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مقایسه روش‌های استخراج، تغلیظ و نگهداری آنزیم‌های هضم‌کننده الیاف گیاهی از محتویات شکمبه گوسفند

نوع مقاله : مقاله پژوهشی

نویسندگان

1 استادیار گروه علوم دامی، مؤسسه آموزش عالی علمی کاربردی جهاد کشاورزی، تهران، ایران

2 دانشیار گروه علوم دامی، دانشکده کشاورزی، دانشگاه آزاد اسلامی واحد علوم و تحقیقات، تهران، ایران

3 کارشناس، گروه علوم دامی، مؤسسه آموزش عالی علمی- کاربردی جهاد کشاورزی، تهران، ایران

چکیده

این تحقیق به منظور بررسی تولید آنزیم‌های هضم‌کنندة الیاف در شکمبه و روش‌های مختلف استخراج و تغلیظ آن‌ها از محتویات شکمبة دام‌های کشتارشده انجام شد. به همین منظور، از دو روش هوموژن‌سازی و سونیکیشن برای آزادسازی آنزیم‌های متصل به مواد جامد محتویات شکمبه و از روش‌های رسوب دادن پروتئین‌ها با اتانل، تری‌کلرو استیک اسید و سولفات آمونیم، همچنین خشک کردن با برودت به منظور تغلیظ آنزیم‌های هضم‌کنندة الیاف استفاده شد. به منظور بررسی اثر دما و مدت زمان نگهداری بر فعالیت آنزیم‌ها از روش‌های نگهداری در نیتروژن مایع، فریزر 70- و 20- درجة سانتی‌گراد استفاده شد. میانگین فعالیت ویژة آنزیم‌های سلولاز و زایلاناز به ترتیب 5/7 و 5/16 واحد به ازای هر میلی‌گرم پروتئین موجود در محلول بود. میزان آزادسازی و استخراج آنزیم از محتویات شکمبه در روش هوموژن‌سازی بیشتر از روش سونیکیشن بود. فعالیت و بازیافت آنزیم‌ها در روش‌های تغلیظ با برودت و رسوب دادن با سولفات آمونیم بیشتر بود. روش نیتروژن مایع برای نگهداری طولانی‌مدت و روش فریزر 70- برای نگهداری میان‌مدت مناسب‌تر بود. نتایج این تحقیق نشان داد که محتویات شکمبة دام‌های کشتارشده منبع مناسبی از آنزیم‌های هضم‌کنندة الیاف گیاهی است و می‌توان با به‌کارگیری روش‌های استخراج و تغلیظ مناسب از آن‌ها استفاده کرد.

کلیدواژه‌ها

عنوان مقاله [English]

Investigation of methods of extraction, concentration and conservation of rumen content fibrolytic enzymes

نویسندگان [English]

  • Hossein Manafi Rasi 1
  • Mohammad Chamani 2
  • Salman Afshar 3

1 Assistant Professor, Department of Animal Science, Institute of Scientific-Applied Higher Education of Jihad-e-Agriculture, Tehran, Iran

2 Association Professor, Department of Animal Science, Agriculture Faculty, Science and Research Branch, Islamic Azad University, Tehran, Iran

3 Research Expert, Department of Animal Science, Institute of Scientific-Applied Higher Education of Jihad-e-Agriculture, Tehran, Iran

چکیده [English]

This study was carried out to determine the potential of rumen microorganisms in samples taken from slaughterhouse to produce fibrolytic enzymes and to compare different methods for extraction, concentration and conservation of them.  According to the results, the average specific activity of cellulase and xylanase were 7.5 and 16.5 U/mg protein respectively. The application of homogenizing and sonication methods to extract rumen content enzymes showed that the former had better performance in the liberation of enzymes. To determine optimum time length for the maximum release of enzymes from solid phases, various time lengths including one, 1.5 and two min were examined. Based on this experiment, one minute appeared the best. To attain a high level of concentration of enzymes obtained from rumen liquor, various procedures were applied. The results indicate that freeze drying and precipitation of enzyme using ammonium sulphate were the best methods, while trichloroacetic acid precipitation turned out to be the most inappropriate method due to improper effect on enzymatic activity. In order to examine the effect of temperature and time length on enzymes activities, some methods including liquid nitrogen, freezing in -70° and freezing in -20° were applied. The comparison of different methods of enzymes conservation indicated that applying liquid nitrogen for long term as well as keeping in the freezer (-70°) for medium term purposes were the best.

کلیدواژه‌ها [English]

  • Cellulase
  • concentration
  • Conservation
  • extraction
  • rumen
  • Xylanase
مراجع
1 . Abd El-Nasser NH, Mahdy EM, Shousha WG and El Sayed GH (2010) Purification and Partial Characterization of Extracellular Cellulase Free Xylanase from Streptomyces rochei. Applied Science Research. 6(9): 1373-1378.
2 . Bayer EA, Morag E, Wilchek M, Lamed R, Yaron S and Shoham Y (1995) Cellulosome domains for novel biotechnological application. In: Petersen, S.B., Svensson, B. and Pedersen, S. (Eds) Carbohydrate Bioengineering. Progress in Biotechnology, Vol. 10. Elsevier, Amsterdam, the Netherlands. Pp. 251-260.
3 . Bayer EA, Lamed R and Himmel ME (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Current Opinion in Biotechnology, 18: 237-245.
4 . Bayer EA, Morag E and Lamed R (1994) The cellulosome – a treasure-trove for biotechnology. Trends in Biotechnology. 12: 379-386.
5 . Bedford MR and Partridge GG (2001) (Eds) Enzymes in Farm Animal Nutrition CAB International, Wallingford, UK, 406 pp.
6 . Beguin P and Lemaire M (1996) The cellulosome: an exocellular, multiprotein complex specialised in cellulose degradation. Critical Reviews in Biochemistry and Molecular Biology, 31: 201-236.
7 . Bhat MK and Bhat S (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances. 15: 583-620.
8 . Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analytical Biochemistry, 72: 248-54.
9 . Byrne H, Christou NV, Verma DP and Maclachlan GA (1975) Purification and characterization of two cellulases from auxin-treated pea epicotyls. Biological Chemistry, 250(3): 1012-1018.
10 . Chen PJ, Wei TC, Chang YT and Lin LP (2004) Purification and characterization of carboxymethyl cellulase from Sinorhizobium fredii. Botanical Bulletin of Academai Sinica, 45: 111-118
11 . Chi WK, Ku CH and Chang CC (1994) Two-step cell disruption for the extraction of membrane-Associated recombinant protein from Saccharomyces cerevisiae. Annals of the New York Academy of Sciences, 721(1): 365-373.
12 . Choi H, Laleye L and Fetal A (1997) Release of amino peptidase from Lactobacillus casei sp. casei by cell disruption in microfluidizer. Biotechnology Techniques. 11(7): 451-453.
13 . Cumming R and Iceton H G (2001) Cell disintegration and extraction techniques. In: Protein purification techniques: a practical approach. Oxford University Press, Oxford. Pp. 83-110.
14 . Doi RH, Kosugi A, Murashima K, Tamaru Y and Han SO (2003) Cellulosomes from Mesophilic Bacteria. Journal of Bacteriology,185(20): 5907-5914.
15 . Festenstein GN (1957) Cellulolytic enzymes from sheep-rumen liquor microorganisms. Biochemical  Journal, 69 (4):  562-567.
16 . Ghose TK (1987) Measurement of cellulase activities. Pure and Applied Chemistry, Vol. 59, No. 2, pp. 257-268.
17 . Hurst PL, Nielsen J, Sullivan PA and Shepherd MG (1977) Purification and properties of a cellulase from Aspergillus niger. Biochemical Journal, 165: 33-41.
18 . Hwang BJ and Chu G (1996) Trichloroacetic acid precipitation by ultracentrifugation to concentrate dilute protein in viscous solution. Biotechniques. 20: 982-984.
19 . Illanes A (2008) Enzyme production. In: ILLANES, Andrés. Ed. Enzyme Biocatalysis: Principles and Applications. United Kingdom; Springer. Pp. 60-89.
20 . Jiang ZQ, Deng W, Li XT, Ai ZL, Li LT and Kusakabe I (2005) Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86. Enzyme and Microbial Technology, 36: 923-929.
21 . Kang MK, Maeng P and Rhee YH (1996) Purification and characterization of two xylanases from Alkalophilic Cephalosporium sp. strain rym-202. Applied and Enviromental Microbiology, 62(9): 3480-3482.
22 . Kitts WD and Underkofler LA (1954) Digestion by rumen microorganisms. hydrolytic products of cellulose and the cellulolytie enzymes. Agricultural Food Chemistry, 2: 639-645
23 . Koppolu A and Davis Clements L (2004) Ruminal waste stream as a source of industrial chemicals. Resource Recovery and Conservation, 41(3): 215-226.
24 . Lamed R, Setter E and Bayer EA (1983) Characterization of a cellulose binding, cellulase-containing complex in Clostridium thermocellum. Biochemical Journal, 156: 828-836.
25 . Leser EW and Asenjo JA (1994) Protein recovery, separation and purification. Selection of optimal techniques using an expert system. Memorias do Instituto Oswaldo Cruz, 89(1): 99-109.
26 . Lei J, Lin H and Fountoulakis M (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography A. 1023: 317-320.
27 . Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry, 31: 426-428.
28 . Nakamura S, Wakabayashi K, Nakai R, Aono R and Horikoshi K (1993) Purification and Some Properties of an Alkaline Xylanase from Alkaliphilic Bacillus sp. Strain 41M-1. Applied and Enviromental Microbiology, 59(7): 2311-2316.
29 . Noziere P and Michalet-Doreau B (1994) Effect of extraction method on activities of polysaccharide-depolymerase enzymes in the microbial population from the solid phase in the rumen. Reproduction Nutrition Development, 34: 281-288.
30 . Scopes RK (1994) Protein Purification, Principles and Practice, 3rd ed. Springer Advanced Texts in Chemistry, Springer-Verlag, N.Y.
31 . Selinger LB, Forsberg CW and Cheng KJ (1996) The rumen: a unique source of enzymes for enhancing livestock production. Anaerobe. 2: 263-284.
32 . Shawn D (1996) Protein Purification Protocols, Second Edition, Methods in Molecular Biology, P. Cutler, Ed. Humana Press. 59: 101-117.
33 . Sibtain A, Bashir A, Saleem H, Saadia M and Jamil A (2009) Production and purification of cellulose degrading enzymes from a filamentous fungus Trichoderma harzianum. Pakistan Journal of Botany, 41(3): 1411-1419.
34 . Sivaraman T, Kumar TK, Jayaraman G and Yu C (1997) The mechanism of 2, 2, 2-trichloroacetic acid-induced protein precipitation. Journal of Protein Chemistry, 16: 291-297.
35 . Stanley RW and Kesler EM (1958) Preparation and some basic properties of cell-free cellulolytic extracts of rumen fluid. Journal of Dairy Science. 42: 127-136.
36 . Wateewuthajarn K and Pinphanichakarn P (2000) Purification and characterization of xylanases from Streptomyces sp. PC22. Science Research of Chulalongkorn University. 25(2): 245-258
37 . Widjaja A, Lestari E, Tanjung A, Widiawan, Alfian and H. Ogino. 2009. Optimized production of xylanase from fungal strains and its purification strategies. Journal of Applied Sciences in Environmental Sanitation, 4(3): 219-232.
38 . Wood TM (1992a) Microbial enzymes involved in the degradation of the cellulose component of plant cell walls. In: Rowett Research Institute Annual Report, 10-24.
تولیدات دامی
دوره 16، شماره 2 - شماره پیاپی 2
پاییز و زمستان
مهر 1393
صفحه 167-178
فایل ها
هم رسانی
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