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activity inhibition lipoliticheskih enzymes hitozanom in heterogenous solutions, on border of section of phases

Inhibition studying low-molecular hitozanom lipoliticheskoj activity has been spent on three lipazah - pork pancreatic lipaze, lipaze from S rugosa and lipaze from wheat sprouts in a heterogenous phase, on border an oil-water.

Dependences of initial speeds of hydrolysis of an olive oil lipazami from concentration of a substratum in absence and in presence ingibitora are described by Michaelis-Menten equation, after linearizatsii which in co-ordinates Lajnuivera-Berka the maximum speeds of hydrolysis and constant Mihaelisa Kt have been defined. On fig. 16 dependence of anatropic initial speed of hydrolysis olive is shown
Oils pork pancreatic lipazoj from concentration of a substratum in absence and in presence hitozana. Similar dependences are received and for lipazy from sprouts of wheat and for lipazy from S rugosa.

Fig. 16. The schedule of dependences in anatropic co-ordinates of initial speeds of hydrolysis of an olive oil pork pancreatic lipazoj from concentration of a substratum in absence (1) and at presence ingibitora (2) at pH 6,7 and ZGS.

The projection of straight lines to an axis of abscisses has allowed to define, that hitozan is a competitive inhibitor lipoliticheskih enzymes. The values of molecular constants of speed found from these dependences, kcat and constants Mihaelisa, Kt are presented in table 20.

Table 20.

Kinetic constants of reaction of hydrolysis of an olive oil lipoliticheskimi enzymes

Enzyme Kt, mol K^atjCek Kcat/Km,

mol '-sek ' 1

Pancreatic lipaza the pork 0,24*10 ' 3 19,4 81*103
Candida rugosa 0,27*10 ' 3 P, 4 42,2*103
From wheat sprouts 0,2*10 ’ 3 1,7 8,5 • 103

Efficacyy gidrolaznogo actions pancreatic lipazy on an olive oil, expressed bimolekuljarnoj a constant kcat/Km, in 10 times
Above, than for vegetative lipazy, and in 5 times above, than for lipazy from S rugosa. The difference in efficacyy gidrolaznogo on a substratum between pancreatic lipazoj and lipazoj from S rugosa compounds actions about two times. It is caused by distinctions in sizes as kcat, and Km.

From given tab. 21 follows, that low-molecular hitozan ingibiruet lipoliticheskuju activity, constants of inhibition Kj variate within 70-160 microns, depending on ingibiruemogo enzyme.

Table 21.

Inhibition constants lipoliticheskih enzymes

Low-molecular hitozanami and monogljukozaminom

Enzyme Constants of inhibition Kj, mol
hitozan N-suktsinil

hitozan

monogljukozamin
Pancreatic lipaza the pork 7,7*10 ' 5 1,1*10 "5 4,3*10 ’ 2
Candida rugosa 1,52’КГ4 3,3*10 ’ 4 1,1*10 ' 2
From wheat sprouts 1,14*10 "4 4,2*10 ‘ 4 0,36*10 ' 2

Values of constants of inhibition vegetative lipazy and lipazy from S rugosa one order.

The inhibition constant hitozanom pancreatic lipazy 10 times less also has compounded 77 microns. The augmentation of constants of inhibition hitozanom vegetative and fungal lipaz in comparison with the constants found at hydrolysis of a water-soluble substratum, will be co-ordinated with data that at a finding lipoliticheskih enzymes in region of section of phases, a lipid - water, efficacyy of linkage with ingibitorom is essential increases at the expense of considerable prevalence open konformatsii enzyme.

Activity inhibition lipoliticheskih enzymes monogljukozaminom

In quality ingibitora lipoliticheskoj activity has been studied and monogljukozamin. Earlier it has been shown, that monogljukozamin not ingibiruet
lipoliticheskie enzymes [243]. Representation of the experimental data received by us in co-ordinates Lajnuivera-Berka (fig. 17.) has allowed to define constants of inhibition studied lipaz.

1/S, mmol ' 1

Fig. 17. The schedule of dependences in anatropic co-ordinates of initial speeds of hydrolysis of an olive oil pork pancreatic lipazoj from concentration of a substratum in absence (1) and in the presence of (2) monogljukozamina at pH 6,7 and 37°С.

Efficacyy of an inhibiting effect monogljukozamina has appeared on three order more low, in case of inhibition pancreatic lipazy and on two order more low, in case of inhibition vegetative lipazy and fungal lipazy. Inhibition constants, To; have compounded 10-40 mm (tab. 21). The augmentation of values of constants of inhibition is bound, apparently, to participation in a complex formation with lipazami only the unique part monogljukozamina. The given conclusion the found values of free energy, AG confirm complex formations monogljukozamina with lipazami.

The free energy contribution, AG in a complex formation between lipazami and monogljukozaminom has compounded-2,32 kcal/mol for pancreatic lipazy,-2,41 kcal/mol for lipazy of sprouts of wheat and-2,7 kcal/mol for lipazy from S rugosa. Inhibition constants lipaz low-molecular hitozanom with free amino groups compound from 70 - 160 mkmol, that corresponds to values AG-5,8 kcal/mol for the pork pancreatic
lipazy,-5,0 kcal/mol for lipazy from sprouts of wheat and-5,4 kcal/mol for lipazy from S rugosa. The difference in values of free energy of a complex formation with lipazami monogljukozamina and hitozana differs twice, that most likely testifies that at a hydrophobic interaction there is a linkage at least two parts hitozana with lipoliticheskimi enzymes, and in case of interaction monogljukozamina, the unique part is bound.

Thus, we have shown, that low-molecular hitozan depresses activity lipoliticheskih enzymes, both in homogeneous solutions, and on border of section of phases. Low-molecular hitozan is a competitive inhibitor pork pancreatic lipazy, lipazy from C.rugosa and lipazy from wheat sprouts. hitozan ingibiruet specific activity lipazy C.rugosa and wheat sprouts in homogeneous water solutions with values To; an order of 1 mm. Inhibition constants, Kj pork pancreatic lipazy, lipazy from C.rugosa and lipazy from wheat sprouts on border of section of phases an oil-water, have compounded 77 microns, 152 microns and 114 microns, accordingly. At a finding lipoliticheskih enzymes in region of section of phases, a lipid - water, efficacyy of linkage ingibitora with enzyme is essential increases at the expense of considerable prevalence open konformatsii lipaz. The size of effect of inhibition depended on time preinkubatsii hitozana with lipazami and reached a maximum at time preinkubatsii 30 minutes Though hitozan is polikationom, the contribution of electrostatic interactions to its complex formation with lipazami is insignificant.

Constants of inhibition of many known ingibitorov lipoliticheskih enzymes, the same order, as the found constants of inhibition hitozanom [238-239]. However the majority ingibitorov applied to treatment of excess weight and diseases accompanying it possess a number of by-effects [249-250].

hitozan thanks to presence of a positive charge and the constitution
Is the multifunctional bond possessing variety of unique properties: high compatibility with animal fabrics, biodegradiruemostju, low toxicity, etc. [15-21,25]. It discovers the big prospects of use low-molecular hitozana in quality ingibitora lipoliticheskih enzymes for preventive maintenance and treatment of infringements lipidnogo a metabolism and diseases accompanying it as a part of biologically awake additives.

Conclusions

1. The technological scheme of processing kutikuly larvae wax is developed ask Galleria mellonella with reception chitin-melaninovogo of a complex and hitozana various molecular mass. Optimum conditions for each stage of the scheme of processing are defined.

2. It is shown, that hitozan possesses antitubercular activity. Low-molecular samples hitozana show the maximum antibacterial activity. With augmentation of molecular mass the antitubercular effect hitozana decreases. It is established, that there are enough 10 minutes of interaction of cages mikobaktery with hitozanom that the antibacterial effect was observed. Introduction of negatively charged bunches in a molecula hitozana conducts to loss of antitubercular properties.

3. The consecutive fractionating of an extract secured of wax is spent ask. After two hromatograficheskih clearing stages - waterproof and metal-chelate affine specific lipoliticheskaja activity increases in 29 times. With augmentation lipoliticheskoj activity as a result of a consecutive fractionating of an extract antibacterial activity, concerning atypical shtamma M smegmatis increases also.

4. It is established, that hitozan is an effective competitive inhibitor lipoliticheskih enzymes, both in homogeneous water solutions, and on border of section of phases. Kinetic constants of inhibition lipaz hitozanom have compounded 70-160 microns.

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A source: Ostanina EKATERINA SERGEEVNA. TECHNOLOGY of PROCESSING WAX ASK, STUDYING of ANTITUBERCULAR PROPERTIES hitozana And INTERACTIONS With LIPOLITICHESKY ENZYMES. The dissertation on competition of a scientific degree of a Cand.Biol.Sci. Shchelkovo -. 2007

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