CS265540B1 - Process for preparing pure alpha-amylaze from biological materials - Google Patents

Abstract

The solution concerns a new way isolation and purification of alpha-amylase in industrial production from biological materials. It is based on the use of isoelectric existence the point of this enzyme where it is autofocusing by acting unidirectional the current at a voltage of 200 to 1,000 V divides, from a mixture of enzymes and proteins in solution with conductivity 100 to 800 pS.cnT1 at pH 5.82 to 6.12. This fraction is subjected to a separating fraction using different molecular techniques weight, preferably gel chromatography. Repeating or confusing both techniques super clean enzyme needed mainly in industrial production.

Description

The invention relates to a process for the isolation and purification of the alpha-amylase enzyme (EC 3.2.1.1) in industrial production, which uses the existence of the isoelectric point of this enzyme as a criterion of its purity.

According to the prior art, alpha-amylase has been produced by several processes, including precipitation with ammonium sulfate, ethanol, acetone, various gel and ion exchange chromatography systems, or electrophoresis in various configurations. Their common disadvantage was laboriousness, difficulty: the technical equipment while achieving higher purity was hampered by the fact that at the final stage of isolation the protein mixture was approximately the same molecular weight where the major part was the enzyme, but the admixture content was + 5-10%. These ballast proteins reduce specific activity and cause other side effects, such as gradual degradation of the isolate.

In our inventions CS AO 211 856 and 234 801, in principle, a new method of automatic isoelectric focusing is protected by directing a direct current in a uniform solution of a split substance which is mechanically stabilized, as well as a simple device for carrying out the method.

The above-described drawbacks of enzyme isolation and purification are substantially eliminated by the invention, which consists in adjusting the conductivity of a solution of biological material containing alpha-amylase activity to 100 to 800 pS.cm 1 by dialysis or ultrafiltration and adjusting the protein content to The 1 to 30 g / l solution is autofocused with a direct current at a voltage of 200 to 1000 V, the solution forming the fraction with the highest enzymatic activity, preferably at a pH of 5.82 to 6.18, is isolated and subjected to a separation technique using different molecular weights. , preferably by gel chromatography, depending on the desired purity, one or both of the separation techniques are repeated at least once.

The invention is suitable for obtaining super-pure alpha-amylase on a technological scale in the manufacture of this enzyme for the needs of several industries.

EXAMPLE

The bacterial culture of Bacillus subtilis is centrifuged and the supernatant obtained is concentrated in a vacuum evaporator. The conductivity of the concentrated supernatant is adjusted to 100-800 µS.cm by dialysis or ultrafiltration with the addition of Mcllvari buffer 0.01 mol / l pH 6.0 containing citrate ions which stabilize alpha-amylase activity. The protein content is adjusted to 10-30 g / ^{l by} dilution with buffer. The treated solution is filled with a 20-cell autofocusing apparatus and, after closing, a 200 to 1000 V direct current controlled current is applied so that the product of current and voltage does not exceed 3 W. The autofocusing is performed at + 4 ° C. The autofocusing process is complete when the current drops to zero at 1,000 volts. The split mixture automatically generates an ascending pH gradient along which the individual protein fractions are formed into peaks according to their isoelectric points. Alpha-amylase is found in the fractions with the highest enzymatic activity, mainly in chambers with a pH between 5.82 to 6.18, the enzymatic activity being determined based on the increasing oxidation activity of the enzyme-cleaved starch. Proteins from this range are pooled and loaded onto an 80x50 cm column packed with a Spheron P-40 molecular sieve, equilibrated with McIlvain buffer. Chromatography is carried out at 4 ° C at a flow rate of 120 ml / h and 30 ml fractions are collected. Upon completion of gel filtration, Lawry protein content and alpha-amylase activity were determined in all fractions as described above. By gel filtration, the isolated protein is separated into four peaks by autofocusing, where pure alpha-amylase is found on the third peak from the beginning of fraction collection. The individual steps can be repeated according to the degree of purity required, which is defined both by the electrophoretic presence of a single line and by the specific activity to be achieved. at least 3 U.mg according to international assessment. This results in up to 99.99% purity of the enzyme.

Example 2

Technical alpha-amylase, which contains an average of 60% pure enzyme, is dissolved in distilled water so that the protein content is 10 to 30 g.l -1. Thereafter, the solution is subjected to ultrafiltration in such an arrangement (e.g., the AMICON system) to eliminate all molecules up to 40,000 molecular weight therefrom. The resulting solution is devoid of a substantial amount of protein and as such is applied to the autofocusing apparatus and divided as described in example no. 1. After uniting the active fractions, the obtained alpha-amylase solution is again subjected to ultrafiltration but in such a way as to eliminate all molecules having a molecular weight below 60,000. The alpha-amylase, which has a molecular weight of about 54 to 56,000, is present in the filtrate. is applied to the autofocusing apparatus of a smaller content and re-focused under the conditions described in Example no. 1. Upon completion of focusing, chambers with a pH between 5.82 to 6.18 contain super-pure alpha-amylase that meets the purity criteria described above.

In addition to the aforementioned separation techniques using different molecular weights of the resolved substances, autofocusing can also be combined with electrophoresis or salting-out.

Claims (1)

Method for isolating alpha-amylase from biological materials, characterized in that by conducting dialysis or ultrafiltration, the conductivity of the biological material solution is adjusted to 100 to 800 pS.cm ¹ and the protein content is adjusted to 1 to 30 g and the solution is autofocused by direct current at 200 to 1000 volts until the current drops to zero and the solution forming the fraction with the highest enzymatic activity, preferably at pH 5.82 to 6.18, is isolated and subjected to a separation technique using a different molecular weight of the separated substances, preferably by gel chromatography wherein one or both of the separation techniques are repeated at least once depending on the desired purity.