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Production of a recombinant protein using Pichia pastoris grown in kraft evaporator condensate Hoy, Preston Yee Ming


Increasingly, environmental regulations are guiding industries to adopt practices which minimize water use and associated effluent discharge. In the pulp and paper industry, this has resulted in a trend to recycle waste streams as process water within various mill operations. Since instituting environmentally sound practices is often a balance between satisfying regulatory requirements and plant economics, any process which may satisfy both goals should be investigated further. In this study, the feasibility of cultivating a recombinant strain of the methylotrophic yeast, Pichia pastoris, on combined evaporator condensate from a Kraft mill was investigated. The use of a recombinant yeast in this application is novel and the reasoning behind this research is two-fold. Firstly, to facilitate complete methanol removal from a waste stream, rendering it suitable for re-use within the mill or discharge. Secondly, to generate a product of potential economic value in the pulp and paper industry. A literature review was conducted to generate a list of Pichia transformants expressing potentially useful products. Through a selection process that rated each candidate based on a criteria set, a recombinant Pichia strain expressing lipase from Geotrichum candidum was selected. A series of shake flask experiments were performed to gauge the effect of various media compositions on yeast growth. This was followed by fed-batch cultivations in a 1.8 L reactor which was monitored and controlled via a program written in Lab VIEW [National Instruments, Austin, Texas]. Feeding was automated using a feedback algorithm loosely based on the SCF technique which used DO patterns to initiate cycling. Cell densities of 8-12 g/L (dry weight) were reached within the reactor grown on a condensate/methanol feed. Lipase activity was determined titrimetrically and was found to occur only in the presence of yeast-peptone. Maximum enzyme activities for the reactor trials were in the range of 10.8 - 13.9 μmol/min/mL. Protein concentrations for the two final runs were measured at 57 and 48 mg/L, yielding respective specific activity values of 220 and 140 jjmol/min/mg. These values are roughly 6-7 times lower than cited in literature and is thought to have been resultant from various factors including proteolytic deactivation. This study has demonstrated the possibility of growing a recombinant yeast in a kraft waste effluent to produce a useful product.

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