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  • The advent of the genomic era has allowed

    2021-10-14

    The advent of the genomic era has allowed greater detailed investigation of enzymatic properties and has shown that specific enzyme activity is linked to the substrate utilization profiles of the isoenzymes [30], [31]. Not surprisingly, the functional four β-galactosidases from A. niger F0215 shared similar structures (Fig. 1 and Fig. 2), similar molecular weights between 106,500 and 111,840 (Table 2), yet there was substantial variation in the kinetic profiles (temperature and pH optima, substrate affinity). This might explain the multiple form patterns of the A. niger β-galactosidase reported in the initial study of Widmer and Leuba [12]. Many potential applications of these ASA 404 synthesis have been identified. Two are particularly attractive: (1) hydrolysis of lactose in dairy products or administration as a digestive supplement to prevent or treat lactose intolerance [32] and (2) transglycosylation of lactose to produce the valuable prebiotic GOS. All four β-galactosidases hydrolyzed and transgalactosylated lactose but with unique kinetic properties. pH is an important factor affecting the kinetics of lactose hydrolysis and GOS synthesis. In many cases, similar pH optima are reported for GOS synthesis and galactoside hydrolysis [18], [33]. In contrast, the A. niger β-galactosidases optimally synthesized GOS in acidic pH values (Fig. 3), although lactose hydrolysis occurred optimally at higher pH values (Table 3). These enzymes have been reported to be useful in the modification of the glycosyl side chains on xylan, pectin, xyloglucan, and arabinoxylan amongst other polymers [29]. The potential of A. niger β-galactosidases to modify polymers requires further investigation.
    Conclusion In addition to the known LacA in A. niger, three new β-galactosidases belonging glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. A. niger has at least four β-galactosidase family members with remarkably different biochemical properties.
    Financial support This study was funded in part by the National Natural Science Foundation of China (grant numbers 31601407, 31370076), the National Natural Science Foundation of Fujian Province, China (grant number 2016J01157), the Priming Scientific Research Foundation of Fuzhou University (XRC-1564).
    Conflict of interest
    Introduction Chitosan is a polyaminosaccharide composed of β-(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units. It is produced by the partial deacetylation of chitin, a major constituent of crustacean's shells which represent a waste material from the seafood processing industry [[1], [2], [3]]. Chitosan has several attractive properties, such as its natural origin, antibacterial activity, non-toxicity, easy modification, biodegradability, and low cost [[1], [2], [3], [4]]. These properties have led to its application in a wide variety of fields including medicine [5,6], wastewater treatment [7], agriculture [8], and biotechnology [[9], [10], [11], [12]]. One biotechnological use of chitosan is as carrier for enzymes immobilization [[10], [11], [12]]. The immobilization of enzymes is an effective and powerful tool allowing biocatalyst reusability, easy handling, prolonged lifespan, easy separation from products and flexibility of reactor design and operation [[13], [14], [15]]. Additionally, immobilization may improve the biocatalyst stability and modify its catalytic properties [[13], [14], [15], [16], [17]]. The presence of primary amino and hydroxyl groups in chitosan has allowed the immobilization of enzymes using different methodologies, including adsorption [[18], [19], [20]], and covalent bonding [[19], [20], [21], [22], [23], [24]]. Covalent bonding to a carrier has the advantage that the enzyme is tightly fixed, minimizing its leaching in aqueous media and contamination of the product with protein [25]. Additionally, multipoint covalent bonds between the enzyme and the carrier may cause a high degree of catalyst stabilization, since relative distances among the amino acid residues involved in the immobilization may be maintained during conformational changes induced by distorting agents [15,17,[25], [26], [27]]. The covalent immobilization of enzymes in chitosan has been carried out usually after the reaction of the polymer amino groups with the bi-functional reagent glutaraldehyde [11,[19], [20], [21],24]. The reaction of glutaraldehyde with chitosan allows the support activation with aldehyde groups that may react with the enzyme through its amino groups (ε-amino group of lysine residues and terminal amino group), and eventually with other surface functional groups (thiols, phenols, and imidazoles) [28]. Chemistry of the reaction between glutaraldehyde and amino groups is not fully understood and, according to the pH and concentration, the reaction mechanism may involve Schiff bases, nucleophilic substitution of amino groups, and Michael addition [28,29]. Furthermore, the reaction of glutaraldehyde with chitosan allows also the crosslinking of different polymer chains, resulting in the improvement of the support mechanical resistance and avoiding its solubilization in aqueous acidic media due to its cationic nature (pKa of the amino groups ~6.5) [11]. Another bi-functional reagent used for the activation and crosslinking of chitosan is epichlorohydrin that may react with the amino and hydroxyl groups of chitosan according to reaction conditions [12,[30], [31], [32]]. The derivatization of chitosan with epichlorohydrin allows the support activation with epoxide groups [[33], [34], [35]]. Part of these epoxide groups may be spontaneously hydrolyzed into diols under reaction conditions [35] and, consequently, a carrier with several functional groups may be obtained (epoxides, diols, and primary and secondary amino groups). Hydrolysis of residual epoxy groups into diols and their further oxidation to short-chain aldehyde groups results in a support with a more chemically homogeneous surface for the multipoint covalent immobilization of enzymes through the ε-amino groups of their lysine residues [22,23].