การทำให้อัลคาไลน์โปรตีเอสที่ผลิตจาก alkalotolerant Bacillus sp. B12 บริสุทธิ์และศึกษาสมบัติต่าง ๆ

Purification and Characterization of an Alkaline Protease from Alkalotolerant Bacillus sp. B12

Name: พิมล จำนงค์

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Name: Pimon Jamnong

keyword: Alkalotolerant Bacillus sp.

; Alkaline Protease

Abstract: Thirty Bacillus strains were cultured in a soybean mealmedium at an alkaline condition and an alkalotolerant Bacillus sp.B12 was found to be able to grow and produced the highestprotease level. This strain was a Gram-variable, rod-shaped,spore-forming, oxygen required bacterium and produced catalase.The strain B12 protease was highly stable in commercialdetergents such as P1 (Fab perfect), P2 (Breeze power), L1 (Fabperfect), L2 (Fine line) and L3 (Vanish) when incubated at 50 0Cfor 1 hr. Although Bacillus sp. B12 could grow in the 0.5%soybean meal medium and produced high protease at pH 7.0 to10.0, the most suitable condition for production of alkalineprotease was pH 9.0 at 37 0C, and shaken at 250 rpm for 24 hr(0.44 Unit/ml). Alkaline protease produced by this strain wasfound to increase along with the cell growth. The crude proteasehad an optimum pH from 8.0 to 11.0 and maintained its stabilityover a broad range of pH between 6.0 and 11.0 for 1 hr. Theoptimum temperature for crude protease activity was from 30 to80 0C. The protease was stable between 30 and 60 0C for 1 hr.To evaluate the protease action in improving the washingefficiency of the detergents, the prices of white cotton cloth soiledwith cow blood stain were washed with L2 and L3 detergents inthe presence of the crude protease from Bacillus sp. B12. Itshowed the increases in bright values from 77.22 and 71.73 to80.24 and 75.83, respectively and the decreases in red valueswhen compared to the washing only with detergents L2 and L3.Therefore, its properties had a great potential for application indetergent industry.The alkaline protease from Bacillus sp. B12 was purified by0-20% saturated ammonium sulfate precipitation and DEAEToyopearlchromatography, eluted with 0.0-0.15 M NaCl in Tris-HCl buffer, pH 8.0. Analysis of the purified protease by SDSPAGErevealed that its molecular weight was 32 kilodaltons. Thepurified protease had the highest activity at pH 10.0 andmaintained its stability between pH 8.0 and 11.0. The optimumtemperature for the purified protease was between 50 and 70 0C.The protease was stable from 30 to 50 0C for 1 hr. It wascompletely inhibited with 10 mM phenylmethylsulfonylfluoride,indicating that it belongs to the family of serine protease. It wasalso inhibited by sodium dodecyl sulfate (0.5 and 1.0%).Hydrogen peroxide (0.5 and 1.0%) did not totally inactivate theprotease activity, whereas the purified protease activity wasenhanced by the presence of either Triton X-100 or Tween-80.The purified protease showed the highest rate on hydrolysis ofcasein, followed by skim milk, bovine serum albumin, gelatin,collagen Type I and keratin, but it did not hydrolyse elastin. Thepurified protease could be active in any commercial detergentsand was highly stable in L2 and L3 detergents but was not stablein P1, P2, P3 (Pao handforce), L1 and L4 (Home) detergents.Kinetic studies of this enzyme for casein and skim milk showedthat Vmax/Km were 2.00 and 1.16 ?g.ml-1.min-1/mg.ml-1, respectively,indicating that Bacillus sp. B12 alkaline protease could hydrolysecasein better than skim milk.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: รศ.ดร. กนก รัตนะกนกชัย

Role: อาจารย์ที่ปรึกษา

Created: 2545

Issued: 2005-09-20

Modified: 2006-05-10

วิทยานิพนธ์/Thesis

ISBN: 9749636929

CallNumber: BCT69

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวเคมี

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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