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1.
Brundiek H Saß S Evitt A Kourist R Bornscheuer UT 《Applied microbiology and biotechnology》2012,94(1):141-150
The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with […] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant
expression of two versions of lipase UM03410: the full-length form (lipUMf) and an N-terminally truncated form (lipUMs). For
comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate
choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics
of the recombinant lipase are its pH optimum in the range of 6.5–7.0 and its temperature optimum at 55 °C. As is typical for
lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most
preferred one. More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity. 相似文献
2.
Bioinformatic analysis of the Yarrowia lipolytica CLIB122 genome has revealed 18 putative lipase genes all of which were expressed in Escherichia coli and screened for hydrolyzing activities against p-nitrophenyl-palmitate. One positive transformant containing an ORF of 1,098 bp encoding a protein of 365 amino acids was
obtained. To characterize its enzymatic properties, the lipase gene was functionally expressed in Pichia pastoris. The resulting lipase exhibited the highest activity towards p-NP-decanoate at pH 7 and 35°C. In addition, the new lipase had a lower optimal temperature and pH compared to other Y. lipolytica lipases. It was noticeably enhanced by Ca2+, but was inhibited by PMSF, Hg2+ and Ni2+. The new lipase displayed the 1,3-specificity for triolein. 相似文献
3.
Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and
expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity
chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal
pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg,
respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC
is a thermostable lipase to long-chain p-nitrophenyl esters.
P. Wei and L. Bai contributed equally to this work. 相似文献
4.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
5.
Fábio Cristiano Angonesi Brod Márcia Regina Pelisser Jean Borges Bertoldo Javier Vernal Carlos BlochJr Hernán Terenzi Ana Carolina Maisonnave Arisi 《Molecular biotechnology》2010,44(2):110-119
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim
of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant
fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as
determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42°C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC2, pNPC4, pNPC10, pNPC12, pNPC14, pNPC16, pNPC18). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95°C, 77%
of the initial activity was retained. 相似文献
6.
Rosa María Camacho Juan Carlos Mateos Orfil González-Reynoso Lilia Arely Prado Jesús Córdova 《Journal of industrial microbiology & biotechnology》2009,36(7):901-909
The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases
and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using
intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities
using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and
lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h−1 and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l−1 using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature,
pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface
design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect
between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal
values for growth rate and productions of esterase and lipase: 0.086 h−1 (at 42.5°C, pH 7.4, and 3.6 mol l−1 NaCl), 2.3 U l−1 (at 50°C, pH 7.5, and 4.3 mol l−1 NaCl), and 0.58 U l−1 (at 50°C, pH 7.6, and 4.5 mol l−1 NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l−1 NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained
50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the
first report evidencing the synthesis of esterase and lipase by H. marismortui. 相似文献
7.
Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant
of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher
activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function
relationship. Five other mutants also lost thermostability (20–40%) but exhibited enhanced thermoactivity (6.3–79-fold). The
mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65°C for 24 h. In comparison to r03Lip,
the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant
lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role
in the thermostability and activity of this thermoalkalophilic lipase. 相似文献
8.
Marina Royter M. Schmidt C. Elend H. Höbenreich T. Schäfer U. T. Bornscheuer G. Antranikian 《Extremophiles : life under extreme conditions》2009,13(5):769-783
Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography.
Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between
40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups
play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described
lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known
lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents,
and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols,
and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates
for kinetic resolution of synthetically useful compounds. 相似文献
9.
Jeon JH Kim JT Kim YJ Kim HK Lee HS Kang SG Kim SJ Lee JH 《Applied microbiology and biotechnology》2009,81(5):865-874
To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount
and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and
the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed
the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase
together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and
25°C, respectively, and rEML1 displayed more than 50% activity at 5°C. The activation energy for the hydrolysis of olive oil
was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols
acyl-group chains with long chain lengths of ≥8 carbon atoms and displayed hydrolyzing activities toward various natural oil
substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example
which developed a new cold-active lipase from a deep-sea sediment metagenome. 相似文献
10.
Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive
for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free
and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable
up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more
thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable
of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents,
serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments
inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and
temperatures. Applications of free and immobilized lipases for esterification were also presented. 相似文献
11.
Aijun Zhang Renjun Gao Naibing Diao Guiqiu Xie Gui Gao Shugui Cao 《Journal of Molecular Catalysis .B, Enzymatic》2009,56(2-3):78-84
A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced, and overexpressed as an N-terminus His-tag fusion protein in E. coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily I.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily I.1 and I.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 °C and 9.0, respectively. Its activity was found to increase in the presence of metal ions such as Ca2+, Sn2+ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives. 相似文献
12.
A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T1/2 of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality
of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated
higher thermostability than its wild type predecessor. To explore the structure–function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme
by 2-folds when compared with WT. The mutant enzyme showed enhanced T1/2 of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher
affinity for substrate (decreased K
m
) in comparison to the wild type. The k
cat and catalytic efficiency (k
cat/K
m
) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part
of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic
residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure. 相似文献
13.
Bacillus
thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene,
chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their
stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size
of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher
chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal
enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded
that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed
in the native chitinase, and for improving its activity. 相似文献
14.
Rodolfo Quintana-Castro Pilar Díaz Gerardo Valerio-Alfaro Hugo S. García Rosamaría Oliart-Ros 《Molecular biotechnology》2009,42(1):75-83
The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed
in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding
for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant
lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate
specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained
fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the
recombinant lipase without compromising the proper folding of the protein. 相似文献
15.
Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4 °C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5′ and 3′ regions of the coding sequence of the related protein.This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25 °C. 相似文献
16.
Mateus G. Godoy Melissa L. E. Gutarra Aline M. Castro Olga L. T. Machado Denise M. G. Freire 《Journal of industrial microbiology & biotechnology》2011,38(8):945-953
In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases,
these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor
bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research
Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation
(SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was
used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs–Plackett-Burman
(variable screening) followed by central composite rotatable design (CCRD)—were carried out, attaining a considerable increase
in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy
was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases
in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct
characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0
and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with
distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas
of biotechnology. 相似文献
17.
Takashi Kamijo Akihiro Saito Sadaharu Ema Inchi Yoh Hiroko Hayashi Ryo Nagata Yoshiho Nagata Akikazu Ando 《Antonie van Leeuwenhoek》2011,99(2):179-187
An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of
the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned
from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids,
exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively.
The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly,
enhanced by supplementing with 1 mM CoCl2, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil. 相似文献
18.
Snehasish Basu Arunava Roy Abhrajyoti Ghosh Amit Bera Dhrubajyoti Chattopadhyay Krishanu Chakrabarti 《Biodegradation》2011,22(1):153-161
After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the
pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of
35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5–9.0. Both Ca2+ and Mn2+ ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn2+ and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from
Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic
Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1
pectate lyase. 相似文献
19.
Yaping Lu Qian Lin Jin Wang Yufan Wu Wuyundalai Bao Fengxia Lv Zhaoxin Lu 《Journal of industrial microbiology & biotechnology》2010,37(9):919-925
A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce
the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced
287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ
(sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity
reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the
native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one. 相似文献
20.
Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained
by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually
with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted
with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were
11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively.
The specific activity and catalytic efficiency (k
cat/K
m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was
used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min. 相似文献