Optimization Of Inulinase Production From Yeast Biology Essay
Inulinase or I?-fructan fructanohydrolase hydrolyzes inulin, a polyose of I?- ( 2,1 ) -linked fructose residues attached to a terminal glucose molecule, to fructose and fructo-oligosaccharides, both of which are of import ingredients in nutrient and pharmaceutical industry ( Vandamme and Derycke, 1983 ) . It is chiefly used in confectionery industry where fruit sugar is preferred over sucrose because it is sweeter and does non easy crystallise ( Rubio and Navarro, 2006 ) . Inulinases are encountered in workss and many micro-organisms. Microorganisms are the best beginning for commercial production of inulinases because of their easy cultivation and high outputs of the enzyme. It has been found that the micro-organisms which can bring forth high degree of inulinases include Aspergillus spp. , Penicilium spp. , Arthrobacter spp. , Bacillus spp, Clostridium spp, Pseudomonas spp, Arthrobacter spp, Staphylococcus spp, Xanthomonas spp, Kluyveromyces spp, Cryptococcus spp, Pichia spp, Sporotrichum spp, and Candida spp ( Chi et al. , 2009 ) . It besides has been confirmed that barm strains can bring forth more inulinase than fungous and bacterial strains. Among the barms, Pichia sp. , two strains of Kluyveromyces fragilis, Cryptococcus aureus, and Kluyveromyces marxianus have high potency for bring forthing commercially acceptable outputs of the enzyme ( Gong et al. , 2007 ; Sheng et al. , 2008b ) . As many enzymes of industrial significance are regulated by medium composings and agitation status, the survey of this ordinance is of import in the commercial production of such enzymes. Optimization of agitation status and media composings to hold a balanced proportion is really of import to acquire optimal microbic growing and enzyme output.
Major betterments in the productiveness of a agitation procedure can be achieved by modifying the parametric quantities like physicochemical and nutritionary parametric quantities, to which being are exposed. The increasing potency of inulinase applications prompts testing for new inulinase bring forthing micro-organisms that can run into the conditions favourable for industrial applications. Therefore, the present survey was undertaken to research the inulinase production ability of a freshly isolated strain of Kluyveromyces marxianus YS-1.
To analyze the optimal conditions, such as C beginning, N beginning,
different hint component, wetting agent, pH, temperature, inoculums age, inoculants size and incubation clip for a freshly isolated strain of Kluyveromyces marxianus YS-1.
Inulin consists of additive I?-2,1-linked polyfructose ironss exposing a terminal glucose unit ( Vandamme and Derycke, 1983 ) . The fructose units in this mixture of additive fructose polymers and oligomers are each linked by I? -2,1 bonds. A glucose molecule typically resides at the terminal of each fruit sugar concatenation and is linked by an I±-1,2 bond, as in saccharose ( Molina et al. , 2005 ) . Fructans serve as storage polymers in many members of the Compositae such as Cichorium intybus ( chicory ) , Inula Helenium ( Inula helenium ) , Taraxacum officinalis ( blowball ) , and Helianthus tuberosus ( Jerusalem artichoke ) . All fructans found in the magnoliopsids, every bit good as some liliopsids, are of this type. By comparing, fructans composed predominately of additive fructose units bound by a I? ( 2 6 ) glycosidic bond are typically levans that are produced by many dirt and unwritten bacteriums, barms and Fungis. The molecular expression of inulin is GFn, with G being a terminal glucosyl unit, F stand foring the fructosyl units and “ n ” stand foring the figure of fructosyl units. The basic GF2 trimer in inulin and the shortest fructan of the inulin type is 1-kestose. The same bonds link the resulting fructosyl units, i.e. I? ( 2 cubic decimeter ) as that in 1-kestose, Figure 1. Short concatenation fractions of fructooligosaccharides such as 1-kestose, the major GF, compound in chicory roots or Jerusalem Cynara scolymus, and neokestose in onion do non differ analytically ( Van Loo et al. , 1995 ) . Further, fructan ironss linked to either of these of course happening trisaccharides have the I? ( 2 1 ) constellation, connoting that with the exclusion of one glycosidic linkage within the basic trisaccharide there is no difference between a fructan molecule based on 1-kestose or neokestose ( Crow, 2000 ) .
[ Fm ]
[ GFn ]
[ GF ]
Figure 1 Chemical Structure of saccharose, inulin and oligofructose
Beginning: Crow ( 2000 )
Inulin is a mixture of oligomers and polymers of fructose holding changing grades of polymerisation ( DP ) , but typically holding a DP scope from three ( matching to GF2 ) to about 60 with a modal concatenation length of about nine. Native inulin from Compositae frequently is made up of about 1 – 2 per centum I? ( 2 6 ) ramification with short side-chains ( Van Loo et al. , 1995 ) . In add-on to holding predominately additive ironss of the GFn-type, native inulin extracted from fresh chicory roots besides has been shown to incorporate about 1- 1.5 % ( Fn ) compounds on dry solids, those composed of homopolymers of fructose bound by a I? ( 2 1 ) linkage ( Van Loo et al. , 1995 ) . The n represents the figure of fructosyl medieties in the homopolymers. Both GFn and Fm have really similar physicochernical belongingss except that Fm type merchandises are cut downing, due to the presence of a cut downing fructose-end group, whereas GFn compounds are non. An apprehension of different footings used to depict fructose-containing polymers is of import as more commercial merchandises become available. Oligofructose was introduced as a equivalent word for fructo-oligosaccharides ( FOS ) . The oligofructose merchandise is a partial enzymatic hydrozlyate of inulin incorporating predominately molecules of the Fm-type ( homopolymers of fructose bound by a I? ( 2 1 ) glycosidic linkage holding no terminal glucose ) , Figure 1. Oligofructose has been defined by the IUB-IUPAC Joint Commission on Biochemical Nomenclature and the AOAC as fructose oligosaccharides incorporating 2-10 monosaccharose residues connected by glycosidic linkages ( Niness, 1999 ) . As antecedently stated, inulin, as extracted from Compositae root, is a polydisperse fructan with concatenation lengths runing from 2 to 60 units with a average DP of A± 9. Commercial chicory inulin is composed of about 2 % monosaccharoses, 5 % disaccharides and 93 % inulin.
Neosugar is a FOS mixture of kestose ( n=2 ) , nystose ( n=3 ) and IF-B-fructofuranosyl nystose ( n=4 ) . Basically these are sucrose molecules to which one to three extra fructose units have been added. The development and isolation of neosugar was foremost reported in the Nipponese literature in 1983 ( Oku et al. , 1984 ) . Neosugar can be isolated from brans of triticale, wheat and rye or unnaturally by the action of the fungous enzyme I?-fructofuranisidase on saccharose, the enzyme being a merchandise of the fungus Aspergillus Niger ( Fishbein et al. , 1988 ) . Using the most widely available and recognized terminology, all FOS and inulins are fructans. Those inulin molecules holding a grade of polymerisation of & lt ; 10 fructose units by and large are considered to stand for FOS.
Fructooligosaccharides ( FOS ) have been accepted as functional sweetenings similar to other microbic oligosaccharides. Recent involvement in the procedure development for the production of Fructooligosaccharides has concentrated on high content commercial merchandises. Inulin, being a polyfructan, has been widely investigated as a utile beginning for production of ultra- high fruit sugar sirup. However, the attempt to use inulin for production of functional sweetenings is a more recent attack ( Cho et al. , 2001 ) .
Inulin is hydrolyzed by enzyme known as inulinases. The inulinases are classified among the hydrolyzes and mark on the I?-2,1-linked of inulin and hydrolyse it into fructose and glucose. They can be divided into exoinulinases and endoinulinase. The exoinulinases catalyze remotion of the terminus fruit sugar residues from the non-reducing terminal of the inulin molecule while the endoinulinases hydrolyze the internal linkages in inulin to give inulotriose, inulotetraose, and inulopentaose.
Type of inulinases
Endo-inulinases are specific for inulin. It hydrolyzes inulin by interrupting bonds between fructose units that are located off from the terminals of the polymer web, to bring forth oligosaccharide.
Exo-inulinases split terminal fructose units in saccharose, rafffinose and inulin to emancipate the fruit sugar.
Figure 2 Mechanism of inulinase
Beginning: Worawuthiyanun ( 2005 )
Production of inulinase
Inulinase is found in filiform Fungis, barms, bacteriums. The production of inulinases is affected by type of the being medium constituents and procedure parametric quantities used for agitation.
Pichia guilliermondii strain 1 ( the aggregation figure: 2E00048 at
Marine Microorganisms Culture Collection of China ) , isolated from the surface of a marine alga, was found to release a big sum of inulinase into the medium ( Gong et al. , 2007 ) . Under the optimum conditions, over 61.5A± 0.4 U/ml of inulinase activity is produced within 48 H of agitation in the shaking flasks. The consequences indicate that 2.0 % ( w/v ) of added NaCl is the most suited for the inulinase production by the Marine barm strain 1 ( Gong et al. , 2007 ) . Particularly when the Marine barm strain is grown in the medium prepared with saltwater, the inulinase activity reaches the highest activity ( 61.5A±0.4 U/ml ) .
In order to insulate the inulinase overproducers of the P. guilliermondii
strain 1, its monoploid cells were treated by utilizing UV visible radiation and LiCl. One mutation ( M-30 ) that produces 115A±1.1 U/ml of inulinase activity was obtained ( Guo et al. , 2008 ) . It found that glucose repression on the inulinase production by the mutant M- 30 was relieved in some grade compared to that by its parent strain when added glucose concentration was more than 20.0 g/l ( Yu et al. , 2008 ) .
Response surface methodological analysis ( RSM ) was used to optimise the
medium composings and cultivation conditions for the inulinase production by the mutant M-30 in the submersed agitation ( Yu et al. , 2008 ) . After the optimisation, 127.7A±0.6 U/ml of inulinase activity is reached in the liquid civilization of the mutant M-30 whereas the predicted maximal inulinase activity of 129.8 U/ml is derived from RSM arrested development. Under the same conditions, its parent strain merely produces 48.1A±0.4 U/ml of inulinase activity. This is the highest inulinase activity produced by the barm strains reported so far. The consequences demonstrate that the inulinase production can be greatly improved by the familial alteration of the natural manufacturers.
RSM was besides used to optimise the medium composings and
cultivation conditions for the inulinase production by the inulinase overproducer ( the mutation M-30 ) from P. guilliermondii strain 1 in solid-state agitation ( SSF ) ( Guo et al. , 2008 ) . Under the optimized conditions, 455.9A± 1.2 U/g of dry substrate ( soman ) of inulinase activity is reached in the solid-state agitation civilization of the mutant M-30 whereas the predicted maximal inulinase activity of 459.2 U/gds is derived from RSM arrested development. Under the same conditions, its parent strain merely produces 291.0A± 0.7 U/gds of inulinase activity. This besides is the highest inulinase activity in the civilization of solid-state agitation produced by the barm strains reported so far.
The Marine barm strain C. aureus G7a ( the aggregation figure:
2E00002 at Marine Microorganisms Culture Collection of China ) isolated from deposit of South China Sea was found to release a big sum of inulinase into the medium ( Sheng et al. , 2007 ) . Under the optimum conditions, over 85.0A±1.1 U/ml of inulinase activity is produced within 42 H of agitation at shake flask degree. It is worthy to detect that 4.0 % ( w/v ) of added NaCl and 0.6 % ( w/v ) of added MgCl2A·6H2O is the most suited for the inulinase production ( 84.1A±0.6 U/ml ) by the Marine barm ( Sheng et al. , 2007 ) . The optimisation of procedure parametric quantities for the high inulinase production by the Marine barm strain C. aureus G7a in SSF is carried out utilizing RSM ( Sheng et al. , 2008a ) . Under the optimized conditions, 420.9A±1.3 U/g of dry substrate of inulinase activity is reached in the solid-state agitation civilization of strain G7a within 120 Hs whereas the predicted maximal inulinase activity of 436.2 U/g of dry weight is derived from RSM arrested development.
Two strains of K. marxianus ( A1 and A2 ) isolated from “ aguamiel ”
( agave sap ) and one strain of Kluyveromyces lactis volt-ampere. lactis ( P7 ) isolated from “ pulque ” ( its fermented merchandise ) show good inulinase manufacturers ( less than 32 U/ml ) in the liquid medium incorporating different concentrations of inulin and they have low susceptibleness to catabolic repression ( Cruz-Guerrero et al. , 2006 ) .
It has been shown that the inulinase production by K. marxianus
ATCC 16045 is strongly influenced by blending conditions in the liquid medium. The disc impeller at 450 revolutions per minute and aeration at 1.0 volume/volume per minute led to an activity of 121 U/ml, while the pitched blade was shown to be the best impeller for this procedure, taking to the best production. The maximal shear emphasis for the inulinase production was about 0.22 Pa ( Silva-Santisteban and Filho, 2005 ) .
The maximal inulinase activity of 59.5 U/ml is produced by
Kluyveromyces sp. Y-85 within 24 H of agitation when infusion of Jerusalem Cynara scolymus, urea, beef infusion, and maize steep spirits concentrations in the liquid medium are 8.0 % , 2.0 % , 0.2 % , and 4.0 % , severally ( Wei et al. , 1998 ) . The agitation in 15-l fermentor and scaling-up in 1,000-l tower fermentor were carried out and the inulinase activity in the scaling-up is 68.9 U/ml ( Wei et al. , 1999b ) . After the cells of Kluyveromyces sp. Y- 85 were treated by ethyl methane sulfonate, one mutation named Kluyveromyces sp.Y-85 K6 which is immune to catabolite repression of glucose was obtained ( Wei et al. , 1999a ) . Unfortunately, no farther work on the mutation has been reported since so.
Maximal inulinase production ( 55.4 U/ml ) by a freshly isolated strain
of K. marxianus YS-1 was obtained at an agitation rate of 200 revolutions per minute and aeration of 0.75 volume/ volume per minute in a moved armored combat vehicle reactor ( 1.5 cubic decimeter ) with a agitation clip of 60 H ( Singh et al. , 2007 ) . In another survey, inulinase activity of 50.2 U/ml was obtained from the same barm strain under agitation ( 200 revolutions per minute ) and aeration ( 0.75 volume/volume per minute ) at 30A°C after 60 H of agitation in 1.5 cubic decimeter of fermentor when natural inulin ( 4.0 % ) from root tubers of Asparagus officinalis was used as the production medium. Inulinase output in the bioreactor is about six times higher than the basal medium used ab initio in shingle flask ( Singh and Bhermi, 2008 ) .
It was found that the barm K. marxianus volt-ampere. bulgaricu produces big
sums of extracellular inulinase activity when grown on inulin, sucrose, fructose, and glucose as C beginnings. The degrees of inulinase in sucrose-limited chemostat civilizations are strongly dependent on the dilution rate. When the dilution rate D is 0.05 per hr, a upper limit of 107 U/ml is obtained, bespeaking that the inulinase production by this barm strain is regulated by the residuary sugar in the uninterrupted civilizations ( Kushi et al. , 2000 ) .
When K. marxianus NRRL Y-7571 is grown in the civilization incorporating
sugar cane bagasse as support and C beginning and maize steep spirits as nitrogen addendum, the extracellular inulinase concentration reaches 391.9 U/g of dry fermented bagasse ( Bender et al. , 2006 ) . The usage of soya bean bran decreases the clip to make the maximal activity from 96 to 24 H and the maximal productiveness achieved K. marxianus NRRL Y-7571 is 8.87 U/gds per hr ( Mazutti et al. , 2006 ) . The mean inulinase activity produced by a freshly isolated strain Kluyveromyces S120 in SSF is 409.8 U/g initial dry substrate in the optimum medium ( Chen et al. , 2007 ) .
The inulinolytic Fungi Penicillium sp. TN-88 showed a high
endoinulinase productiveness of 9.9 U/ml in the civilization filtrate when grown in a liquid medium incorporating inulin as the C beginning at 30A°C for 4 yearss ( Nakamura et al. ,
1997 ) .
The fungus Aspergillus Niger NK-126 shows good growing on a
medium incorporating 40 % ( v/v ) of blowball pat root infusion composed of 50-g tap roots blended with 200-ml H2O and 2 % barm extract medium and produces 55 U/ml of inulinase activity in 96 H at 30A°C and 150 revolutions per minute during the liquid agitation. The consequences besides suggest that the blowball pat root infusion in the liquid medium induces the endoinulinase synthesis in A. niger NK-126 ( Kango, 2008 ) .
Although it has been reported that barms can bring forth more inulinase than
bacteriums and filiform Fungis, Streptomyces sp. GNDU 1 was still used to bring forth high degrees of extracellular inulinase ( 0.552 U/ml ) after 24 H at pH 7.5, temperature 46A°C, in the presence of 1.0 % inulin in the liquid medium ( Gill et al. , 2006 ) .
Solid-state agitation for the synthesis of inulinase from the
bacteria Staphylococcus sp. was besides carried out utilizing the medium incorporating wheat bran, rice bran, coconut oil bar, and maize flour, separately or in combinations. Under the optimized conditions, the extracellular inulinase concentration reaches a extremum in 48 H with Staphylococcus sp. ( 107.6 U of inulinase per gm prohibitionist fermented substrate ) while the barm K. marxianus ATCC 52466 produces the highest degree of the extracellular inulinase in 72 H ( 122.9 U/gds ) . The consequences once more demonstrate that the barms can bring forth more inulinase than bacteriums ( Selvakumar and Pandey, 1999 ) .
Table 1 summarizes the inulinase micro-organism and their inulinase
activity. It can be clearly seen from the consequences in Table 1 that most of the inulinase micro-organism that are being used are barms, particularly the strains of Kluyveromyces, Cryptococcus, and Pichia. They besides produce much higher inulinase activity than any other micro-organisms.
Table 1 Inulinase micro-organisms and Inulinase activity
Type of cultivation