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  1. Abdullah N, Chase HA
    Biotechnol Bioeng, 2005 Nov 20;92(4):501-13.
    PMID: 16080185
    Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
  2. Lam SS, Liew RK, Cheng CK, Rasit N, Ooi CK, Ma NL, et al.
    J Environ Manage, 2018 May 01;213:400-408.
    PMID: 29505995 DOI: 10.1016/j.jenvman.2018.02.092
    Fruit peel, an abundant waste, represents a potential bio-resource to be converted into useful materials instead of being dumped in landfill sites. Palm oil mill effluent (POME) is a harmful waste that should also be treated before it can safely be released to the environment. In this study, pyrolysis of banana and orange peels was performed under different temperatures to produce biochar that was then examined as adsorbent in POME treatment. The pyrolysis generated 30.7-47.7 wt% yield of a dark biochar over a temperature ranging between 400 and 500 °C. The biochar contained no sulphur and possessed a hard texture, low volatile content (≤34 wt%), and high amounts of fixed carbon (≥72 wt%), showing durability in terms of high resistance to chemical reactions such as oxidation. The biochar showed a surface area of 105 m2/g and a porous structure containing mesopores, indicating its potential to provide many adsorption sites for use as an adsorbent. The use of the biochar as adsorbent to treat the POME showed a removal efficiency of up to 57% in reducing the concentration of biochemical oxygen demand (BOD), chemical oxygen demand COD, total suspended solid (TSS) and oil and grease (O&G) of POME to an acceptable level below the discharge standard. Our results indicate that pyrolysis shows promise as a technique to transform banana and orange peel into value-added biochar for use as adsorbent to treat POME. The recovery of biochar from fruit waste also shows advantage over traditional landfill approaches in disposing this waste.
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