**6. Perspectives**

Function and application of chitooligosacharides frequently depend on their size, and, therefore, the degrees of polymerization and acetylation. Substrate-enzyme synergisms determine the molecular weight of the generated COS. Gutierrez-Román *et al.* 2014 [174] tested three chitolytic enzymes ChiA, ChiB and ChiC, alone and in combination. In addition, three chitanases were tested in synergism with a chitobiase and a non-catalytic binding protein. When evaluated individually, ChiA was unable to hydrolyze chitin while ChiB and ChiC were able to degrade chitin and generate chitin monomers and dimers. When enzymes were tested pairwise (ChiA-ChiB, ChiA-ChiC, and ChiB-ChiC) the production of dimers and trimers was much higher, and monomers significantly lower than those seen with ChiB or ChiC individ‐ ually. However, higher concentrations of COS were obtained when the authors tested the four enzymes in combination with non-catalytic binding protein acting on chitin.

Further studies must be focused on the action of the enzymes on substrates with different degrees of polymerization and acetylation and N-acetylation pattern to improve the compre‐ hension of that synergism. In addition, researches involving synergism of non-catalytic binding proteins and hydrolytic enzymes should be developed in order to increase the understanding of oligomers syntheses [127]. Consequently, to produce size-specific chitooli‐ gosaccharides by enzymatic hydrolysis, further studies on genetic modification are necessary to overproduce enzymes and non-catalytic binding proteins, which will have a great impact on the quality of oligomers obtained and on the productivity of industrial processes.

Another important challenge in the development of biotechnological processes that employ agro-food industry residues as raw material is the direct fermentation of those raw materials. Obviously, direct fermentation of raw materials is closely related with the aforementioned aspects, since fermentative processes involve microbial growth and enzymatic hydrolysis, and process conditions that in many cases are different from physiological conditions. Moreover, it is important to give attention to screening of new enzymes from extremophile microorgan‐ isms, which usually catalyze reactions under non-physiological conditions such as high salinity, high temperature and low water activity [175].

As important part of the biotechnological process, bioreactors and enzyme (free or immobi‐ lized) are essential and need special attention to improve yields and productivities. Free enzymes in batch systems are the most conventional technology employed in the production of oligosaccharides by enzymatic hydrolysis. However, it has several important drawbacks, because enzymes are unstable, can be employed once and accumulation of products usually reduces their activity. These drawbacks are related directly to the quality of the product and the yield of the process. Development of novel technologies in order to solve those snags employing immobilized enzymes in column reactor and membrane systems have been studied. Column reactor packing with immobilized enzymes allows continuous production of oligosaccharides and has important advantages, such as increased operational stability of the enzyme and reduced accumulation which otherwise could lead to enzyme inhibition. The poorer affinity of immobilized enzymes is the main disadvantage of the application of column reactors at industrial scale. Studies should be directed towards the improvement of enzymesupport affinity. Membrane reactors are considered a new and attractive technology to produce oligosaccharides, in which enzymes are confined in the reaction side and continuously reused, with obvious implications for the efficiency and economy of the process. Low-cost and low-energy consumption are other important advantages to increase its utilization. The main limitation for industrial application of membrane reactors are fouling and polarization phenomena, which decrease considerably permeate flux, containing the produced oligosac‐ charides [176]. The main challenge to be studied in order to implement this technology advantageously in the industry is how to reduce the effect of these problems without affecting the stability of enzymes.
