Patent Application: US-77648497-A

Abstract:
aminopeptidases originating from aspergilli niger are disclosed , which can be produced in a fermentation broth or a liquid concentrate thereof substantially free of endoprotease . such aminopeptidases may be advantageously employed in the food industry , e . g . in the preparation of bread doughs or cheese .

Description:
phenylalanine paranitroanilid was dissolved in 7 . 5 mm hcl at a concentration of 0 . 9 mm . 1 ml of that substrate solution was mixed with 1 . 5 ml 0 . 1m phosphate buffer ph 7 . 2 . at t = 0 , 0 . 5 ml enzyme was introduced and left for reaction at 20 ° c . 1 ml 1n hcl was added 15 minutes later . a blank was run with 1n hcl being introduced at t = 0 . optical density was determined for the blank ( od blank ) and for the assay ( od assay ) at 400 nm . activity was calculated as follows : ## equ1 ## 2 -- leucine - aminopeptidase ( leu - ap ) leucine paranitroanilid was dissolved in water at a concentration of 9 mm . 1 ml of that substrate solution was mixed with 1 . 5 ml 0 . 1m phosphate buffer ph 7 . 2 . at t = 0 , 0 . 5 ml enzyme was introduced and left for reaction at 20 ° c . 1 ml 1n hcl was added 15 minutes later . a blank was run with 1n hcl being introduced at t = 0 . optical density was determined for the blank ( od blank ) and for the assay ( od assay ) at 400 nm . activity was calculated as follows : ## equ2 ## 3 -- valine - aminopeptidase ( val - ap ) valine paranitroanilid was dissolved in water at a concentration of 9 mm . 1 ml of that substrate solution was mixed with 1 . 5 ml 0 . 1m phosphate buffer ph 7 . 2 . at t = 0 , 0 . 5 ml enzyme was introduced and left for reaction at 20 ° c . 1 ml 1n hcl was added 15 minutes later . a blank was run with 1n hcl being introduced at t = 0 . optical density was determined for the blank ( od blank ) and for the assay ( od assay ) at 400 nm . activity was calculated as follows : ## equ3 ## 4 -- endoprotease ( pu ) this activity is measured by the hydrolysis of casein at ph 6 . 0 , 4 ° c . for 1 h . one pu is the amount of enzyme needed to liberate the equivalent of 1 μmole tyrosine per minute after precipitation of the remaining proteins with trichloracetic acid . 200 aspergillus niger strains , isolated from different sources or obtained from culture collections , were grown in a medium containing 15 g / l potato flour , 20 g / l bactopeptone , 7 g / l yeast extract , 4 g / l potassium dihydrogenphosphate , 0 . 5 g / l magnesium sulfate , 0 . 5 g / l calcium chloride , 0 . 5 g / l zinc chloride . ph was 4 . 8 . after 24 h . preculture at 240 rpm 30 ° c . and 96 h culture at 275 rpm 30 ° c ., supernatants were collected and assayed for leucine -, phenylalanine - and valine - aminopeptidase activity as described above . several aspergillus niger strains showed high production potentials for at least one of these enzymatic activities , as shown in table 1 ( each value is a mean value from four individual results ): table 1______________________________________ aminopeptidase activities instrain supernatants endopeptidasenumber leu - ap / 1 phe - ap / 1 val - ap / 1 pu / ml______________________________________1053 25 170 32 & lt ; 0 . 11085 23 135 48 0 . 11103 37 285 40 0 . 11108 60 435 29 0 . 11444 40 192 50 0 . 11497 25 105 75 0 . 11502 16 44 63 0 . 1______________________________________ amongst the above strains , strains 1108 and 1502 were obtained from a culture collection and were deposited under the accession numbers nrrl 3112 and cbs 115 . 39 , respectively , strain nrrl 3112 has been used for the production of amyloglucosidase , α - amylase and glucoamylase . strains cbs 115 . 39 has been used for the production of amylase or lipase . some strains from the screening described in example 1 have been fermented in laboratory fermentors ( 10 liters ). results obtained with strain 1502 are presented in this example . spores of aspergillus niger strain no . 1502 were collected on pda - plates after 7 - 10 days of incubation at 30 ° c . an inoculum step was performed in a shake flask in a medium composed of glucose ( 20 g / l ) and corn steep ( 20 g / l ) at ph 4 . 8 over 24 h . the main fermentation was performed according to a batch process . the following nutrients were used : 100 g / l maltodextrins , 40 g / l soy bean flour , 40 g / l hydrolysed casein , 5 g / l corn steep , 2 g / l gelatin , 2 g / l potassium dihydrogenphosphate , 1 . 3 g / l sodium nitrate , 1 g / l ammonium chloride , 0 . 01 g / l iron sulfate and 0 . 5 g / l antifoaming agent . all nutrients were firstly mixed together except the maltodextrins . ph was adjusted to 4 . 8 ± 0 . 1 . the fermentor was then sterilized at 125 ° c . for 40 minutes . the maltodextrin solution was sterilized separately and added to the sterile but cooled fermentation medium . the main fermentation was run in a laboratory fermentor which was filled with 6 liters of the medium described above and inoculated with the inoculum flask . stirring and air provision were adjusted to maintain the dissolved oxygen concentration as high as possible . the temperature was maintained at 30 ° c . the fermentation was stopped when all the nutrients had been consumed , i . e . after about 130 hours . the fermentation broth was filtered to remove all microorganisms . aminopeptidase and endoprotease activities were measured in the filtrate : uf concentration was then performed to formulate liquid aminopeptidase , glycerol ( 50 %) being the stabilizing agent . the resulting solution called ` peptidase l2 ` had the following activities : these results show that the selected aspergillus niger strain grown under our selected conditions produces aminopeptidases without substantial amounts of endoprotease . leu - ap and phe - ap activities were determined in peptidase l2 ( see example 2 ) using different buffers to screen a ph range from 2 . 5 to 9 . 0 . the ph profile of leucine - aminopeptidase from aspergillus niger is shown in fig1 . the ph profile of phenylalanine - aminopeptidase from aspergillus niger is shown in fig2 . the figures show that leu - ap is active in the ph range from 5 to 8 . 5 , whereas phe - ap is active in the ph range from 5 . 5 to 9 which is similar to aminopeptidases from other aspergillus species . leu - ap and phe - ap activities were determined in peptidase l2 ( see example 2 ) using different incubation temperatures to screen a temperature range from 5 to 70 ° c . the resulting temperature profiles are shown in fig3 . the results show that each enzyme has a different optimal temperature , i . e . 50 ° c . for leu - ap and 60 ° c . for phe - ap . normal cheese milk was inoculated with starter cultures and renneting was executed with an average dosage of animal rennet ( 1 : 15 . 000 mcu ; control 1 ) to the first experimental batch of milk 25 phe - ap units per thousand liters of milk were added . in a second trial , the animal rennet was substituted by microbial rennet ; the acid protease from mucor miehei . the experimental lot contained again 25 phe - ap units of aminopeptidase per 1000 liters of milk . a third experiment was performed , according to experiment 1 , in which the aminopeptidase was substituded with a commercial enzyme preparation derived from a . oryzea . this preparation consists of an endopeptidase / exopeptidase activity . cheese making parameters were maintained conform the procedure applied for semi hard cheese for all four cheese lots . after the subsequent brining of the cheese the ripening was performed at 8 ° c . related to experiment 1 , during the course of maturation , a difference was noted in terms of flavour and aroma development between experimental cheeses and control cheese to such an extent that the experimental cheese had obtained most of their required organoleptical properties after three ( 3 ) weeks whereas the control cheeses had obtained a similar qualification after six ( 6 ) weeks . the level of free amino acids after three weeks of maturation was shown to be twice as high in the experimental cheese ; after six weeks of ripening the levels were comparable again . this suggests that the product is ready for sale three weeks earlier without decreasing the keeping quality of the cheese . in this experiment we showed that the ripening can be accelerated by the initial increase of the liberation of amino acids whereas no overripening has been observed . for experiment 2 , the organoleptics of the cheeses differed to the extent that the bland cheese flavour with a slight tendency to bitterness of the control cheese was overcome in the experimental cheeses in the presence of aminopeptidase . the texture of the cheese was found to be somewhat smoother as well . the results of experiment 3 showed both a higher level of free amino acids and at the same time a higher bitter score in the organoleptic evaluation , suggesting that the endopeptidase activity enhances bitterness . normal cheese milk was inoculated with starter cultures and coagulant was added ( acid protease from endothia parasitrica ). to the experimental lot of milk an enzyme mixture consisting of 9000 units endoprotease ( from neutral protease b 500 ) and 15 phe - ap units of aminopeptidase , calculated per 1000 liters of milk , were added . cheesemaking parameters were kept conform the recipe during the rest of the experiment . after brining the cheeses were pre - incubated at 13 ° c . for 20 days to prepare the texture of curd and subsequently incubated at 19 ° c . for another 35 days in order to stimulate propionic acid fermentation . the results surprisingly showed absence of bitterness , a well known defect of the utilisation of this neutral protease in cheesemaking , absence of holes ( eyes ) but with good and typical taste and flavour development , a good gratable cheese with excellent melting properties at a lower dry matter content of the cheese ; 60 . 5 % in the control versus 57 . 8 in the experimental cheese . a repetition of this trial was performed in which the coagulant was substituted with fromase ® and in which the pre - incubation at 13 ° c . lasted 10 days and the incubation at 19 ° c . lasted 20 days . the total ripening time was halved . the results of this trial were largely comparable with the first one . 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