The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli

Zock, Joseph; Cantwell, Cathleen A.; Swartling, James R.; Hodges, Roland; Pohl, Tonya; Sutton, Kimberly; Rosteck, Paul R.; McGilvray, D. I.; Queener, Stephen W.

doi:10.1016/0378-1119(94)90630-0

Cited by 86 publications

(57 citation statements)

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“…The 1.8 kb product was cut from the gel and the DNA extracted using a Qiaex kit. The ®nal products were ligated with the vector generated by BamHI-XbaI digestion of pNB106R (Zock et al, 1994). This library was used to transform competent E. coli TG1 cells, as described (Moore & Arnold, 1996).…”

Section: Dna Shufflingmentioning

confidence: 99%

“…Bacillus subtilis p-nitrobenzyl (pNB) esterase catalyzes the hydrolysis of the para-nitrobenzyl esters of various cephalosporin-type antibiotics, a necessary step in their large-scale synthesis (Zock et al, 1994). Using four generations of sequential random mutagenesis and screening, we evolved a series of pNB esterases up to 30 times more active towards hydrolysis of the pNB ester of loracarbef (LCNpNB) in aqueous dimethylformamide (Moore & Arnold, 1996).…”

Section: Introductionmentioning

confidence: 99%

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Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences 1 1Edited by J. Wells

Moore

Jin

Kuchner

et al. 1997

Journal of Molecular Biology

146

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Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-®delity DNA shuf¯ing and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuf¯ing con®rmed important features of the recombination process: rapid ®xation and accumulation of bene®cial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The ®ve to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.# 1997 Academic Press Limited

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Section: Dna Shufflingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences 1 1Edited by J. Wells

Moore

Jin

Kuchner

et al. 1997

Journal of Molecular Biology

146

View full text Add to dashboard Cite

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“…( Table 1) as those shown among the microbial lipolytic enzymes, LipA, LipB, YtpA, and PnbA of B. subtilis, and PldB and Aes of E. coli [8][9][10][11][12][13][14] . This suggests that YkoN has a lipolytic activity, though overall homology of YkoN with those of the lipase/esterase is not significant.…”

Section: Resultsmentioning

confidence: 70%

“…The results suggested that ykoN is not involved in the glucosylated diacylglycerides metabolismbut that it is involved in other function. Deep inspection of the amino acid sequence of YkoN revealed a pentapeptide sequence (Gly-Gly-Ser-Met-Gly) that is compatible with the Gly/Ala-X-Ser-X-Gly motif of lipase/esterase flanked with four hydrophobic residues (Ile-Ile-Ile-Thr) on the N-terminal side of the motif Amino acid sequences aligned are YkoN (BSORF), LipA (BSORF), LipB 10) , YtpA 11) and PnbA/EstB 12) from B. subtilis, and PldB 13) and Aes 14) from E. coli.…”

Section: Resultsmentioning

confidence: 83%

“…Among these the amino acid sequence of the product (YkoN) of ykoN has a weak homology with that of ugtP (ypfP) that is responsible for the synthesis of glucosylated diacylglycerates in this organism 5,7) , however, YkoN is found not to be involved in the synthesis of glucosylated diacylglycerates. The amino acid sequence of YkoN has a pentapeptide sequence (Gly-Gly-Ser-Met-Gly) that is compatible with the Gly/Ala-X-Ser-X-Gly motif of lipase/esterase as those shown among the microbial lipolytic enzymes, LipA, LipB, YtpA, and p-nitrobenzyl esterase (PnbA) of B. subtilis and lysophospholipase L2 (PldB) and Aes of E. coli [8][9][10][11][12][13][14] . We have purified His- Tagged YkoN and have shown that YkoN has an esterase activity.…”

Section: Introductionmentioning

confidence: 93%

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Characterization of a Novel Esterase YkoN from Bacillus subtilis Marburg

Matsuura

Usami

Hara

et al. 2007

J. Jpn. Soc. Extremophiles

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The product YkoN of the gene of unknown function, ykoN, of Bacillus subtilis Marburg has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and thus YkoN is expected to have a lipase or esterase activity. To characterize the expected enzyme activity the plasmid having a modified ykoN that include the sequence for His(x6) tag at its C-terminus of YkoN, which has 373 amino acid residues, was constructed. His-tagged YkoN protein of 39 kDa was induced in Escherichia coli BL21 (DE3) cells harboring chaperon plasmid pGro7 and purified to near homogeneity by using gel filtration and Ni-agarose. When p-nitrophenyl-esters of different fatty acid chain length were examined, the purified YkoN hydrolyzed the esters of fatty acid with short chain length (4-6 carbon atoms) preferentially. The esters of fatty acid with longer chain (C ≥ 10) were hydrolyzed inefficiently. The activity required no divalent cations and was not affected by addition of EDTA. The optimal pH for the activity was from pH 7.4 to pH 8.6. These results indicate that YkoN is a novel esterase which hydrolyzes the esters of fatty acid with short chain length.

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