The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:4119 |
| Date | January 2014 |
| Creators | Morrison, David Graham |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | 164 p., pdf |
| Rights | Morrison, David Graham |
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