Production of a recombinant protein using Pichia pastoris grown in kraft evaporator condensate

• Main Author: Hoy, Preston Yee Ming
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/15696

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.