Expression, characterization and formulation of recombinant bromelain /
Bromelain is a proteolytic enzyme obtained from the stems and fruits of pineapples (Ananas comosus) with abundant therapeutic, industrial and analytical applications. The challenges related to the development of bromelain technology on industrial scale include cost of production and downstream proce...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur:
Kulliyyah of Engineering, International Islamic University Malaysia,
2013
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| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
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| Summary: | Bromelain is a proteolytic enzyme obtained from the stems and fruits of pineapples (Ananas comosus) with abundant therapeutic, industrial and analytical applications. The challenges related to the development of bromelain technology on industrial scale include cost of production and downstream processing as well as the purity of the enzyme. In this research work, the recombinant stem bromelain has been expressed in E. coli BL21. The expressed ~43 kDa active bromelain was purified by a single step affinity chromatography and confirmed by SDS-PAGE analysis and Western blotting. A 3.7-fold lab-scale purification and 64 % recovery yield of the enzyme were achieved. Sequential optimization approach based on statistical experimental design including one-factor-at-a-time (OFAT) method was used to improve the recombinant bromelain production in shake flask cultures (1.7 U/mg). Response surface methodology (RSM) based on face-centered central composite design (FCCCD) developed cultivation conditions for production of recombinant bromelain at 0.15 % (w/v) L-arabinose, 27 ºC post-induction temperature and 8 hrs post-induction time. This led to maximum crude bromelain activity of 9.6±0.02 U/mg. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). The purified enzyme exhibited maximum activity at pH range of 5-8 on four substrates studied with optimum temperature of 45 ºC. It was totally inhibited by E-64 (10μM) but only slightly inhibited by non cysteine protease inhibitors and activated by all the sulphur-containing reducing reagents studied. Kinetic studies on the enzyme yielded lower lower values of values of values of values of Ki and Kand Kand Km coupled with higher coupled with higher coupled with higher coupled with higher coupled with higher coupled with higher coupled with higher coupled with higher kcat/Km ratio for recombinant bromelainratio; implying that it had more affinities towards the inhibitor used and all substrates(with the exemption of pGlu-phe-LeuLeuLeu-pNA) than commercial bromelain.NA). The mechanism of the enzyme inhibition by E-64 (0-5 μM) was found to be competitive in nature. For the formulation of the enzyme, the effects of some spray drying operating variables on bromelain had been investigated using full factorial design (FFD). On statistical analysis, the specified optimum conditions established (inlet temperature, 110ºC; aspirator setting, 90 (%); and pump setting, 12.5 (%) yielded the maximum activity of 78.08±0.07 U/ml, residual activity of 92.64±0.09 (%) and recovery yield of 89.28± 0.08 (%) for the spray dried recombinant bromelain. Analysis of variance (ANOVA) exhibited greater value of coefficient of determination, R2 (0.999); suggesting good conformity between the experimental and the theoretical values predicted by the model. The formulated bromelain was further subjected to in vitro testing. The enzyme exhibited more cytotoxic activity against murine melanoma (Tm5) tumor cell lines than in Chinese hamster ovary (normal CHO cell line) with the values that are almost comparable to that of cisplatin. In addition, recombinant bromelain also inhibited nitric oxide (NO) production at the highest concentration (400 μg/ml) and thus, the enzyme exerted good anti-inflammatory effects on the studied cell line. |
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| Physical Description: | xvii, 191 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 154-177). |