High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-celldensity Cultivation

Zhou Ronghua,Liao Xianqing,Liu Fang,Dong Qing,Chen Wei,Wang YaPing,Rao Ben 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.4

Chitin is a linear homopolymer of acetylated β- (1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) from Aspergillus oryzae NKY2017 obtained from Hu’bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed in Pichia pastoris and subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels of CCHA expression in multi-copy strains, with strain CCHA4 chosen for high-density fermentation and enzyme-activity experiments. High-density fermentation achieved a CCHA yield of 22,500 U/mL, and temperature and pH optimization resulted in the highest CCHA activity at 40°C and 4.0, respectively. We used this enzyme for a large-scale preparation of oligosaccharides: 4 g enzyme could convert 150 kg chitosan into oligosaccharides in 24 h at 40°C. These results demonstrated abundant CCHA expression in P. pastoris and suggested the efficacy of CCHA for use in industrial applications.

Study on transmission error and torsional stiffness of RV reducer under wear

Ronghua Zhang,Jianxing Zhou,Zheng Wei 대한기계학회 2022 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.36 No.8

In order to discuss the influence of wear dynamic evolution on the degradation of core transmission performance, transmission error and meshing torsional stiffness of RV reducer, taking BX-40E reducer as the research object, based on Langkali-Nikraves contact force model and Archard wear theory, the wear coefficients with different position conditions were obtained through equivalent wear experiments. The RV transmission dynamic model considering wear, meshing stiffness, meshing force, meshing tooth pairs and transmission error linkage was established by using centralized parameter method and dynamic subsystem method. Compared with the traditional tooth profile wear simulation, which distribution and size of the wear depth curves were more and more different with wear dynamic evolution, theoretically concluded that the dynamic update of wear coefficient is necessary and correct for the accurate simulation of tooth surface wear. The transmission error shows a "double hump" distribution mapped to the wear depth of the tooth profile, and deteriorates with the wear accumulation. The equivalent torsional stiffness of cycloid pin teeth decreases piecewise approximately linearly due to wear. In the previous stage, the reduction rate is small because the tooth detachment first occurs at the tooth top and root, and the single tooth torsional stiffness near the tooth top and root is smaller. The research results provide a new idea and theoretical basis for improving the transmission accuracy, stability holding ability, wear reduction and life extension of RV reducer.

Segment Training Based Individual Channel Estimation for Multi-pair Two-Way Relay Network with Power Allocation

( Xiandeng He ),( Ronghua Zhou ),( Nan Chen ),( Shun Zhang ) 한국인터넷정보학회 2018 KSII Transactions on Internet and Information Syst Vol.12 No.2

In this paper, we design a segment training based individual channel estimation (STICE) scheme for the classical two-way relay network (TWRN) with multi-pair sources (MPS) and amplify-and-forward (AF). We adopt the linear minimum mean square error (LMMSE) channel estimator to minimize the mean square error (MSE) without channel estimation error, where the optimal power allocation strategy from the relay for different sources is obtained. Then the MSE gains are given with different source pairs among the proposed power allocation scheme and the existing power allocation schemes. Numerical results show that the proposed method outperforms the existing ones.

Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains

Rao Ben,Zhou Ronghua,Dong Qing,Liao Xianqing,Liu Fang,Chen Wei,Liu Xiaoyan,Min Yong,Wang YaPing 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.4

L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to α-aketoglutaric acid (α-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM in Pichia pastoris to produce α-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed on P. pastoris to achieve the conversion. We constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PGLOD(1-3)-AGα1 and PAAO(1-3)-AGα1, respectively. The following results showed that expression of GLOD(1-3)- AGα1 and AAO(1-3)-AGα1 in multi-copy strains increased as designed and strain PGLOD3-AGα1 and PAAO3-AGα1 was chosen for high-density fermentation and enzyme activity experiments. By using a multi-copy expression approach and high-density fermentation, we achieved a GLOD expression yield of 688.5 U/g dry cell weight and AAO expression yield of 626.7 U/g dry cell weight. By using displayed GLOD, the average production rate of L-glutamic acid to α-KG was 6.22 g/L/h and the highest α-KG titer (124.5 g/L) was converted from 135 g/L L-glutamic acid. By using displayed AAO, the average production rate of L-glutamic acid to α-KG was 5.78 g/L/h and the highest α-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes on P. pastoris are suitable for use in industrial applications.

Cloning of porcine chemerin, ChemR23 and GPR1 and their involvement in regulation of Lipogenesis

( Jianfeng Huang ),( Jian Zhang ),( Ting Lei ),( Xiaodong Chen ),( Yan Zhang ),( Lulu Zhou ),( An Yu ),( Zhilong Chen ),( Ronghua Zhou ),( Zaiqing Yang ) 생화학분자생물학회 (구 한국생화학분자생물학회) 2010 BMB Reports Vol.43 No.7

Chemerin is a novel adipokine which is abundant in adipose tissue to promote adipocyte differentiation and with significant relativity to BMI and insulin sensitivity. We report here the molecular characterization of porcine chemerin and its receptors ChemR23 and GPR1, as well as their transcriptional regulation during lipogenesis. Chemerin was mainly expressed in liver, intestine, kidney and adipose tissue, consistent with the expression pattern of GPR1, but not ChemR23, which was predominantly present in spleen and temperately in adipose tissue. We further investigated the lipogenesis-related transcriptional activation of PPARγ and KLF15 on chemerin and its receptors. The data showed that KLF15, but not PPARγ, can up-regulate the mRNA level of chemerin, ChemR23 and GPR1, which was consistent with the results of luciferase assay that confirmed the effect of KLF15 on ChemR23 promoter. Taken together, our data provide basic molecular information for the further investigation on the function of chemerin in lipogenesis. [BMB reports 2010; 43(7): 491-498]

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.