Optimization Of Dextranase Application During Cane Sugar Manufacturing And Improvement Of Its Industrial Efficiency

Dextran is one of the most significant impurities present in sugar production process. Thepresence of dextran in the canesugar factories and refineries leads to many technical possessingdifficulties, however, from the processing point of view, the most damaging effects of elevateddextran concentrations in a technical sucrose solution are foreseen in the crystallization process.All these possessing difficulties caused by dextrans lead, in the end, to the sugar losses and theseeconomic losses are continuous throughout the process. Therefore, in recent years many studieshave been undertaken to minimization of dextran effects in the sugar factory by controllingmicroorganisms and reducing its molecular weight during the manufacturing process. To date, theuse of the dextranase enzyme is the most efficient method for hydrolyzing dextrans at canesugarfactories and refineries.The objectives of this research are to (1) optimize the addition and investigate the conditionsof using dextranase in cane sugar manufacturing,(2) characterize the effects of thebiodegradation of dextran using dextranase enzyme on the crystallization process and the qualityof the final sugar crystal and (3) find novel methods to improve dextranases activity to decreasetheir industrial application costs.To begin with, the effects of dextran at differents molecular weight (Mw) and theconcentrations of on the rheological and glass transition properties of supersaturated sucrosesolution were investigated. Three dextrans of various Mw, namely100,000g/mol,500,000g/moland2,000,000g/mol, were admixed in concentrations between (1000-10000ppm) with (60%-75%w/w) sucrose solution. The results indicated that both the apparent viscosity and dynamicmodulus increased with an increase in dextran concentrations and they demonstrated strongdependence on its Mw. Glass transition temperature (Tg) of the samples were measured bydifferential scanning calorimetry, and their dependence on dextran Mw and concentration wasanalyzed by the Fox and expanded Gordon-Taylor models. It was found that, the higher the Mwand concentration of the dextran, the greater the increase in Tg. The expanded Gordon-Taylorequation has proved useful in predicting the Tg of different dextrans and sucrose solutionmixtures.In the further step, dextran extracted from deteriorated sugarcane was characterized using themore recently available techniques such as (1H,13C) and two-dimensional (COSY and HMQC)NMR spectral analysis, methylation GC-MS and MALDI-TOF mass spectrometry, the structureof sugarcane dextran (SC-Dex). dextran was extracted from deteriorated sugarcane by alcoholprecipitation and purified by gel filtration chromatography. Total acid hydrolysis and enzymatic degradation were utilized to confirm the purity of separated polysaccharide. On the basis of allspectra, SC-Dex showed a branched polysaccharide that contained only D-glucose residues in-(1-(13) branches. Methylation analysis-(13) branching levels was4.37%. Several structural fragments wereidentified from MALDI-TOF spectrum with peak-to-peak mass difference of162gmol-1, whichconfirmed that the repeat unit in SC-Dex was D-glucose. The surface morphology of SC-Dex,revealed the spherically shaped and porous structure. Using HPSEC-MALLS-RI system, theaverage molecular weight of SC-Dex was estimated to be1.753x106gmol1with an index ofpolydispersity value of1.069.The influence of dextranase enzyme on the molecular weight (Mw) parameters of remainingdextran and intrinsic viscosity after different enzymatic treatments at different steps during sugarmanufacturing was investigated. A spectrophotometric method was used to determine the relativeactivity of dextranase and the result has been confirmed by measured reducing sugar using HPLCsystem. For comparison, the action patterns of concentrated and diluted enzymes wereadditionally included in the experiments. Additions of dextranase to juice were much moreefficient and economical to reduce the Mw of remaining dextran than adding it to evaporatorsyrups. Addition of dextranase at juice pH5.5showed similar minimum Mwwith the lowestintrinsic viscosity, observed at55.0°C, and the enzymes activity was decreased after20°Brix.The highest dextran removal was observed at dextranase concentration at100ppm/juice whichwas resulted in80.29%removal dextran in the juice, Moreover, the higher the level ofconcentrated dextranase applied to the juice, the more the removal of dextran occurred. Inaddition, the longer the availability of the residence time in the factory, the lower dextran Mw hasbeen observed. To reach a satisfactory level of dextran hydrolysis, it was necessary to correct thedose of dextranase enzyme according to the losses of activity caused by the high°Brix.After optimizing the dextranase application, the Influence of hydrolysis of dextran catalyzedby dextranase enzymes during sugar manufacturing on the rate of sucrose crystallization andgrowth rate of sucrose crystals in pure sucrose solution at different temperatures was investigated.To elucidate the influence of hydrolysis of dextran on the growth rate of sucrose crystals, dextranat Mw, of2,000,000g/mol (T2000), was admixed in concentrations between (1000-10000ppm)with (55%-70%w/w) sucrose solution, however, three different concentrations of dextranaseenzyme (50,75,100ppm) were applied for the emzymatic hydrolysis. After enzymatic hydrolysisof dextran T2000presence in the sucrose solution, the growth rate of the sucrose crystal wasincreased, in addition, more perfect crystal surfaces was observed compared with that reported inthe presence of dextran T2000. From the results it could be shown that an increase of crystallization rate of up to73.56%after hydrolysis of dextran T2000using dextranase enzyme at100ppm,compared to crystallization rate with pure sucrose solution in the presence of dextran T2000. Sucha positive influence of enzymatic hydrolysis of dextran using dextranase enzyme could decreasethe crystallization time in the sugar house and thus decreases the production costs of sugarmanufacturing.Based on the above result, that dextranase could minimize of dextran effects during thecrystallization process, further investigations to find novel methods for improving dextranasesactivity to decrease their industrial application costs were carried out. Sequently, the effect ofultrasound irradiation (US) on the enzymatic activity and enzymatic hydrolysis kinetic parametersof dextran catalysis by dextranase were investigated. US has improved the catalytic kineticsactivity of dextranase at all the reaction conditions studied. The maximum activity of dextranasewas observed when the sample was treated with US at25kHz,40W for15min, under which theenzyme activity increased by13.43%compared the routine thermal incubation at50°C.Experimental Kinetics results, demonstrated that, both the Vmaxand KMvalues of dextranaseincreased with US-treated compared with the incubation at50°C. Likewise, both the catalyticand specificity constants are higher under the effects of US field, indicating that, the substrate isconverted into the product at an increased rate when compared with the incubation at50°C. Inaddition, to date, no research work is available on the combination of other methods with US toimprove the enzymes activities. Therefore, we decided to study the effect of combination of othermethods with the ultrasonic irradiation on the enzymes activities.After the enhancement of dextranase activity by means of US, the effect of combination ofUS and high hydrostatic pressure (US/HHP) on the enzymatic activity and enzymatic hydrolysiskinetic parameters of dextran catalytic by dextranase were investigated. Furthermore, the effectsof US/HHP on the structure of dextranase were also discussed with the aid of fluorescencespectroscopy and circular dichroism (CD) spectroscopy. The maximum hydrolysis of dextran wasobserved under US (40W at25kHz for15min) combined with HHP (400MPa for25min), inwhich the hydrolysis of dextran increased by163.79%compared with the conventional thermalincubation at50°C. Results also showed that, Vmaxand KMvalues, as well as, kcatof dextranaseunder US/HHP treatment were higher than that under US, HHP and thermal incubation at50°C,indicating that, the substrate is converted into the product at an increased rate when comparedwith the conventional thermal incubation at50°C. Compared to the enzymatic reaction under US,HHP, and conventional thermal incubation, dextranase enzymatic reaction under US/HHPtreatIn addition, Fluorescence and CD spectra reflected that US/HHP treatment had increased the num-helix by19.80%and reducedrandom coil by6.94%upon US/HHP-treated dextranase protein compared to the control, whichwere helpful for the improvement of its activity.The synergetic effects of ultrasonic and microwave irradiation sock (US/MIS) on hydrolysis ofdextran by dextranase were finally studied. The US treatments were performed at fixed power of50W/40kHz, and the microwave irradiation shock (MI-S) was applied at different powerconditions, of10-140W at2450MHz at sock rate of20sec/min. The hydrolysis of dextran underUS/MI-S was significantly higher than those performed under US, MI-S and conventionalthermal incubation at all condition studied. The maximum hydrolysis rate was observed whenUS/MI-S (US of50W combined with MI-S of60W at sock rate of20sec/min for25min) wasused in which the dextran hydrolysis increased by163.58%compared with routine conventionalheating. Results also showed that, Vmax and KM values of dextranase under US/MI-S treatmentwere higher than those under US, MI-S and conventional thermal incubation. Higher kcatand(Kcat/Km) values were obtained under US/MI-s in comparison with the values in the respectiveconventional heating. Compared to the enzymatic reaction under US, MI-S, and conventionalheating incubation, dextranase enzymatic reaction under US/MI-S treatment showed decreases inOn the other hand, CD spectrareflected that-sheet and random coil content were decreased by the mean of the US/MI-s andUS treatments and increased under MI-s treatment compared to control. However, fluorescencespectra demonstrate the rate of degradation of its secondary structure was slower than that ofenzyme tertiary structure. Therefore, US/MI-s treatment results in reordered secondary structureof dextranase, which were helpful for the improvement of its activity.These results, therefore, indicated that, the combination of US with HHP or MI-Streatments could be used as a novel technique method for improving the industrial efficiency ofdextranases in many industrial applications including sugar manufacturing processes. This studywill serve as a basis for the future work in this area of research.