Small-Scale Production of Burkholderia cepacia ATCC21808 Lipase Adapted to High-Throughput Screening

Background Neurofibromatosis type 1 (NF1) is characterized by low‐grade tumors of the central and peripheral nervous system. There is also an increased risk of developing malignant tumors. Glioblastoma is an uncommon, malignant tumor of children that is even less frequently observed in children with NF1. Procedure We performed a retrospective review of patients with NF1 and glioblastoma to determine specific clinical and pathologic indicators of overall prognosis. Results Five patients were identified from the CHB/DFCI database for whom pathologic and imaging studies were available. All pathologic specimens demonstrated vascular proliferation and necrosis. All samples stained positively for p53. Chromogenic in situ hybridization (CISH) for epidermal growth factor receptor (EGFR) copy numbers was increased, PTEN copy numbers were normal and the promoter of the O(6)‐methylguanine‐DNA methyltransferase (MGMT) gene was unmethylated in the one patient evaluated. In the same time period, there were 56 patients without NF1 diagnosed with glioblastoma who were treated at our institution. Although the small sample size precludes formal statistical analysis, the 2‐year survival of patients with NF1 is 60% with median overall survival of 9.25 years compared to non‐NF1 patients with a 2‐year survival of 25% and median overall survival 1.08 years. Conclusions This study provides preliminary evidence that children with NF1 may be at risk for glioblastoma, but that these patients have an increased survival compared to children without NF1. Additional molecular studies will be required to determine if the pathogenesis of these tumors differs from glioblastoma in children without NF1. Pediatr Blood Cancer 2010;54:890–896 © 2010 Wiley‐Liss, Inc.

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“…BCL was incubated with diethyl chlorophosphate dissolved in isopropanol. Residual lipase activity was measured using a pNPB assay 78…”

Section: Resultsmentioning

confidence: 99%

“…Lipase activity was determined using pNPB assay78 (Sigma‐Aldrich). 20 μL of reaction was mixed with 175 μL of Tris HCl buffer (100 m M , pH = 7.5) and 5 μL of pNPB (40 m M in solution in 2M2B).…”

Section: Methodsmentioning

confidence: 99%

The role of the surgeon in whether patients with lymph node‐positive colon cancer see a medical oncologist

Luo

Giordano

Zhang

et al. 2007

Cancer

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“…Site-directed mutagenesis: By using the plasmid pFLAG-ATS-LipHp as a template, the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used according to the manufacturer's instruction to introduce 19 other amino acids at positions 266, [25] 17 and 287. The primers employed are L17 forw (5'-CTC GTG CAC GGG XXX ACG GGC ACC GAC-3') and L17 rev (5'-GTC GGT GCC CGT XXX CCC GTG CAC GAG-3'), where X corresponds to the 19 other amino acids (L17A forw = GCC; L17A rev = GGC; L17C forw = TGC; L17C rev = GCA; L17D forw = GAC; L17D rev = GCT; L17F forw = TTC; L17F rev = GAA; L17G forw = GGC; L17G rev = GCC; L17H forw = CAC; L17H rev = GTG; L17I forw = ATC; L17I rev = GAT; L17M forw = ATG; L17M rev = CAT; L17N forw = AAC; L17N rev = GTT; L17P forw = CCC, L17P rev = GGG; L17Q forw = CAG; L17Q rev = CTG; L17R forw = CGC; L17R rev = GCG; L17S forw = AGC; L17S rev = GCT; L17T forw = ACC; L17T rev = GGT; L17V forw = GTC; L17V rev = GAC; L17W forw = TGG; L17W rev = CCA; L17Y forw = TAC; L17Y rev = GTA; L17E forw = GAG; L17E rev = CTC; L17K forw = AAG; L17K rev = CTT).…”

Section: Methodsmentioning

confidence: 99%

“…Following cell lysis, the mutants were screened for their ability to hydrolyse pNPB (Scheme 1 A), a substrate easily monitored by spectrophotometry at 405 nm and often used to determine lipase activity. [25] The variability of pNPB hydrolytic activity found amongst the variants is shown in Figure 2. Overall, ten variants out of 57 appeared more active on pNPB than the wild-type BCL.…”

Section: Preliminary Screening On Pnpb Substratementioning

confidence: 98%

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Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

et al. 2009

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Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2-substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild-type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)-2-chloro ethyl 2-bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15-fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.

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Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

et al. 2009

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The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so-called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position-restrained MD simulations. Our conclusions indicate that the sole mobility of alpha9 helix side-chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by alpha5 helix movement. The role of selected alpha5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase.

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Small-Scale Production of Burkholderia cepacia ATCC21808 Lipase Adapted to High-Throughput Screening

Cited by 5 publications

References 21 publications

The role of the surgeon in whether patients with lymph node‐positive colon cancer see a medical oncologist

The role of the surgeon in whether patients with lymph node‐positive colon cancer see a medical oncologist

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

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