With the progressive implementation of Low-Carbon Policies based on Life Cycle Assessment (LCA) methodologies to assess climate change mitigation, biofuels' potential environmental advantages could finally be translated into economic revenues, especially for residue-based production chains. This implies, however, tackling environmental hotspots along the Life Cycle, such as the use of enzymes, an important input in cellulosic ethanol production. While relevant, however, aspects of enzyme manufacturing, crucial to the LCA results, are often oversimplified in databases or even undisclosed, which might render the accounting for this flow unreliable. A potential solution for this is to model enzyme production, disclosing aspects such as carbon source uptakes, energy consumption and purification steps, all of which may carry significant environmental burdens, especially for fossil-reliant procedures. Integrating these steps into the biorefinery process design as an on-site operation, however, would allow access to renewable energy and carbon sources for enzyme production, potentially reducing their burden in the final biofuel LCA profile, when compared with off-site supply. This work, then, aims to evaluate the effect of inserting on-site enzyme production within a stand-alone second-generation (2G) biorefinery, using sugarcane straw as feedstock, to produce ethanol and electricity, as opposed to off-site enzyme supply from manufacturers. The results show that adopting on-site production can reduce the overall environmental impact profile for cellulosic ethanol, in comparison to off-site supply, even though off-site data present significant variability among databases and literature. This trend persisted for varied enzyme dosages in the saccharification process and for different inventories to model off-site supply. The reference inventories for offsite supply lack transparency and details, while also displaying large discrepancies between impact indicators, indicating the need to pursue more consistent, reliable, and complete inventories for its modelling in LCA. Compared to first-generation (1G) sugarcane ethanol, 2G-ethanol (with on-site enzyme production) demonstrated a reduction in the Global Warming Potential category of around 80\%. However, relevant trade-offs for 2G ethanol (with off-site enzyme supply) were observed - regarding ecotoxicity, mineral scarcity, eutrophication and even the potential GHG reduction - when different enzyme inventories are used.