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PubMed | Guangdong Vtr Bio Technology Co., China Agricultural University, CAS Wuhan Institute of Hydrobiology, Sichuan Academy of Animal Science and Sichuan Agricultural University
Type: | Journal: Fish & shellfish immunology | Year: 2016

This study investigated the effects of exogenous lipase supplementation on the growth performance, intestinal growth and function, immune response and physical barrier function, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). A total of 450 grass carp (255.020.34g) were fed five diets for 60 days. There were 5 dietary treatments that included a normal protein and lipid diet containing 30% crude protein (CP) with 5% ether extract (EE), and the low-protein and high-lipid diets (28% CP, 6% EE) supplemented with graded levels of exogenous lipase supplementation activity at 0, 1193, 2560 and 3730U/kg diet. The results indicated that compared with a normal protein and lipid diet (30% CP, 5% EE), a low-protein and high-lipid diet (28% CP, 6% EE) (un-supplemented lipase) improved lysozyme activities and complement component 3 contents in the distal intestine (DI), interleukin 10 mRNA expression in the proximal intestine (PI), and glutathione S-transferases activity and glutathione content in the intestine of young grass carp. In addition, in low-protein and high-lipid diets, optimal exogenous lipase supplementation significantly increased acid phosphatase (ACP) activities and complement component 3 (C3) contents (P<0.05), up-regulated the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and hepcidin) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor 1) and signaling molecules inhibitor protein-B (IB) and target of rapamycin (TOR) (P<0.05), down-regulated the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor , interleukin 8, interferon 2, and interleukin 1), and signaling molecules (nuclear factor kappa B p65, IB kinase , IB kinase ) (P<0.05) in the intestine of young grass carp. Moreover, optimal exogenous lipase supplementation significantly decreased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents (P<0.05), improved the activities of anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR), glutathione content, and the activities and mRNA levels of antioxidant enzymes (copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase) (P<0.05), up-regulated signaling molecule NF-E2-related factor 2 (Nrf2) (P<0.05), down-regulated signaling molecules (Kelch-like-ECH-associated protein 1a, Kelch-like-ECH-associated protein 1b) (P<0.05) in the intestine of young grass carp. Furthermore, optimal exogenous lipase supplementation significantly elevated the mRNA levels of tight junction proteins (Occludin, zonula occludens 1, Claudin b, Claudin c and Claudin 3) (P<0.05), down-regulated the mRNA levels of tight junction proteins (Claudin 12 and Claudin 15a) (P<0.05), down-regulated signaling molecules myosin light chain kinase (P<0.05) in the intestine of young grass carp. In conclusion, dietary lipid could partially spare protein, and the low-protein and high-lipid diet could improve growth, intestinal growth and function, immune response and antioxidant capability of fish. Meanwhile, in high-fat and low-protein diets, optimal exogenous lipase supplementation improved growth, intestinal growth and function, intestinal immunity, physical barrier, and regulated the mRNA expression of related signal molecules of fish. The optimal level of exogenous lipase supplementation in young grass carp (255-771g) was estimated to be 1193Ukg(-1) diet.


Liu Y.,Jiangnan University | Sun J.,Jiangsu University | Rao S.,Yangzhou University | Su Y.,Jiangnan University | And 4 more authors.
Food and Chemical Toxicology | Year: 2013

Se-polysaccharide from Catathelasma ventricosum (SPC-2) was purified by DEAE-52 and Sephadex G-100 column chromatography. The average size of SPC-2 was 1.6×105 Da, and it was mainly composed of glucose (87.4%) with the conformation of β-pyran ring. The branched structure of SPC-2 was proved intuitively by atomic force microscope (AFM). The antidiabetic potential of SPC-2 was tested in STZ-induced diabetic mice. After STZ-induced diabetic mice being administered of SPC-2 for 30days, SPC-2 treatment significantly reduced the levels of malondialdehyde (MDA) and low-density lipoprotein cholesterol (LDL-C) that were increased by the STZ treatment. Further, the SPC-2 treatment led to increased activity of antioxidant enzymes in liver and kidney and high-density lipoprotein cholesterol (HDL-C) that were decreased by the STZ. The results of histopathology also showed SPC-2 protected tissues (pancreas, liver and kidney) against peroxidation damage and maintained tissue integrity. © 2013 Elsevier Ltd.


Li D.,South China University of Technology | Qin X.,Southwest University | Wang J.,Guangdong VTR Bio Technology Co. | Yang B.,South China University of Technology | And 3 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

A lipase from Rhizopus oryzae (rProROL) was produced by recombinant Pichia pastoris in high productivity with a maximum lipase activity of 15,900 U/mL, and was employed to hydrolyze soybean oil for the production of diacylglycerol (DAG). Changes in triacylglycerol (TAG) conversion and DAG content were observed under various factors, like pH, water content, temperature and enzyme loading. A polynomial equation (R2 = 0.9797) between DAG content and TAG conversion was modeled and showed good agreement with the experimental results. The highest content of DAG at 32.09% was observed when TAG conversion was between 68% and 73%. Besides, 13C nuclear magnetic resonance (NMR) was used to identify the 1,2(2,3)-DAG and 1,3-DAG, and during hydrolysis, rProROL was discovered to show 1,3-regioselectivity in the hydrolysis. Moreover, rProROL exhibited high hydrolytic activity and capacity in the accumulation of DAG, indicating that rProROL is a prospective enzyme which could be used in the oils and fats industries. © 2015 Elsevier B.V. All rights reserved.


PubMed | Hunan University and Guangdong VTR Bio Technology Co
Type: Journal Article | Journal: Genetics and molecular research : GMR | Year: 2016

Myostatin (MSTN) is an important member of the transforming growth factor- (TGF-) superfamily and is a muscle growth inhibitor. In the present study, we cloned the Chinese perch MSTN cDNA sequence and analyzed its expression patterns under various conditions. The MSTN full cDNA sequence was 3347 bp long, including an open-reading frame of 1131 bp, which encoded 376 amino acids. Sequence analysis demonstrated that the MSTN shared a highly conserved signal peptide, a TGF- functional peptide, a hydrolytic site (RARR), and nine conservative cysteine residues with other members of the TGF- superfamily. Sequence alignment and phylogenetic tree analyses indicated that the MSTN had a close relationship with teleostean fish, but they are far separated from mammals. Real-time polymerase chain reaction analysis revealed that the MSTN was strongly expressed in the skeletal muscle and heart tissues. Temporal expression analysis demonstrated that the MSTN gene was expressed in very low levels, from 20 to 90 dph (post-hatching development), and was at its highest level at 150 dph (P < 0.05). The fasting-re-feeding experiment showed that the expression of the MSTN gene was initially decreased in response to a single meal, after seven days of fasting, and subsequently increased significantly, and finally decreased back to its original level. Together, our results provided valuable knowledge regarding the regulation of MSTN gene expression in Chinese perch.


Wang J.-R.,Guangdong VTR Bio Technology Co. | Li Y.-Y.,Guangdong VTR Bio Technology Co. | Liu D.,Guangdong VTR Bio Technology Co.
Journal of microbiology and biotechnology | Year: 2015

The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZαA and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60 °C and kept 45% enzyme activity after 1 h incubation at 70 °C. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).


Wang J.-R.,South China Agricultural University | Wang J.-R.,Guangdong VTR Bio Technology Co. | Lin J.-F.,South China Agricultural University | Guo L.-Q.,South China Agricultural University | And 3 more authors.
World Journal of Microbiology and Biotechnology | Year: 2014

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 μg/g, when the concentration of MeJA was 300 μM after 72 h induction. © 2013 Springer Science+Business Media Dordrecht.


Li Y.-Y.,Guangdong VTR Bio Technology Co. | Zhong K.-X.,Guangdong VTR Bio Technology Co. | Hu A.-H.,Guangdong VTR Bio Technology Co. | Liu D.-N.,Guangdong VTR Bio Technology Co. | And 2 more authors.
Protein Expression and Purification | Year: 2015

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10 kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75 °C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20 °C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80 °C for 10 min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22 °C, respectively. The maximum xylanase activity of Mxyn2p was 13,000 nkat/mg (9.88 g/l) in fed-batch cultivation after 168 h induction with methanol in a 50 l bioreactor. © 2014 Elsevier Inc.


PubMed | Guangdong VTR Bio Technology Co.
Type: | Journal: BioMed research international | Year: 2016

A series of strategies were applied to improve expression level of recombinant endo--1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo--1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792U/mL when the induction temperature was 22C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris.


Wang J.-R.,Guangdong VTR Bio Technology Co. | Li Y.-Y.,Guangdong VTR Bio Technology Co. | Xu S.-D.,Guangdong VTR Bio Technology Co. | Li P.,Guangdong VTR Bio Technology Co. | And 2 more authors.
International Journal of Molecular Sciences | Year: 2014

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0). © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Wang J.,Guangdong VTR Bio Technology Co. | Li Y.,Guangdong VTR Bio Technology Co. | Liu D.,Guangdong VTR Bio Technology Co.
BioMed Research International | Year: 2016

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris. © 2016 Jianrong Wang et al.

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