Production and characterization of protease by Bacillus licheniformis on skim latex serum fortified media /
Protease from Bacillus licheniformis (ATCC 12759) can be produced using a readily available agro-industrial residue as potential substrate. Skim latex serum effluent is an abundant and inexpensive liquid waste from natural rubber industry that provides various organic compounds for microbial growth....
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
IKulliyyah of Engineering, International Islamic University Malaysia,
2015
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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: | Protease from Bacillus licheniformis (ATCC 12759) can be produced using a readily available agro-industrial residue as potential substrate. Skim latex serum effluent is an abundant and inexpensive liquid waste from natural rubber industry that provides various organic compounds for microbial growth. The present study utilized skim latex effluent as a basal medium to cultivate B. liceniformis for extracellular protease production. Statistical based experimental designs were adopted to optimize the physicochemical factors for the maximization of protease production. Screening the eleven factors such as lactose, galactose, casein, KH2PO4, MgSO4.7H2O, LB broth, skim latex serum, inoculums size, agitation, initial pH, and temperature for protease production was performed using Plackett-Burman design prior to optimization. Four variables (galactose, skim latex serum, agitation and pH) were identified as the most critical factors and selected for further optimization to enhance protease production using Face Centered Central Composite Design (FCCCD) under Response Surface Methodology (RSM). The protease production was found to increase from 2 U/ml to 19.35 U/ml approximately a nine fold increase as compare to the original medium. The validation of developed model was established to verify the adequacy and accuracy of the model, and the results showed that predicted value agreed well with experimental value with error less than 20 %. ANOVA of the quadratic model showed a significant of the model (p = 0.0002) with high determination coefficient (R2 = 0.9537) indicating a satisfactory fit of the model with experimental data. Following the optimization strategy, the sequential purification steps of the optimized media were conducted using ammonium sulphate precipitation, dialysis and ion exchange chromatography. The results revealed that the enzyme activity increase to 2.28 fold of purification compare to the crude enzyme. Assessment of the purified protein by SDS PAGE showed a single band with molecular mass of about 47 kDa. The enzyme was stable at temperature range of 35 oC to 65 oC and also at pH 6.0 and 7.0 for 60 min. The stimulatory effects on protease activity were observed in the presence of Mn2+and Ca2+ , while inhibitory effects were found in the presence of Cu2+, Zn2+, Mg2+, and EDTA. This indicated that the produced protease might be a metallo protease. In the case of detergent application, the enzyme exhibited the stability toward surfactants (Triton X100, Tween 20, SDS), solvents (acetone, chloroform, hexane and toluene), oxidizing agent (H2O2) and Tesco Everyday Value® detergent with the residual activity around 80 %. It also demonstrated the removal activity of blood stain completely with supplementation of the 7 mg/ml detergent solution. The characteristics of produced protease suggest that it may be used as a potential additive for detergent formulation as well as laundry detergent and clinical waste treatment. |
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| Physical Description: | xviii, 172 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 139-155). |