Molecular Characterization of Cold-Inducible β-Galactosidase from Arthrobacter sp. ON14 Isolated from Antarctica

( Xiang Xiao ),( Feng Ping Wang ),( Xu Ke ),( Xi Xiang Tang ),( Ying Bao Gai ),( Muhammad Aamer Mehmood ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.3

A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated from Antarctica, was shown to exhibit a high β-galactosidase activity at a low temperature. A genomic library of ON14 was constructed and screened for β-galactosidase genes on functional plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as the substrate. Two different β-galactosidase genes, named as galA, galB, were found in ON14. Computational analyses of the genes revealed that the encoded protein GalA belongs to family 2 of glycosyl hydrolysases and is a cold-active protein, whereas GalB belongs to family 42 of glycosyl hydrolysases and is a mesophilic protein. Reverse transcription analyses revealed that the expression of galA is highly induced at a low temperature (4℃) and repressed at a high temperature (28℃) when lactose is used as the sole carbon source. Conversely, the expression of galB is inhibited at a low temperature and induced at a high temperature. The purified GalA showed its peak activity at 15℃ and pH 8. The mineral ions Na+, K+, Mg2+, and Mn2+ were identified as enzyme activators, whereas Ca2+ had no influence on the enzyme activity. An enzyme stability assay revealed that the activity of GalA is significantly decreased when it is incubated at 45℃ for 2 h, and all its activity is lost when it is incubated at 50℃.

Automated Breast Ultrasound: Interobserver Agreement, Diagnostic Value, and Associated Clinical Factors of Coronal-Plane Image Features

Guoxue Tang,Xin An,Huiling Xiang,Lixian Liu,Anhua Li,Xi Lin 대한영상의학회 2020 Korean Journal of Radiology Vol.21 No.5

Objective: To evaluate the interobserver agreement, diagnostic value, and associated clinical factors of automated breast ultrasound (ABUS) coronal features in differentiating breast lesions. Materials and Methods: This study enrolled 457 pathologically confirmed lesions in 387 female (age, 46.4 ± 10.3 years), including 377 masses and 80 non-mass lesions (NMLs). The unique coronal features, including retraction phenomenon, hyper- or hypoechoic rim (continuous or discontinuous), skipping sign, and white wall sign, were defined and recorded. The interobserver agreement on image type and coronal features was evaluated. Furthermore, clinical factors, including the lesion size, distance to the nipple or skin, palpability, and the histological grade were analyzed. Results: Among the 457 lesions, 296 were malignant and 161 were benign. The overall interobserver agreement for image type and all coronal features was moderate to good. For masses, the retraction phenomenon was significantly associated with malignancies (p < 0.001) and more frequently presented in small and superficial invasive carcinomas with a low histological grade (p = 0.027, 0.002, and < 0.001, respectively). Furthermore, continuous hyper- or hypoechoic rims were predictive of benign masses (p < 0.001), whereas discontinuous rims were predictive of malignancies (p < 0.001). A hyperechoic rim was more commonly detected in masses more distant from the nipple (p = 0.027), and a hypoechoic rim was more frequently found in large superficial masses (p < 0.001 for both). For NMLs, the skipping sign was a predictor of malignancies (p = 0.040). Conclusion: The coronal plane of ABUS may provide useful diagnostic value for breast lesions.

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.

Molecular Biology and Omics : Cloning and Characterization of a Novel α-Amylase from a Fecal Microbial Metagenome

( Bo Xu ),( Fu Ya Yang ),( Cai Yun Xiong ),( Jun Jun Li ),( Xiang Hua Tang ),( Jun Pei Zhou ),( Zhen Rong Xie ),( Jun Mei Ding ),( Yun Juan Yang ),( Zun Xi Huang ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.4

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for α-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel α-amylase from a gastrointestinal metagenomic library.

Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti

( Bo Xu ),( Li Ming Dai ),( Jun Jun Li ),( Meng Deng ),( Hua Biao Miao ),( Jun Pei Zhou ),( Yue Lin Mu ),( Qian Wu ),( Xiang Hua Tang ),( Yun Juan Yang ),( Jun Mei Ding ),( Nan Yu Han ),( Zun Xi Huang 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1

Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.