Improved Temperature Characteristics of an Aspergillus oryzae GHF11 Xylanase, by In Silico Design and Site-directed Mutagenesis

Die Hu,Jianfang Li,Qin Wu,Jia Zang,Jianqing Cheng,Minchen Wu 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.6

To improve the temperature characteristics of a mesophilic glycoside hydrolase family (GHF) 11 xylanase AoXyn11A from Aspergillus oryzae, both introduction of a disulfide bridge and the substitution of a specific amino acid were carried out by in silico design and site-directed mutagenesis. Based on the analysis of a known crystal structure of thermophilic xylanase TlXynA from Thermomyces lanuginosus, and the alignment of primary structures between AoXyn11A and TlXynA, one mutant AoXyn11AM with a disulfide bridge (Cys108–Cys152) was designed by replacing the Ser108 and Asn152 of AoXyn11A with Cys residues, respectively. Additionally, based on the analysis of amino acid B-factor values, another mutant AoXyn11AM-G22A was predicted by substituting Gly22 of AoXyn11AM (having the maximum B-factor value of 69.25 Å, with the corresponding Ala23 of TlXynA. Thereafter, two mutant xylanase-encoding genes, Aoxyn11AM and Aoxyn11AM-G22A, were constructed by site-directed mutagenesis. Aoxyn11A and two mutant genes were expressed in E. coli BL21(DE3) respectively, and three expressed recombinant xylanases, reAoXyn11A, reAoXyn11AM and reAoXyn11AM-G22A, were purified to homogeneity. The temperature optima of reAoXyn11AM and reAoXyn11AM-G22A were 60 and 65°C, respectively, being 5 and 10°C higher than that of reAoXyn11A. Their thermal inactivation half-lives at 50°C were 1.8- and 8.4-folds longer than that of reAoXyn11A. There were no obvious alterations after mutations in specific activity and enzymatic properties, except for the temperature characteristics.

芥川龍之介「蜘蛛の糸」論 ―「悪人正機説」との関わりを中心に―

胡逸蝶(Hu, Yi-Die) 한국일본문화학회 2022 日本文化學報 Vol.- No.95

There are many previous studies discussing the famous work Kumo No Ito of Ryunosuke Akutagawa from the perspective of Buddhism, and pointing out the deficiency of Akutagawa’s cognition of Buddhism. Meanwhile, these studies also show that under the background of The Times, almost no Japanese literati can correctly understand Buddhist thoughts. However, the reality that Akutagawa actually has his own unique thoughts on Japan’s local Buddhism which is different from the orthodox Buddhism seldom arises public attention. Therefore, this paper will clarify the materials in Kumo No Ito which talks about the relief of the wicked, the doctrines of Jodo Shinshu which expands rapidly after Edo Period, and especially the relationship among the thoughts of Akunin-Shoki, thus re-exploring Akutagawa’s understanding of Buddhism.

Nuclear DNA content variation of three Miscanthus species in China

Xi Li,Die Hu,Manman Luo,Ming Zhu,Xinwei Li,Fan Luo,Jianqiang Li,Juan Yan 한국유전학회 2013 Genes & Genomics Vol.35 No.1

In order to estimate the variation in nuclear genome size in Miscanthus, flow cytometry of nuclei stained by propidium iodide was carried out using 36 populations of three Miscanthus species: M. lutarioriparius, M. sacchariflorus and M. sinensis, which were sampled from cold northern to warm and humid southern and central China, as well as near the sea level in eastern China to mountains in western China. The DNA content of diploid was 4.37 ±0.02 pg/2C in M. lutarioriparius, 4.37 ± 0.01 pg/2C in M. sacchariflorus, and 5.37 ± 0.03 pg/2C in M. sinensis,respectively. There was no intraspecific variation in the three Miscanthus species at the diploid level, suggesting that the genome size was stable within species and the diverse environments did not induce variation in genome size at the diploid level. However, tetraploid populations were found in M. lutarioriparius and M. sacchariflorus, and their genome sizes were 8.56 and 8.54 pg, respectively, which are lower than expected values (8.74 pg), indicating the genome downsizing after polyploidization in the genus. Our results showed that the plant height of M. lutarioriparius was the highest one among the three species and the species was more closely related to M. sacchariflorus than M. sinensis. The intra-species genomic variation and inter-species differentiation in Miscanthus species provide important genetic and genomic information for the development of Miscanthus,especially for the endemic species, M. lutarioriparius,(together with Miscanthus 9 giganteus) which are now emerging as a key bio-energy crop because of their high yields and strong adaptability.

PLCE1 rs2274223 Polymorphism and Susceptibility to Esophageal Cancer: a Meta-analysis

Guo, Li-Yan,Yang, Ning,Hu, Die,Zhao, Xia,Feng, Bing,Zhang, Yan,Zhai, Min Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.21

Purpose: To investigate and study the relationship between the PLCE1 rs2274223 gene polymorphism and susceptibility to esophageal cancer by meta-analysis. Materials and Methods: The literature was searched in Wanfang, CNKI, PubMed, CBM, Web of Science, MEDLINE, EMBASE, Springer, Elsevier and Cochrane databases from the date of January $1^{st}$ 2004 to April $1^{st}$ 2014 to collect case-control studies on the PLCE1 polymorphism and susceptibility to esophageal cancer. For the population genotype distributions of both esophagus cancer and control groups, their odds ratios (ORs) and 95% confidence intervals (CIs) were taken as effect indexes. Disqualified studies were excluded. Odds ratios of PLCE1 rs2274223 genotype distributions in the group of patients with esophageal cancer and the group of healthy control were calculated. The metaanalysis software, RevMan5.0, was applied for heterogeneity test, pooled OR and 95% confidence intervals. Sensitivity analysis and publication bias were also explored. Results: A total of twelve case-control studies were included, covering a total of 9, 912 esophageal cancer cases and 13, 023 controls were included. The pooled odds ratio of PLCE1 rs2274223 genotype GA vs AA was 1.29 (95%CI=1.17~1.43), p<0.01, GG vs AA was 1.65 (95%CI=1.32~2.05), p<0.01, GG/GA vs AA was 1.30 (95%CI=1.16~1.46), p<0.01 and GG vs GA/AA was 1.48 (95%CI=1.22~1.80), p<0.01. The PLCE1 rs2274223 polymorphism was thus associated with risk of esophageal cancer in all genetic models. In the stratified analysis by ethnicity, and source of controls, no significantly increased risk was observed for white persons. There was no obvious publication bias detected. Conclusions: This meta-analysis showed there was a significantly association between PLCE1 rs2274223 polymorphism and esophageal cancer in yellow race populations. Due to some minor limitations, our findings should be confirmed in further studies.

PLCE1 Gene in Esophageal Cancer and Interaction with Environmental Factors

Guo, Li-Yan,Zhang, Shen,Suo, Zhen,Yang, Chang-Shuang,Zhao, Xia,Zhang, Guo-An,Hu, Die,Ji, Xing-Zhao,Zhai, Min Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.7

Objective: To study the PLCE1 gene rs2274223 polymorphism with regard to esophageal cancer and its interaction with diet, lifestyle, psychological and environmental factors in Southwest Shandong province. Materials and Methods: A case series study (case-case) was conducted. Questionnaire data were collected and 3 ml-5ml venous blood was drawn for DNA extraction among the qualified research subjects. PLCE1 gene polymorphism was detected after PCR amplification of DNA. SPSS 13.0 software was used for statistical analysis of the data. Results: The three genotypes A/A, A/G and G/G PLCE1 gene rs2274223 was 31, 16 and 4 cases, accounting for 60.8%, 31.4%, 0.08% respectively. The difference of three genotypes (AA/GA/GG) proportion between negative and positive family history of patients was statistically significant, ${\chi}^2=6.213$, p=0.045. There was no statistically significant relationship between PLCE1 gene rs2274223 polymorphism and smoking, drinking, ${\chi}^2=0.119$, p=0.998, and ${\chi}^2=1.727$, p=0.786. There was no linkage of the three rs2274223 PLCE1 gene genotypes (AA/GA/GG) proportion with eating fried, pickled, hot, mildew, overnight, smoked, excitant food, eat speed, salt taste or not (p>0.05). or with living environment pollution and nine risk factors of occupational exposure (p>0.05). There was no statistically significant difference in TS scores between different genotype of rs2274223 PLCE1 gene. Conclusions: The PLCE1 rs2274223 polymorphism has a relationship with family history of esophageal cancer, but does not have any significant association with age, gender, smoking, alcohol drinking, food hygiene, eating habits, living around the environment and occupation in cases.