Every year, an average of 5.3 million tons of sugar are produced in Mexico. According to the national statistical organizations, there are 12 million Mexicans that depend economically from the production of sugar. Cane sugar mills are located in 15 of the 32 Mexican states. The cities with sugar cane industrial activity have their infrastructure, services, culture and education based on this industry. The sugar industry generates more than 450 thousand jobs and benefits approximately 2.2 million people. However, this sector is facing some rising problems: a decrease in the international sugar prices ( FAO & OECD ), and a decrease in internal consumption because of the replacement of sucrose by fructose and artificial sweeteners. After NAFTA was passed in 2008, high fructose corn syrup no longer has an import tariff for its entrance into Mexico and is more industrially viable compared to sucrose. In this context, it is possible to identify some new opportunities in this industry: the diversification of the uses of sugar cane products and by-products, and the development of new technologies for more sustainability of the sugar cane industry.
Inverted sugar contains fructose and glucose in equal proportions. This product has a greater demand than pure glucose as a food and drink sweetener due to many useful physical and functional attributes of fructose including sweetness, flavor enhancement, humectancy, color and flavor development, freezing-point depression, and osmotic stability. (Hanover & White, 1993). Sugarcane bagasse constitutes the fibrous residue of sugar cane after undergoing conventional milling, which contains about 50% cellulose, 25% hemicellulose and 25% lignin (Pandey et al., 2000). Bagasse is of low economic value and constitutes an environmental problem to sugar mills and surrounding districts because many mills burn large portions of the bagasse. (Lavarack et al., 2002) However, it can serve as an ideal substrate for microbial processes for the production of value-added products (Pandey et al., 2000), specifically as an ideally inexpensive and abundantly available source of sugar for fermentation into fuel ethanol (Yanase et al., 2005). The implementation of new technologies that enable the production of inverted sugar from sucrose, and the use of sugarcane bagasse as a bio-fuel source could benefit 3 principal areas: a food industry, green energy and the environment. Introduction of enzymatically-inverted sugar could profit sugar mills by generating a diversification of sugarcane products, and enzymatic hydrolysis of cellulosic components of sugarcane bagasse could contribute to the production of bio-ethanol, in this way, reducing CO2 emission during the burning process of the excess bagasse after milling.
A cellulase was considered for the new application of sugarcane bagasse. Clostridium thermocellum endoglucanase CelD is an enzyme that belongs to the family E cellulases. Family E includes, beside C.thermocellum CelD, a number of cellulases such as Butyrivibrio fibrisolvens cellodextrinase Cedl, C. thermocellum endoglucanase CelF, Cellulomonas fimi endoglucanase CenB, Clostridium stercorarium Avicelase I, Persea americana endoglucanase, Dictyostelium discoideum endoglucanase, Cellulomonas fimi endoglucanase CenC, and Pseudomonas fluorescens var. cellulosa endoglucanase A. (Chauvaux, Beguin & Aubert, 1992).
The second enzyme considered was an invertase. Zymomonas mobilis is a gram negative bacterium that produces ethanol from glucose, fructose and sucrose (Swings & DeLey, 1977) at a rate three to four fold higher, and at a higher final yield compared to the traditionally used yeast strains (Rogers et al., 1982). Almost 60 % of the extracellular sucrase activity of Zymomonas mobilis is the result of the activity of the extracellular SacC. This sacC gene expressed in Escherichia coli BL21 exhibited sucrase activity of 1948 - 2672 U/mg while the un-induced strain expressed 12.8 – 24.6 U/mg . One unit of sucrase was defined as the amount of enzyme releasing 1umol of reducing sugar per minute. It is a monomer in its native state, with a molecular weight of 46 kDa. (Gurunathan S & Gunasekaran P, 2004)
Industrial production of invert sugar is usually based on the acid or enzymatic hydrolysis of sucrose. Acid hydrolysis is based on the application of strong mineral or weak organic acids. The disadvantage of acid hydrolysis is the possible presence of impurities in the product introduced by uncontrollable parameters during conversion. On the other hand this conversion can also be achieved by enzymatic action of invertase on sucrose with a conversion efﬁciency of almost 100% without the inherent disadvantages of acid hydrolysis. (Safarik et al., 2009) As an alternative method to the traditional chemical process to produce inverted sugar, cell surface display was suggested. Cell surface display is a technique to display proteins on the surface of bacteria, fungi, or mammalian cells by fusing them to surface anchoring motifs. This technique has a wide range of biotechnological and industrial applications, including development of vaccines, peptide and antibody libraries, bioremediation, whole-cell-biosensors, and whole-cell-biocatalysis. When proteins are expressed in the outer membrane of E. coli, the cell envelope acts as their matrix. This display is achievable thanks to several displaying systems as outer membrane porins, lipoproteins, GPI-anchored-proteins, fimbriae, and autotransporters. (Jana S & Deb JK, 2005; Lee SH et al., 2004) Displaying proteins on the cell surface also makes preparing or purifying them unnecessary in many instances. Whole cells displaying the molecule of interest can be used in industrial process reactions or analytical assays and then can be simply recovered by centrifugation. (Joachim J & Meyer TF, 2007)
The Tat (twin-arginine translocation) system functions to translocate folded proteins across the membrane while the Sec secretory pathway translocates unfolded substrates. The Sec translocase is comprised of the SecYEG translocation channel and the accessory components SecA, SecDFYajC, and YidC. (Yuan J et al., 2010 & Yuan J et al ., 2010) Type II secretory system and the type V autotransporter system are natural translocation systems to import/export substrates through the periplasm and membrane. The type II secretory system can take both Tat and Sec system while the type V autotransporters use the Sec system. Sec secretory pathway is composed of an N-terminal Sec-dependent signal peptide, a passenger domain, and a translocator domain that is predicted to form a β-barrel. (Rutherford et al., 2006) Both lpp and phoA signal peptides are natural Sec-dependent E. coli signal peptides which permits the translocation of the outer membrane proteins by the type II and the type V system, respectively.
In this project, the natural passenger domain of the autotransporter estA from Pseudomonas sp was replaced by a cellulase (CelD) and an invertase (SacC) as means to display them at the bacterial surface by the translocator domain of the estA protein. Also, these enzymes were fused with an integral outer membrane fragment of ompA protein (BBa_K103006) to display them at the outer membrane of E. coli. Using the signal peptide of a protein which is naturally transported to the cytoplasm (signal peptide of phoA and lpp), we intend to export CelD and SacC to the external surface of E. coli. The sequence of CelD used to construct our genetic frame was modified according to Chauvaux et al., substituing Asp523 by Ala, since that mutation increases the specific activity of CelD to 224% . (Chauvaux S et al., 1992) Our genetic construct will be able to immobilize an invertase (sacC) and a cellulase (celD) on the outer membrane of E. coli, fusing the enzymes with fragments of ompA and estA. This chassis will be capable of transforming sucrose into fructose or hydrolyzing a complex material of sugarcane bagasse into reducing sugars without further downstream primary recovery, purification, or immobilization steps to obtain enzymes; thus reducing the amount of unit operations and cutting production costs.
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