1.Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei.
Manchi ZHU ; Xian ZHANG ; Zhi WANG ; Wenxuan LIN ; Meijuan XU ; Taowei YANG ; Minglong SHAO ; Zhiming RAO
Chinese Journal of Biotechnology 2021;37(9):3242-3252
L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics*
;
Bacillus subtilis/genetics*
;
Industrial Microbiology
;
Protein Engineering
;
Rhizomucor/enzymology*
;
Sequence Alignment
2.Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host.
Xinyuan YANG ; Yi RAO ; Mengxi ZHANG ; Jiaqi WANG ; Wenyuan LIU ; Dongbo CAI ; Shouwen CHEN
Chinese Journal of Biotechnology 2023;39(3):1096-1106
L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Bacillus licheniformis/metabolism*
;
Asparaginase/genetics*
;
Bacillus/genetics*
;
Protein Sorting Signals
;
Promoter Regions, Genetic/genetics*
;
Bacillus subtilis/genetics*
;
Bacterial Proteins
3.Asparagine synthetase activity in pediatric acute lymphoblastic leukemia.
Hua JIANG ; Long-Jun GU ; Hui-Liang XUE ; Jing-Yan TANG ; Jing CHEN ; Ci PAN ; Jing CHEN
Chinese Journal of Contemporary Pediatrics 2006;8(4):272-274
OBJECTIVETo study the cellular activity of asparagine synthetase in different types of childhood acute lymphoblastic leukemia (ALL).
METHODSThe cellular activity of asparagine synthetase was detected by HPLC-FLD and Protein measurement in 28 ALL children (7 cases of T-ALL and 21 cases of B-lymphoid lineage ALL) before chemotherapy.
RESULTSThe asparagines synthetase activity levels in T-ALL children were significantly higher than those of the B-lymphoid lineage ALL patients, with the median activity level of 9.3 nM Asn/mg protein/hr vs 5.2 nM Asn/mg protein/hr (P < 0.05). The distribution of the asparagine synthetase activity demonstrated a polymorphism in either T-ALL or B-lymphoid lineage ALL patients.
CONCLUSIONSThe cellular activity of asparagines synthetase in ALL patients is presented with a polymorphism distribution. The asparagines synthetase activity levels in T-ALL are significantly higher than in B-lymphoid lineage ALL.
Asparaginase ; therapeutic use ; Aspartate-Ammonia Ligase ; genetics ; metabolism ; Child ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Humans ; Male ; Oncogene Proteins, Fusion ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; enzymology ; RNA, Messenger ; analysis
4.Enhanced expression of halp gene confers cellular resistance to H2O2 induced senescence.
Yun LING ; Hai-jing LIU ; Lin HOU ; Bo ZHANG
Chinese Medical Sciences Journal 2006;21(1):1-5
OBJECTIVETo investigate the H2O2-induced expression of human histone acetyltransferase-like protein (hALP), a telomerase regulation-associated gene, and its effects on the stress-triggered cellular senescence.
METHODSThe induced expression of hALP was measured by semi-quantitative RT-PCR and immunofluorescent histochemistry after treatment of HeLa cells by H2O2. The effects of hALP expression on cellular responses to H2O2 were analyzed by MTT, flowcytometry, and SA-beta-gal staining, respectively.
RESULTShALP mRNA could be dose-dependently induced by treatments of 0.2-1.6 mmol/L H2O2, and the induction could be observed after 6 hours and kept for 36 hours in the presence of 0.4 mmol/L H2O2. Meanwhile, the immunofluorescent staining showed marked stronger nuclear intensity of hALP protein in H2O2-treated HeLa cells. In the treatment of H2O2, the ectopic expression of hALP enhanced continuous growth and overcame G2/M arrest as well as decreased senescence-associated beta-gal staining. On the contrary, the transfected clones with antisense or blank vector and original He-La cells presented growth suppression, G2/M delay and higher percentage of SA-beta-gal activities in the presence of H2O2.
CONCLUSIONSThe expression of hALP could be up-regulated by treatment of H2O2, and elevated expression could enhance cellular resistance to H2O2-induced cellular senescence. The data might be of references to elucidation of basic biological function of hALP gene and its associated telomerase activity.
Asparaginase ; biosynthesis ; genetics ; Autoantigens ; biosynthesis ; genetics ; Cell Cycle ; Cell Proliferation ; Cellular Senescence ; drug effects ; Dose-Response Relationship, Drug ; HeLa Cells ; Humans ; Hydrogen Peroxide ; administration & dosage ; pharmacology ; Oxidants ; administration & dosage ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Up-Regulation
5.An analysis of induced expression and function of telomerase-regulation associated hALP gene on genotoxic agents.
Hai-Jing LIU ; Yun LING ; Lin HOU ; Bo ZHANG
Chinese Journal of Pathology 2005;34(11):732-736
OBJECTIVETo confirm the responses and function of hALP, a telomerase-regulation associated gene, in DNA damage.
METHODSHeLa and Hep2 cells were treated by genotoxic agents H2O2 and cisplatin, and the induced expression of hALP was measured by quantitative RT-PCR and immunofluorescent histochemistry. The alterations in transcriptional activity of hALP promoter were estimated by luciferase reporter assays. The effects of genotoxic agents on cells in different status of hALP expression were analyzed by MTT method.
RESULTSThe level of hALP mRNA could be increased when treated by 0.2 - 1.6 mmol/L H2O2 and reach a peak in concentration of 0.4 mmol/L. The induction could be observed after 6 h in the treatment of 0.4 mmol/L H2O2 and the higher level can be retained for 36 h. Similarly, cisplatin induced hALP mRNA expression is also dose and time dependent. The immunofluorescent staining showed that the treatment of 0.2 or 0.4 mmol/L H2O2, 0.2 or 0.5 micromol/L cisplatin increased the intensity of hALP protein in cellular nuclei. The luciferase assays demonstrated that both H2O2 and cisplatin could up-regulate hALP promoter activity through its upstream - 705 - +20 nt region. In cell survivor assay, the HeLa cells expressing sense hALP gene could grow continuously in the presence of 0.4 mmol/L H2O2 or 0. 5 micromol/L cisplatin while cells with antisense hALP or control cells were slower in growth.
CONCLUSIONSThe expression of hALP gene could be up-regulated by DNA damage through activating transcription of its promoter, and increase cellular resistance to genotoxic agents.
Antineoplastic Agents ; administration & dosage ; pharmacology ; Asparaginase ; biosynthesis ; genetics ; Autoantigens ; biosynthesis ; genetics ; Cell Proliferation ; Cells, Cultured ; Cisplatin ; administration & dosage ; pharmacology ; DNA Damage ; physiology ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Enzymologic ; HeLa Cells ; metabolism ; Humans ; Hydrogen Peroxide ; administration & dosage ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Telomerase ; genetics ; physiology ; Up-Regulation
6.SLCO1B1c. 521T>C gene polymorphisms are associated with high-dose methotrexate pharmacokinetics and clinical outcome of pediatric acute lymphoblastic leukemia.
Huanian ZHANG ; Xuelian HE ; Jianxin LI ; Yang WANG ; Cheng WANG ; Yujun CHEN ; Changhe NIU ; Ping GAO
Chinese Journal of Pediatrics 2014;52(10):770-776
OBJECTIVETo provide guidance for the high-dose methotrexate (HD-MTX) treatment of pediatric acute lymphoblastic leukemia (ALL), and to understand the impact of SLCO1B1c.521T>C (rs4149056) variant on methotrexate (MTX) pharmacokinetics and clinical outcome in children with ALL.
METHODEighty-two children with ALL in Division of Hematology of Wuhan Children's Hospital from January 2008 to February 2013 were enrolled. All patients were genotyped for rs4149056 single nucleotide polymorphism (SNP) into wild-type group (TT genotype) and variant group (TC/CC genotype). According to the ALL-BFM 2000 protocol, all patients received intravenous infusion of MTX every ten days at 3 to 5 g/m(2). Leucovorin rescue was performed after 36 hours of the MTX administration and its dose was adjusted according to the MTX plasma concentration at 48 hours. The concentrations of MTX and its metabolite at 24, 48 and 72 h were determined by high performance liquid chromatography with solid phase extraction. Population pharmacokinetic parameters were estimated by the NLME software. The pharmacokinetics, toxicity and leucovorin rescue was compared. The relapse rate within 5 years and event-free survival were followed up.
RESULTEighty-two pediatric patients were classified into two groups: variant group including 20 TC genotype carriers and one CC genotype carrier, wild-type group included 61 patients with TT genotype. Compared with wild-type group, plasma concentration of MTX at 48 and 72 h increased significantly [48 h: (1.00±1.41) vs.(0.34±0.17) µmol/L, t=2.131, P=0.046; 72 h: (0.31±0.26) vs.(0.08±0.04) µmol/L; t=3.995, P=0.001]. Area under the concentration time curve (AUC48-∝) of MTX significantly increased in variant group [(23.18±19.91) vs.(5.66±2.01) h·µmol/L] (t=4.025, P=0.001). Time above the MTX safety threshold (TC>0.1 µmol/L) increased significantly in variant group [(95.3±22.0) vs.(67.1±7.5) h, t=5.880, P<0.001]. Rescue dosage of leucovorin in variant group was higher than that in wild-type group [(312.7±287.8) vs.(140.6±27.5) mg/m2, t=2.614, P=0.017]. The children carrying rs4149056 C allele suffered from a higher frequency of serious adverse effect [gastrointestinal toxicity: 33% (7/21) vs. 5% (3/61);hepatic toxicity: 24% (5/21) vs. 2% (1/61)]. The difference was statistically significant (χ2=9.275, 8.289, all P<0.05). Hospital stay of variant group was significantly longer than that of wild-type [(4.95±1.43) vs. (4.05±0.22) d, t=2.881, P=0.009]. The relapse rate within 5 years of variant group and wild-type group were 9% (2/21) and 13% (8/61), respectively. There were no significant differences in the event-free survival between the two groups (χ2=0.001, P=0.971).
CONCLUSIONThe SLCO1B1 c.521T>C variant was an important determinant of MTX pharmacokinetics. An appropriate leucovorin dose raise in variant group was beneficial to reducing the serious toxicity and did not affect the long-term clinical outcome.
Alleles ; Antineoplastic Combined Chemotherapy Protocols ; Asparaginase ; Child ; Daunorubicin ; Disease-Free Survival ; Genotype ; Humans ; Leucovorin ; administration & dosage ; Methotrexate ; administration & dosage ; pharmacokinetics ; Organic Anion Transporters ; genetics ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; Prednisone ; Solute Carrier Organic Anion Transporter Family Member 1b1 ; Treatment Outcome ; Vincristine