High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase (original) (raw)
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Kluyveromyces marxianus CDBB-L-278: A wild inulinase hyperproducing strain
Journal of Fermentation and Bioengineering, 1995
Kluyveromyces marxianus CDBB-L-278 is an inulinase hyperproducing strain. It was able to grow in a medium containing inulin as the unique carbon source in the presence of 2-deoxyglucose. It produced up to 3.3 times the activity of the control strain K. marxianus NCYC-1429 in an inulin medium, and 3.6 times in a medium with glycerol as the sole carbon source. Although the strain CDBB-L-278 was able to produce inulinase in the presence of 2-deoxyglucose, it was demonstrated that it is not a de-repressed strain since enzyme production was reduced when the concentration of glucose or fructose was increased in the medium. Since inulinase was produced in a glycerol medium without an inducer, it can be considered that the enzyme production was partially constitutive in K. marxianus CDBB-L-278 as well as strain NCYC-1429. The inulinase from K. marxianus CDBB-L-278 was characterized. It had a higher affinity for inulin than for sucrose. Temperature and pH profiles were different for both of these two substrates. The enzyme was stable to high temperatures, with a half-life of 180 min at 50°C.
Agave syrup as a substrate for inulinase production by Kluyveromyces marxianus NRRL Y-7571
Acta Scientiarum. Biological Sciences, 2016
The factorial planning was used to plan and optimize inulinase production by the yeast Kluyveromyces marxianus NRRL Y-7571. The experiments were conducted using a Central Composite Design (CCD) 22, at different concentrations of agave syrup (3.6 to 6.4%) and yeast extract (2.2 to 3.0%). After 96 hours of fermentation, the best condition for the inulinase production was 5% agave syrup and 2.5% yeast extract, which yielded an average of 129.21 U mL-1 of inulinase. Partial characterization of the crude enzyme showed that the optimal pH and temperature were 4.0 and 60°C, respectively. The enzyme showed thermal stability at 55°C for 4 hours.
Production of inulinase from Kluyveromyces marxianus using Dahlia tuber extract
Brazilian Journal of Microbiology, 2012
Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL-1) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL-1). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL-1) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C-sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL-1) and peptone (13.8 nkat mL-1). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.
1988
From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight-' at D = 0.1 h-l to 2 U mg of cell dry weight1 l at D = 0.8 h-'. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/ repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.
Bioresource technology, 2007
A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl 2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30°C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm 2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.
Inulinase Production by Kluyveromyces marxianus NRRL Y-7571 Using Solid State Fermentation
Applied Biochemistry and Biotechnology, 2006
Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructooligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.
Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation
Applied Biochemistry and Biotechnology, 2006
Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.
Optimisation of inulinase production by Kluyveromyces bulgaricus
Web Science, 2002
Present work is based on observation of effects of pH and temperature of fermentation on the production of microbial enzyme inulinase by Kluyveromyces bulgaricus (former Kluyveromyces marxianus). Inulinase hydrolyses inulin, an oligosaccharide which can be isolated from plants such as Jerusalem artichoke, chicory or dahlia, into pure fructose (1). Fructooligosaccharides have great potential in food industry because they can be used as calorie-reduced and noncariogenic sweeteners. Fructose formation from inulin is a single step enzymatic reaction and yields are up to 95 % fructose. On contrary, conventional fructose production from starch needs at least three enzymatic steps, yielding only 45 % fructose (2). Process of inulinase production was optimised by using experimental design method. pH value of the cultivation medium showed to be the most significant variable and it should be maintained at optimum value, 3.6. The effect of temperature was slightly lower and optimal values are between 30 and 33 ºC. At a low pH value of the cultivation medium, the microorganism was not able to produce enough enzyme and enzyme activities were low. Similar effect was caused by high temperature. Highest values of enzyme activities were achieved at optimal fermentation conditions and the values were: 100.16-124.36 IU/ml (with sucrose as substrate for determination of enzyme activity) or 8.6-11.6 IU/ml (with inulin as substrate), respectively. The method of factorial design and response surface analysis makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions (3). The model based on physiological assumptions is also applied. Assumed is a single enzyme rate determing growth (Monod kinetics) with proportional inulinase production rate. Applied are the models of reversible temperature and acidity inhibition based on thermodynamic equilibrium between active and inhibited enzyme states. Predictions by the two models are compared by ANOVA.
The production of extracellular inulinase (13-1,2-D-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40 ° C. At both temperatures , the final biomass concentration exceeded 100 g.1-I and more than 2 g enzyme. L-1 of culture su-pernatant was produced. The biomass yield on 02 at 40°C was substantially lower than at 30 ° C. Nevertheless , at 40°C a growth rate of 0.20 h-1 could be maintained for a longer period than at 30 ° C. The unexpected higher O2-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40 ° C fermentation took only 33 h as compared to 42 h at 30 ° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.