Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171

( Lixin Kang ),( Sijing Jiang ),( Lixin Ma ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 한국미생물·생명공학회지 Vol.46 No.1

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.

Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171

Kang, Lixin,Jiang, Sijing,Ma, Lixin The Korean Society for Microbiology and Biotechnol 2018 한국미생물·생명공학회지 Vol.46 No.1

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.

Expression and Characterization of a New L-amino Acid Oxidase AAO Producing α-ketoglutaric Acid from L-glutamic Acid

Rao Ben,Liao Xianqing,Liu Fang,Chen Wei,Zhou Ronghua,Ma Lixin,Wang YaPing 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.6

L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can’t obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multicopy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications.

The Asia Cornea Society Infectious Keratitis Study: A Prospective Multicenter Study of Infectious Keratitis in Asia

Khor, Wei-Boon,Prajna, Venkatesh N.,Garg, Prashant,Mehta, Jodhbir S.,Xie, Lixin,Liu, Zuguo,Padilla, Ma. Dominga B.,Joo, Choun-Ki,Inoue, Yoshitsugu,Goseyarakwong, Panida,Hu, Fung-Rong,Nishida, Kohji,Ki Elsevier 2018 American journal of ophthalmology Vol.195 No.-

<P><B>Purpose</B></P> <P>To survey the demographics, risk factors, microbiology, and outcomes for infectious keratitis in Asia.</P> <P><B>Design</B></P> <P>Prospective, nonrandomized clinical study.</P> <P><B>Methods</B></P> <P>Thirteen study centers and 30 sub-centers recruited consecutive subjects over 12-18 months, and performed standardized data collection. A microbiological protocol standardized the processing and reporting of all isolates. Treatment of the infectious keratitis was decided by the managing ophthalmologist. Subjects were observed for up to 6 months. Main outcome measures were final visual acuity and the need for surgery during infection.</P> <P><B>Results</B></P> <P>A total of 6626 eyes of 6563 subjects were studied. The majority of subjects were male (n = 3992). Trauma (n = 2279, 34.7%) and contact lens wear (n = 704, 10.7%) were the commonest risk factors. Overall, bacterial keratitis was diagnosed in 2521 eyes (38.0%) and fungal keratitis in 2166 eyes (32.7%). Of the 2831 microorganisms isolated, the most common were <I>Fusarium</I> species (n = 518, 18.3%), <I>Pseudomonas aeruginosa</I> (n = 302, 10.7%), and <I>Aspergillus flavus</I> (n = 236, 8.3%). Cornea transplantation was performed in 628 eyes to manage ongoing infection, but 289 grafts (46%) had failed by the end of the study. Moderate visual impairment (Snellen vision less than 20/60) was documented in 3478 eyes (53.6%).</P> <P><B>Conclusion</B></P> <P>Demographic and risk factors for infection vary by country, but infections occur predominantly in male subjects and are frequently related to trauma. Overall, a similar percentage of bacterial and fungal infections were diagnosed in this study. Visual recovery after infectious keratitis is guarded, and corneal transplantation for active infection is associated with a high failure rate.</P>

Influence of Water Absorption Behavior on Mechanical and Tribological Performance of Polymer Materials

Shengpeng Zhan,Ruize Li,Tian Yang,Dan Jia,Jiesong Tu,Lixin Ma,Bingxue Cheng,Haitao Duan 한국고분자학회 2023 폴리머 Vol.47 No.3

The physical, chemical and mechanical properties of polymer materials used in water environment for a long time will change due to their water absorption characteristics, which will affect their tribological properties. This work studied the changes of physicochemical, mechanical, and tribological properties for five kinds of polymer materials (polyethylene, polyformaldehyde,polyamide, polytetrafluoroethylene, and poly-ether-etherketone) before and after water absorption. The mechanism of water absorption for polymer materials was revealed by molecular dynamics simulation. The results show that the higher the water absorption of polymer, the larger the swelling volume, the more the surface hardness and compressive strength decrease, and the toughness and friction coefficient of the material increase obviously. The water absorption status of polymer materials is closely related to the polarity of molecular chain. Water molecules are easy to form hydrogen bonds with polymer molecular chains of high polarity, which is the main factor of water absorption.