhou,Jiangang Wei,Lei Huang,Qingxiong Wu,Bao-chun Chen 국제구조공학회 2016 Steel and Composite Structures, An International J Vol.22 No.3

The effects of slenderness ratio, eccentricity and column slope on the load-carrying capacities and failure modes of variable and uniform concrete filled steel tubular (CFST) latticed columns under axial and eccentric compression were investigated and compared in this study. The results clearly show that all the CFST latticed columns with variable cross section exhibit an overall failure, which is similar to that of CFST latticed columns with a uniform cross section. The load-carrying capacity decreases with the increase of the slenderness ratio or the eccentricity. For 2-m specimens with a slenderness ratio of 9, the ultimate load-carrying capacity is increased by 3% and 5% for variable CFST latticed columns with a slope of 1:40 and 1:20 as compared with that of uniform CFST latticed columns, respectively. For the eccentrically compressed variable CFST latticed columns, the strain of the columns at the loading side, as well as the difference in the strain, increases from the bottom to the cap, and a more significant increase in strain is observed in the cross section closer to the column cap.

Cloning, Expression, and Characterization of a New Xylanase from Alkalophilic Paenibacillus sp. 12-11

( Yan Yu Zhao ),( Kun Meng ),( Hui Ying Luo ),( Pei Long Yang ),( Peng Jun Shi ),( Huo Qing Huang ),( Ying Guo Bai ),( Bin Yao ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.8

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at 55℃ and was thermostable at 50℃ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent Km and Vmax values were 1.18 mg/ml and 1,961 ?mol/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

Understanding nanorheology and surface forces of confined thin films

Huang, Jun,Yan, Bin,Faghihnejad, Ali,Xu, Haolan,Zeng, Hongbo 한국유변학회 2014 Korea-Australia rheology journal Vol.26 No.1

Understanding the nanorheology and associated intermolecular/surface forces of fluids in confined geometries or porous media is of both fundamental and practical importance, providing significant insights into various applications such as lubrication and micro/nanoelectromechanical systems. In this work, we briefly reviewed the fundamentals of nanoreheolgy, advances in experimental techniques and theoretical simulation methods, as well as important progress in the nanorheology of confined thin films. The advent of advanced experimental techniques such as surface forces apparatus (SFA), X-ray surface forces apparatus (XSFA) and atomic force microscope (AFM) and computational methods such as molecular dynamics simulations provides powerful tools to study a wide range of rheological phenomena at molecular level and nano scale. One of the most challenging issues unresolved is to elucidate the relationship between the rheological properties and structural evolution of the confined fluid films and particles suspensions. Some of the emerging research areas in the nanorheology field include, but are not limited to, the development of more advanced characterization techniques, design of multifunctional rheological fluids, bio-related nanorheology, and polymer brushes.

Flexural behavior of ultra high performance concrete beams reinforced with high strength steel

Jun-Yan Wang,Jin-Ben Gu,Chao Liu,Yu-Hao Huang,Ru-Cheng Xiao,Biao Ma 국제구조공학회 2022 Structural Engineering and Mechanics, An Int'l Jou Vol.81 No.5

A detailed experimental program was conducted to investigate the flexural behavior of ultra high performance concrete (UHPC) beams reinforced with high strength steel (HSS) rebars with a specified yield strength of 600 MPa via direct tensile test and monotonic four-point bending test. First, two sets of direct tensile test specimens, with the same reinforcement ratio but different yield strength of reinforcement, were fabricated and tested. Subsequently, six simply supported beams, including two plain UHPC beams and four reinforced UHPC beams, were prepared and tested under four-point bending load. The results showed that the balanced-reinforced UHPC beams reinforced with HSS rebars could improve the ultimate loadbearing capacity, deformation capacity, ductility properties, etc. more effectively owing to interaction between high strength of HSS rebar and strain-hardening characteristic of UHPC. In addition, the UHPC with steel rebars kept strain compatibility prior to the yielding of the steel rebar, further satisfied the plane-section assumption. Most importantly, the crack pattern of the UHPC beam reinforced with HSS rebars was prone to transform from single main crack failure corresponding to the normal-strength steel, to multiple main cracks failure under the condition of balanced-reinforced failure, which validated by the conclusion of direct tensile tests cooperated with acoustic emission (AE) source locating technique as well.

A Report of Chigger Mites on the Striped Field Mouse, Apodemus agrarius, in Southwest China

Yan-Ling Chen,Xian-Guo Guo,Tian-Guang Ren,Lei Zhang,Rong Fan,Cheng-Fu Zhao,Zhi-Wei Zhang,Ke-Yu Mao,Xiao-Bin Huang,Ti-Jun Qian 대한기생충학열대의학회 2021 The Korean Journal of Parasitology Vol.59 No.6

Functional roles of Tryptophan residues in diketoreductase from Acinetobacter baylyi

( Yan Huang ),( Zhuo Lu ),( Min Ma ),( Nan Liu ),( Yi Jun Chen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2012 BMB Reports Vol.45 No.8

Diketoreductase (DKR) from Acinetobacter baylyi contains two tryptophan residues at positions 149 and 222. Trp-149 and Trp-222 are located along the entry path of substrate into active site and at the dimer interface of DKR, respectively. Single and double substitutions of these positions were generated to probe the roles of tryptophan residues. After replacing Trp with Ala and Phe, biochemical and biophysical characteristics of the mutants were thoroughly investigated. Enzyme activity and substrate binding affinity of W149A and W149F were remarkably decreased, suggesting that Trp-149 regulates the position of substrate at the binding site. Meanwhile, enzyme activity of W222F was increased by 1.7-fold while W222A was completely inactive. In addition to lower thermostability of Trp-222 mutants, molecular modeling of the mutants revealed that Trp-222 is vital to protein folding and dimerization of the enzyme. [BMB Reports 2012; 45(8): 452-457]

Comparison Study of Percutaneous Kyphoplasty and Bone Cement-augmented Short-segment Pedicle Screw Fixation for Kummell Disease

( Yan-sheng Huang ),( Ding-jun Hao ),( Yong-ai Zhang ) 한국감성과학회 2017 한국감성과학회 국제학술대회(ICES) Vol.2017 No.-

Purpose : The purpose of this study was to compare the efficacy of percutaneous kyphoplasty (PKP) and bone cement-augmented short segmental fixation (BCA+SSF) for treating Kummell disease. Methods : Between June 2013 and December 2015, 60 patients were treated with PKP or BCA+SSF. All patients were followed up for 12-36 months. We retrospectively reviewed outcomes, including Oswestry Disability Index (ODI), visual analogue scale (VAS), and kyphotic Cobb angle. Results : VAS, ODI, and Cobb angle, measured postoperatively and at the final follow-up, were lower than those measured preoperatively in both groups (P< 0.05). VAS, ODI, and Cobb angle measured postoperatively demonstrated no significant differences when compared with those measured at the final follow-up in the PKP group (P >0.05). In the BCA+SSF group, VAS and ODI at the final follow-up were lower than those measured postoperatively (P< 0.05), but no significant difference was found in the Cobb angle (P >0.05). The PKP group had better VAS and ODI than the BCA+SSF group, postoperatively (P< 0.05). No significant difference was found in VAS and ODI at the final follow-up (P >0.05) or the Cobb angle measured postoperatively and at the final follow-up (P>0.05) between the two groups. Operative time, blood loss, and hospital stay in the PKP group were lower than those in the BCA+SSF group (P< 0. 05). No significant difference was found in complications (P >0.05). Conclusions : PKP patients had better early clinical outcomes, shorter operation and hospital admission times, and decreased blood loss, but similar complications, radiographic results, and long-term clinical outcomes compared with BCA+SSF patients.