Patent Application: US-201214363722-A

Abstract:
the present disclosure relates to an enzyme derived from candida bombicola that is capable of lactonizing or polymerizing carbohydrate - containing compounds , lipids , fatty acids , hydroxylated fatty acids , alcohols , dicarboxylic acids or mixtures thereof . hence , host cells comprising the latter enzyme can be used , via the formation of intra - or inter - molecular ester - bounds , to produce , for example , lactonized sophorolipids or polymers of acidic sophorolipids . on the other hand , host cells having lost their capability to produce a functional enzyme disclosed herein can be used to produce 100 % acidic sophorolipids .

Description:
occurrence of a putative lipase gene in the secretome of sophorolipid - producing c . bombicola cells c . bombicola atcc 22214 was cultured for eight days in 150 ml lang medium ( lang et al ., 2000 ). the cells were separated from the medium by centrifugation at 4 , 000 rpm for 15 minutes ( 4 ° c .). the medium liquid was subsequently filtered twice on a 0 . 22 μm filter to exclude any cellular trace . a protease inhibitor cocktail ( complete , edta - free protease inhibitor cocktail tablets , roche ) was added to prevent protein degradation . consequently , the protein fraction in the collected medium was enriched and concentrated to a volume of 30 ml using a stirred ultrafiltration cell ( model 8200 ) with a 10 kda molecular weight cut - off membrane ( sartorius - stedim 14439 - 63 - d ). next , proteins were further concentrated by ultrafiltration in vivaspin ® 15r columns ( mwco 10 kda , vivaproducts ) to a volume of 5 ml . the centrifugation was performed at 4 , 000 rpm at 4 ° c . and the sample was mixed every 15 minutes to avoid precipitation . five μl of the sample was mixed with 20 μl of laemmli buffer , boiled for 5 minutes and loaded on a 12 . 5 % sds - page gel . the gel was stained with coomassie brilliant blue g250 overnight and later destained in 30 % meoh . the total sds - page gel lane was divided into 17 fractions . the gel pieces were destained by 3 × 20 - minute incubation cycles at 30 ° c . with 150 μl of a 50 % acetonitrile ( acn )/ 200 mm ( nh 4 ) 2 co 3 mixture . the proteins were digested in gel ( according to shevchenko et al ., 2007 ) by adding 8 μl of 0 . 002 μg / μl trypsin ( promega ) in 50 mm ( nh 4 ) 2 co 3 during overnight incubation in 37 ° c . the peptides were collected from the supernatant . the gel pieces were further extracted with 60 μl of 60 % acn / 0 . 1 % fa during a 20 - minute incubation at 30 ° c . after that , samples were vortexed for 3 minutes , shortly centrifuged and extraction was repeated with 20 μl of 60 % acn / 0 . 1 % fa . these extracts were pooled with the supernatant and the resulting peptide mixture was dried using vacuum centrifugation and dissolved in 15 μl of 2 % acn / 0 . 1 % fa . five μl of the extracted peptides were loaded on a zorbax ® 300sb - c18 analytical column 150 mm × 75 μm ( agilent ) connected to an agilent 1200 chromatographic system ( agilent , santa clara , calif ., usa ) coupled to an ltq - ftultra mass spectrometer ( thermo fisher scientific , waltham , mass . ), as described in de graaf et al . ( 2010 ). the separation was performed by reversed phase chromatography using a 50 - minute linear gradient ranging from 2 % buffer a to 80 % buffer b at a 300 nl / minute flow rate . the mobile phase buffer a was 99 . 9 % water with 0 . 1 % formic acid . mobile phase b was 99 . 9 % acetonitrile with 0 . 1 % formic acid . the lc eluent was directly coupled to a t riversa n anomate ® esi source ( advion , ithaca , n . y . ), working in the nanolc mode and being equipped with d - chips upon which a 1 . 55 - kv voltage was applied . the ft - icr mass analyzer acquired ms scans at 100 , 000 resolution during the lc separation . the three most intense precursor peptides for each ms scan were automatically selected and fragmented by the ltq ion trap mass analyzer . raw lc - ms / ms data received from the ft - icr ms measurements were analyzed using mascot daemon version 2 . 3 . 2 . to identify proteins , mascot version 2 . 3 . 01 searches were performed against the in - house available primary annotated c . bombicola genome containing 4617 genes , predicted from three contigs using the augustus algorithm , together with its decoy database created on the flight by mascot software . the ms / ms data from the 17 sds - page bands were merged into one single search file . the following search settings were applied : data were imported with the thermo finnigan lcq / deca raw filter , maximum two missed cleavages of trypsin were allowed , and an oxidation ( m ) was set up as variable modification . as instrument parameters , the esi - fticr instrument was selected with a possible ms / ms error tolerance of 0 . 5 da and peptides error tolerance of 10 ppm . all peptides with a significance threshold higher than 0 . 01 and an ion score cutoff lower than 30 were discarded . we accepted proteins with two or more peptide hits within the above criteria . the protein abundance , expressed as an empaivalue ( rappsilber et al ., 2002 ), was calculated by mascot using the number of observable peptides n obsd and the number of the observed peptides n obsbl for a parent ion : protein contents in molar and weight fraction percentages are described as : where m r is the molecular weight of the protein , and σempai is the summation of the empai values for all identified proteins . in the secreted protein fraction ( secretome ) from c . bombicola obtained after eight days of growth , 31 proteins were identified . between them , a putative lipase was found with a protein score of 14222 ( score cut off = 30 ) and seven unique peptides . in fig3 , these peptides are marked . in table 1 , detailed information about those peptides can be found , including mascot scores that indicate the quality of the identification . the mascot software automatically calculates the anempai factor for each protein . for the lipase , the calculated empai factor is 1 . 39 , which corresponds to 3 . 72 mol % and 3 . 17 weight %, respectively , of the protein content of the eight - day - old secretome . creation of a lactonase - negative c . bombicola strain for the production of acidic sophorolipids candida bombicola atcc 22214 was used as the parental or wild - type strain . candida bombicola pt36 , an ura3 autotrophic mutant , was derived from this parental strain and used to construct both the knock - out and overexpression strains . when sophorolipid production was intended , the medium described by lang et al . ( 2000 ) was used . 37 . 5 g / l rapeseed oil was added two days after inoculation . yeast cultures were incubated at 30 ° c . and 200 rpm for a total time of ten days . escherichia coli dh5α cells were used in all cloning experiments and were grown in luria - bertani ( lb ) medium 1 % trypton , 0 . 5 % yeast extract and 0 . 5 % sodium chloride ) supplemented with 100 mg / l ampicillin . liquid e . coli cultures were incubated at 37 ° c . and 200 rpm . bacterial plasmid dna was isolated with the qia prep ® spin mini prep ™ kit ( qiagen ®). all dna sequences were determined at lgc genomics ( berlin , germany ). c . bombicola cells were transformed by electroporation . transformants were selected on synthetic dextrose ( sd ) plates ( 0 . 67 % yeast nitrogen base without amino acids ( difco ) and 2 % glucose ). e . coli cells were transformed as described by inoue et al . ( 1990 ). a total fragment of 1944 by comprising the complete lactonase cds was amplified with the primers lip2for3 and lip2rev3 ( table 2 ) and cloned into the pgem - t ® vector ( promega ). the created vector of 4946 by was digested with mfei and nani , in this way deleting 282 by of the lactonase coding region . the candida bombicola ura3 autotrophic marker ( van bogaert et al ., 2008a ) was amplified with the primers ura3mfeifor and ura3narirev ( table 2 ), harboring the restriction sites for , respectively , mfei and nari in their 5 ′ extensions . the purified pcr fragment of 2064 bp was cut with mentioned restriction enzymes and ligated into the digested vector . the resulting vector of 6717 bp was used as a template to generate the lactonase knock - out cassette with the primers lip2for3 and lip2rev3 . the fragment of 3806 bp contains the ura3 marker with approximately 0 . 8 kb of the lactonase sequence on each site , required for homologues recombination at the lactonase locus . this linear fragment was used to transform candida bombicola pt36 . daily sophorolipid samples were extracted as follows : 3 ml of ethanol was added to 1 ml culture broth and shaken vigorously for 5 minutes . after centrifugation at 9 , 000 g for 5 minutes , the supernatant was collected . at the end of the incubation period , 3 volumes ethanol were added to the culture broth for total extraction of sophorolipids . cell debris was removed by centrifugation at 1500 g during 10 minutes . for further gravimetric analysis , the supernatans water - ethanol mixture of the total extraction was evaporated . two volumes of ethanol were added to dissolve the sophorolipids and the residual hydrophobic carbon source . the mixture was filtrated to remove the water - soluble compounds and was evaporated again . one volume of water was added and set at ph 7 , then 1 volume of hexane was added . after vigorous shaking , the mixture was allowed to separate . the different fractions were collected , evaporated and the mass was determined . the hexane phase will contain residual oil , while the water phase contains the sophorolipids . glucose concentration in the culture supernatans was determined by analysis with the 2700 select biochemistry analyzer ( ysi inc .). colony - forming units ( cfu ) were determined by plating decimal dilutions on agar plates with 10 % glucose , 1 % yeast extract and 0 . 1 % urea , which were incubated at 30 ° c . for three days . sophorolipid samples were analyzed by hplc on a varian prostar hplc system using a chromolith ® performance rp - 18e 100 - 4 . 6 mm column from merck kgaa at 30 ° c . and evaporative light scattering detection ( alltech ). a gradient of two eluents , a 0 . 5 % acetic acid aqueous solution and acetonitrile had to be used to separate the components . the gradient started at 5 % acetonitrile and linearly increased until 95 % in 40 minutes . the mixture was kept this way for 10 minutes and was then brought back to 5 % acetonitrile in 5 minutes . a flow rate of 1 ml / minute was applied . in order to be able to compare and quantify the different samples , dilutions of a standard were analyzed in parallel . liquid chromatography mass spectrometry ( lcms ) analysis was performed by intertek asg ( manchester , uk ) with a micromass ® quattro ultima ™ lims 1107 ( waters ). the detection range was set at m / z 100 to 1000 and the negative ion mode was applied . the same column and lc conditions as for the hplc analysis were used . the lactonase knock - out cassette was constructed as described in the materials and methods section . this linear fragment was used to transform the ura3 - negative candida bombicola pt36 strain . the genotype of the transformants was checked by yeast colony pcr with two primer pairs ( table 2 ). the first combination , kolip2for3 and ura3outbeginrev , verifies the upstream recombination event ; kolip2flankfor binds the genomic dna preceding the integration region and ura3outbeginrev binds the marker gene of the disruption cassette . the second pair checks the downstream part in the same way : kolip2flankrev binds the genomic region , whereas ura3outendfor binds the marker gene . several correct mutants were obtained . if the lactonase takes part in ring closure of the acidic sophorolipids , knocking out the gene should result in a reduction of the relative amount of lactonic sophorolipids . sophorolipid production of the knock - out was compared to the wild - type , both on medium with and without addition of rapeseed oil . cell growth and viability were not affected ; cfu for mutants were not significantly different from the wild - type and this was for the full production period . furthermore , the glucose consumption rate was comparable to the wild - type , indicating that sophorolipid synthesis is taking place ; a biochemical process consuming a lot of glucose . during the stationary phase and at the end of the cultivation time , sophorolipid samples were collected . surprisingly , single knock - out of the lactonase gene resulted in complete absence of any lactonic form in any sample and this was both in the supplemented and non - oil supplemented set - up ( fig4 a - 5b ). the experiments were repeated several times at different times and for the cultivations without addition of oil , between 20 and 30 g / l of sophorolipids were obtained , while , confirmed with the literature , the yield was higher when rapeseed oil was added . in this latter case , between 45 and 60 g / l were obtained . unexpectedly , the yields for the wild - type and the mutant were comparable . this is in contrast to previous manipulations of the sophorolipid biosynthetic pathway . indeed , a simple knock - out of the acetyltransferase gene , leading to the production of non - acetylated sophorolipids , resulted in a production of only 5 g / l of sophorolipids , even when rapeseed oil was added ( saerens et al ., 2011b ). furthermore , when aiming for the production of glucolipids by disabling the second glucosyltransferase of the sophorolipid biosynthetic pathway , a strong decrease of biosurfactant production was again observed ( saerens et al ., 2011a ). as can be seen in the chromatograms of fig5 a and 5b , free fatty acids derived from rapeseed oil are detected , both for the wild - type and the knock - out , meaning that the rapeseed oil triglycerides are still hydrolyzed even if an enzyme with a putative lipase function is disabled . this finding indicates that at least one other lipase enzyme is responsible for the hydrolysis of rapeseed oil triglycerides and that , consequently , rapeseed oil still can serve as a carbon source or hydrophobic substrate for sophorolipid production in the lactonase negative strain . creation of a lactonase overexpressing c . bombicola strain for the production of fully lactonized sophorolipids production of a sophorolipid mixture enriched in lactonic sophorolipid molecules is obtained by the usage of a sophorolipid - producing yeast strain as a host for overexpression of the c . bombicola lactonase gene . for c . bombicola atcc 22214 , for example , an overexpression cassette is created in which the lactonase gene is under control of the constitutive and highly active gki ( phosphoglycerate kinase ) promoter . these overexpression constructs are cloned into a vector already comprising the c . bombicola ura3 selection marker with up - and downstream sequences for recombination in the genome ( van bogaert et al ., 2008a ) and are subsequently used for transformation of a ura3 - deficient c . bombicola strain . expressing the lactonase in this way leads to a sophorolipid mixture remarkably enriched in the lactonic forms . 3 . 1 . 1 strains and culture conditions ; dna isolation and sequencing ; transformation . see sections 2 . 1 . 1 , 2 . 1 . 2 and 2 . 1 . 3 . other media promoting production of acidic sophorolipids such as a medium without addition of citrate were used as well . bioreactor experiments were carried out in a biostat ® b culture vessel ( sartorius - bbi systems ) with a maximum working volume of 1 . 5 to 3 l . temperature ( 30 ° c . ), ph ( 3 . 5 ), stirring rate ( 800 rpm ) and airflow rate ( 1 win ) were controlled by the biostat ® b control unit . 100 ml of an overnight grown shake flask culture was used to inoculate the fermentor . for maintaining ph at 3 . 5 , 5 n naoh was used . there was no correction for a too alkaline ph and fermentations started at ph 5 . 8 and were consequently allowed to drop spontaneously until 3 . 5 . feeding of the hydrophobic carbon source was started 48 hours after inoculation , and from then on , 25 g of rapeseed oil was added every 24 hours . additional glucose was added 150 hours after inoculation . for the wild - type fermentations , the stirring rate had to be lowered to 600 rpm after five days of cultivation to control foam formation as the addition of the hydrophobic carbon source no longer helped . the complete gki promotor sequence was amplified from genomic dna of candida bombicola using the primers p124_for_pgki_extinfuspei and p125_rev_pgki_extlipase . the lactonase gene sequence was amplified from genomic dna using primers p126_for_lipase_extpgki and p127_rev_termlac_extinfubamhi and both fragments were subsequently fused using fusion pcr . a vector ( pgem - t ® _cassette_yegfp ) containing the ura3 auxotrophic marker ( under control of its own terminator ), and up - and downstream sequences for homologous recombination , was cut with the enzymes spei and bamhi . the abovementioned linear fragment was subsequently inserted into the cut vector using the infusion advantage pcr cloning kit ( clontech ®). the resulting vector of 7896 bp is used as a template to generate the lactonase overexpression cassette with the primers p1_for_ura3v and p32_rev_cassette . the fragment of 4904 bp was used to transform the ura3 - candida bombicola pt36 strain and integration occurred at the ura3 locus . the resulting strain thus harbored two copies of the lactonase gene ; one under its own regulatory sequences unaltered in the genome and a second one under control of the strong constitutive gki promoter at the ura3 locus . correct integration of the cassette at the ura3 locus was controlled by performing colony pcr with primers p33_for_checkcassin and p125_rev_pgki_extlipase at the 5 ′ side of the insertion cassette and p35_rev_checkcassin_down and p126_for_lipase_extpgki at the 3 ′ side of the insertion cassette . cfu and glucose were determined as described in section 2 . 1 . 5 . samples ( 1 ml ) are withdrawn from the culture medium during cultivation and sophorolipids are extracted by addition of 440 μl ethyl acetate and 11 μl acidic acid . after vigorously shaking , the fractions are separated by centrifugation , the solvent fraction ( 300 μl ) is diluted with 700 μl ethanol and analyzed on hplc and evaporative light scattering detection . optical density ( od ) of cultures was measured at 600 nm using the jasco ® v 630 bio spectrophotometer ( jasco europe ). growth was also evaluated by determining the cell dry weight ( cdw ). three transformants were selected for further characterization and they all showed identical behavior as compared to the wild - type regarding growth and substrate consumption when cultivated on production medium . sophorolipid production was also followed up during cultivation and results are described below . as illustrated in the background art , citrate is described to influence lactonization . therefore , a modified production medium was used for cultivation of the wild - type and the obtained overexpression mutant . this medium did not contain citrate but was otherwise identical to the one described by lang . ph , glucose consumption and od are depicted in fig6 . the observed ph drop was equal for both the wild - type and the overexpression mutant so possible differences in sl composition cannot be attributed to ph effects . some differences in od values were observed , but this is caused by interference of sls and oil with the absorbance . log cfus were also determined before the cultures were stopped and these were equal to 8 . 31 and 8 . 41 log cfu / ml for the wild - type and the overexpression transformant , respectively . sl biosynthesis was also examined and hplc - elsd chromatograms are depicted in fig7 . overexpression of the lactonase gene clearly leads to lactonization of sophorolipids ( peak eluting at 28 . 2 minutes ( diacetylated lactonic ) and 24 . 4 minutes ( monoacetylated lactonic )) in the absence of citrate , which is not true for the wild - type , for which only acidic sophorolipids ( peak at 18 . 3 ) are produced . these results clearly prove that the action of citrate on the lacton / acidic ratio of sophorolipids is not a result of ph as was suggested by stüwer et al . ( 1987 ). for the wild - type , some kind of regulatory effect on the level of transcription must occur as expression of this gene from the strong , constitutive phosphoglycerate kinase promotor leads to the production of lactonic sophorolipids in the absence of citrate . total extractions suggested higher yields of sophorolipids for the overexpression mutant as compared to the wild - type . the total yield for the overexpression transformant was 35 g ± 1 . 3 g and 17 g ± 0 . 5 g for the wild - type , the remaining oil was equal to 0 . 1 g ± 0 . 0 g for the overexpression strain and 9 g ± 0 . 4 g for the wild - type . a second experiment was set up with the normal production medium optimized for production of lactonic sophorolipids . this was done to assess if overexpression of this already quite abundant enzyme responsible for lactonization would even lead to higher ratios of lactonic / acidic sophorolipids and possibly even higher yields of these diacetylated lactonic sophorolipids . two replicas of both the wild - type and the overexpression transformant were cultivated and samples were extracted with ethanol and ethylacetate as described in the materials and methods section . hplc - elsd chromatograms are depicted in fig8 . the peak eluting at 18 . 8 minutes corresponds to the non - acetylated , acidic sophorolipids , whereas the peak eluting at 28 . 3 minutes corresponds to the diacetylated lactonic sophorolipids . surprisingly , overexpression of the lactonase also leads to a very clear effect on sophorolipid composition on standard production medium as can be concluded when comparing fig8 , row a , with fig8 , row b . while acidic sls are still present in substantial amounts for the wild - type , these structures are not detected for the overexpression transformant . total yields for these experiments were subject to high variation and it was thus decided to perform a bioreactor experiment in which all parameters can be controlled to obtain more reliable results . a bioreactor with the wild - type and the lactonase overexpression mutant were run in parallel . the total sl yield of the successful wild - type cultivation accounted up to 188 . 74 g versus 213 . 42 g for the overexpression mutant . sl production was also visible earlier for the overexpression mutants and po 2 dropped a lot faster for these strains . the sls produced by the overexpression mutant were strongly enriched in the lactonic forms from the start , which confirms the results obtained in 3 . 2 . 2 . at the end of the fermentation , this effect was less pronounced as lactonase expression probably occurred a lot earlier during cultivation for the overexpression mutant . another interesting phenomenon was the absence of foam formation for the overexpression experiments . while the wild - type stirring speed had to be adjusted to 600 rpm after five days of cultivation to prevent foam excess caused by the presence of acidic sls , the overexpression strains could be left at the initial 800 rpm without a risk for too much foam formation . this could be an advantage for industrial applications , where foam formation can be a real burden . the lactonase of c . bombicola is produced extracellularly in p . pastoris . the recombinant protein possesses a his - tag that allows purification with a talon column and later with a sd200 column . the edman degradation and ms analysis were used to confirm the correctness of expressed protein sequence . genomic dna of candida bombicola atcc 22214 was used to pick up the mature form of the lactonase . the primers lacforvextsacli : cgtcgactgtatgagttgagt ( seq id no : 36 ) and lacrevextpsti : gctgcaggactccctttaggcc ( seq id no : 37 ) were used to create a pcr fragment with two additional restriction sites of psti and sali . after gel purification , the pcr product was subcloned into the pcr2 . 1 - topo vector ( invitrogen ®) and propagated in escherichia coli top10 cells ( invitrogen ®). after plasmid purification by chromatography ( qiagen ® midi kit ), the insert was cut out with psti and sali . the expression vector αppiczb ( invitrogen ®) was linearized with the same restriction enzymes and purified from the gel ( innuprep doublepure kit ). the insert was ligated to αppiczb using t4dna ligase and contract was transformed into one shot top10 electrocompetent e . coli . the best clone was purified with chromatography ( qiagen ®) and confirmed by multiple restriction digestion with xmnli + hindiii , ncoi and sfoi + ecorv . sequencing ( at beckman coulter genomics ) confirms the αppiczb_lac construct . the linearized αppiczb_lac construct obtained with saci was transformed into electrocompetent pichia pastoris nrrl - y - 11430 cells . the best expressing mutant of pichia pastoris nrrl - y - 11430 containing the βppiczb_lac construct was grown in 1 l of buffered - glycerol complex ( bmgy ) medium in two 2 - l flasks containing 500 ml medium , for 48 hours at 28 ° c ., 250 rpm . then , bmgy medium was replaced with buffered - methanol complex ( bmmy ) medium containing methanol instead of glycerol . the cell pellets were washed and dissolved in 1 l bmmy medium in sterile conditions . the protein expression in bmmy medium was carried for 48 hours at 28 ° c ., 250 rpm . every 12 hours , 1 % meoh was added for continuous stimulation of protein expression . finally , medium containing the expressed lactonase was separated from the cells by 10 minutes centrifugation at 4000 g . the collected medium ( 1 l ) was filtrated through a 0 . 22 μm bottle top filter ( corning ) and divided in ten dialysis membranes with 6 - 8 kda cut - off ( spectra por ). the dialysis was performed in 10 l of na 2 hpo 4 50 mm ; nacl 300 mm ph 7 . 5 buffer in 4 ° c . for 24 hours . next , the dialyzed medium was pooled , filtrated through a 0 . 22 μm bottle top filter ( corning ), and loaded on a talon superflow column ( ge healthcare ) with a bed volume of 20 ml connected to akta - purifier ( ge healthcare ) system and equilibrated with the same buffer as used for dialysis . after loading the medium , the column was washed with equilibration buffer containing 5 mm imidazole . the his - tag protein was eluted with buffer containing 200 mm imidazole . next , the protein was concentrated to 2 ml using vivaspin ® columns with a mw cut - off 10 kda ( sartorius ). the partially purified lactonase was injected into a superdex ® 200 column ( ge healthcare ) running in 20 mm tris ; 150 mm nacl ph 7 . 5 buffer . the fractions containing the lactonase were collected and stored in − 80 ° c . 20 μl of the concentrated lactonase fraction separated by the superdex ® 200 column , was further separated by 12 . 5 % sds - page . then , the protein was electroblotted on a pvdf membrane . n - terminal sequence determination was performed by automated edman degradation on a procise model 494 sequencer instrument , equipped with an on - line hplc system consisting of a 140c microgradient pump and a 785a programmable absorbance detector ( all from applied biosystems ). the analysis was performed with acid delivery in the gas phase . the sample from sds - page from section 4 . 1 . 2 . 1 was used for ms analysis . sample preparation and analysis are described in 1 . 1 . 3 ., 1 . 1 . 4 and 1 . 1 . 5 . the lactonase from c . bombicola was successfully expressed in p . pastoris . for efficient secretion , the original n - terminus was replaced by the α - factor secretion signal of saccharomyces cerevisiae as intrinsic for the αppiczb vector . this signal sequence is removed upon secretion by the p . pastoris cells . consequently , the recombinant protein differs from the natural one : the shorter n - terminus starts with the amino acids alanine and glycine and the c - terminus contains a his - tag ( see nucleic acid seq id no : 32 and amino acid seq id no : 33 ). fig6 represents an sds - page with the pooled lactonase fraction from sd 200 separation ( used for analysis in section 4 . 1 . 2 . 1 ). a single diffused band is visible at the height of 70 - 75 kda , which is higher from the predicted mw 45 kda of the lactonase . the shift of mass is probably due to the abundant glycosylation typical for the secreted proteins produced by pichia pastoris . both methods , the n - terminal sequencing and ms analysis , confirm the correctness of the recombinant lactonase . in edman degradation , the n - terminal sequence of the lactonase was aglplgytaapa ( seq id no : 38 ), as expected for the recombinant protein ( see seq id no : 33 ). sixteen unique peptides corresponding to the predicted sequence id no : 33 were identified by ms analysis ( table 4 ). production of the recombinant lactonase in pichia pastoris allows performance of a more detailed characterization of this enzyme . first , a mixture of acidic mono -, di - and non - acetylated sophorolipids ( sl ) was tested to confirm that the lactonase is responsible for the sl lactone formation . then , the different sl forms were analyzed separately to deeply investigate the enzyme behavior with those substrates . all those tests were performed at two different ph values ( 3 . 5 and 6 ). the lipase / esterase activity of lactonase was investigated . the colorimetric tests were based on the release of p - nitrophenol upon hydrolysis of three p - nitrophenyl derivatives with different chain lengths , being p - nitrophenyl acetate ( pnpa ) and p - nitrophenyl butyrate ( pnpb ) ( jung and park , 2008 ; lopes et al ., 2011 ). compounds used for enzymatic assay buffer preparation : natrium citrate , natrium hydroxide and hydrochloric acid , were purchased from sigma . different types of sl were obtained by production by c . bombicola strains . if required , specific purification steps or chemical treatments known by the person skilled in the art were applied in order to obtain a specific form . about 100 mm stock solution in water was prepared for the enzymatic assays . because most of the times a sl mixture was used , the average estimated molecular weight of 675 . 4 g / mol and a density of 1 . 05 g / ml were applied . candida antarctica lipase b was purchased from sigma . the substrates pnpa and pnpb , and sodium dodecyl sulphate , triton ® x - 100 , tris as well . the solvents acetonitrile and tetrahydrofuran were received from biosolve and riedel de haën , respectively . sodium chloride was purchased from merck . 5 . 1 . 2 . 1 enzymatic assay with mixture of mono -, di -, non - acetylated acidic sl 135 mg of dried sl was dissolved in 2 ml water to prepare a 100 mm stock solution used in the enzymatic assays . five different purified protein concentrations were prepared : 0 . 6 μg / ml ; 1 . 6 μg / ml ; 3 . 2 μg / ml ; 6 μg / ml ; 9 . 2 μg / ml ( stock concentration 60 μg / ml ). the protein was incubated with about 5 mm of a mixture of di -, mono -, and un - acetylated acidic sophorolipids ( obtained from the lactonase ko mutant ) in a total volume of 1 ml . to buffer the reaction at ph 3 . 5 and ph 6 , 50 mm c 6 h 5 na 3 o 7 was used in both cases . the assay was stopped after overnight incubation at 28 ° c . with rotation . for every condition , a blank reaction was prepared , where enzyme was replaced by the buffer in which enzyme was purified : 25 mm tris , 150 mm nacl , ph 7 . 5 . products from the reaction mixture were extracted with 440 μl ethylacetate and 11 μl acetic acid . from the solvent phase , 400 μl was recovered and analysis on hplc ( see section 2 . 1 . 6 ). the spectra from the sample and blank reaction were compared . 0 . 6 μg / ml purified protein was incubated with ˜ 5 mm of different sl preparations , i . e ., a mix of wild - type sl ; enriched lactone sl foam from a standard production ; pure , chemically prepared non - acetylated acidic sl and non - and mono - acetylated acidic sl produced by the ko lactonase mutant . the conditions for incubation and extraction are the same as described in 5 . 1 . 2 . 1 . for the enzymatic assay with p - nitrophenyl derivates , purified lactonase in 150 mm nacl , 25 mm tris , ph 7 . 5 was used . the concentrations of the working solution , 6 mg / ml , were prepared by vivaspin ® concentrators ( sartorius ), with a mwco of 5 kda . the enzyme concentration was checked using a nanodrop 2000 spectrophotometer ( isogen ). candida antarctica lipase b work solutions were derived from a 1 . 2 mg / ml stock solution in the same enzyme buffer that was used for lactonase . each of the p - nitrophenyl derivates were prepared in a different way due to their stability and solubility . a work solution of 1 mm p - nitrophenyl acetate ( ppna ) was freshly prepared in 5 % acn / 50 mm tris , ph 7 . 3 , just before use . for p - nitrophenyl butyrate ( pnpb ), a 1 mm work solution in 50 mm tris , ph 7 . 3 containing 0 . 2 % triton ® x - 100 and 0 . 43 m tetrahydrofuran ( thf ) was used . in a 96 - well microtiter plate , 200 μl of a substrate work solution was added to 40 μl of either enzyme ( sample ) or enzyme buffer ( blank ). from these wells , 200 μl was transferred to an empty well after mixing . in one scale - down experiment with lactonase , the final measured volume was only 120 spectrofotometric measurements were performed on a microplate reader , model 680xr ( bio - rad ), using a wavelength of 405 nm , in the time drive mode , performing 60 readings with intervals of 30 or 60 seconds at an incubation temperature of 25 ° c . ( in one experiment with lipase b , the interval between two readings was set to 15 seconds ). blanking for the background of the microtiter plate wells and / or yellowish color of the concentrated lactonase solution was done by endpoint measurements of the empty wells . only wells with the same background were used to compare samples with blanks . candida antarctica lipase b was used as a positive control for hydrolyses of all substrates . extracted products after every enzymatic assay with sl were analyzed on hplc . the chromatograms of the sample were compared with a blank experiment and new peaks were searched . fig1 and 11 present the results of the enzymatic assay at ph 3 . 5 and ph 6 with a mixture of mono -, di -, non - acetylated acidic sl . the black arrows mark the formed products ( 0 . 6 μg / ml lactonase ): peaks eluting at 26 . 047 minutes and 27 . 494 minutes , which are not present in the blank . those peaks correspond to the di - acetylated lactonic form of the sl with fatty acids c16 and c18 : 1 , as determined by lc - ms ( 2 . 1 . 6 ). the same pattern was visible in assay with five different purified protein concentrations : 0 . 6 μg / ml ; 1 . 6 μg / ml ; 3 . 2 μg / ml ; 6 μg / ml ; and 9 . 2 μg / ml . assays were set up with non - acetylated acidic sl and mixture of non - and mono - acetylated acidic sl at ph 3 . 5 and ph 6 . in both cases , no sl lactone foam was observed . after enzymatic assays with sl mixture from wt and the enriched lactone sl mixture , no difference in the pattern between blanks and the samples were detected . per tested p - nitrophenyl derivative , a time drive absorption spectrum using a wavelength of 405 nm , at 25 ° c ., was obtained . fig1 a and 12b show the spectra for the 1 mg / ml lactonase in combination with p - nitrophenyl butyrate , in a final test volume set - up of , respectively , 120 and 200 μl . a slow release of p - nitrophenyl can be established , which points to some hydrolysis activity of the enzyme on the c 4 substrate . in the graph presented in fig1 , the λ405 absorption was recorded during 60 minutes for a reaction mixture containing the concentrated lactonase and p - nitrophenyl acetate . the data are corrected for background absorbance of the multi - well plate and the 1 mg / ml enzyme solution . although natural hydrolysis causes release of p - nitrophenyl in the blank , the reaction rate clearly increases in the presence of the enzyme . furthermore , it is obvious that the hydrolysis velocity , catalyzed by the lactonase , is higher in combination with the acetate ( c 2 ) than with the butyrate ( c 4 ) substrate . the enzymatic assays with sophorolipids demonstrate that the lactonase is responsible for lactonization of the sophorolipids . after analysis of hplc chromatograms from the enzymatic assay with different sl forms , it can be concluded that di - acetylated acidic sl are the best substrates for the lactonase . the present disclosure shows that to form a lactonic sl , preferably a low concentration of the enzyme is needed : 0 . 6 μg / ml . moreover , sl lactone was produced in the assays at both ph 3 . 5 and ph 6 , which indicates that the enzyme is active in a wide ph range . the αppiczb construct ( invitrogen ®) with the lactonase ( sequence id no : 32 ) described in section 4 . 1 . 1 . 1 was sent to genscript where site - 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