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  • Isolation and application of cold adapted galactosidase in t

    2021-11-30

    Isolation and application of cold-adapted β-galactosidase in the production of low lactose dairy products would play a critical role in improving access to low lactose dairy products for lactose intolerant individuals [9,11]. Milk contains high levels of K+, Ca2+, and Na+, and β-galactosidase used in the industrial production of low lactose milk must be resistant to these three metal ions. We found that Bgal from Alteromonas sp. ML117 was not strongly affected by these ions. Bgal hydrolyzed 86% of the lactose in milk within 24 h at 10 °C. So far, except for the Paracoccus sp. 32d β-galactosidase [25], which can hydrolyze 91% of lactose after 11 h, the lactose hydrolysis efficiency of other cold-adapted β-galactosidases is similar to our findings. Given that Bgal had high tolerance to ethanol, it may be a promising alternative to production of ethanol from whey using lactose-based feedstocks. In conclusion, a novel β-galactosidase gene from the marine microorganism Alteromonas sp. ML117, Bgal, was identified and successfully expressed in E. coli BL21 (DE3) cells. This enzyme exhibited high activity at low temperatures, specificity to lactose. Bgal may have advantageous applications in the food industry, in particular in the production of low lactose milk.
    Acknowledgements This research was funded by Central Public-interest Scientific Institution Basal Research Fund, YSFRI, CAFS (No. 20603022018006) and the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2016ASKJ14).
    Introduction α-Galactosidases (EC 3.2.1.22) are exo-acting glycosidases that could catalyze the hydrolysis of terminal non-reducing α-D-galactose residues in various α-D-galactosides. They are widely distributed in microorganisms, plants and animals. Based on the amino Quercitrin sequence similarity, α-galactosidases have been classified into glycoside hydrolase (GH) families 4, 27, 31, 36, 57, 97 and 110 in CAZy database (http://www.cazy.org/). α-Galactosidases have a great application potential in many industrial processes such as soybean product processing [1], beet sugar production [2] and pulp manufacturing [3]. Raffinose family oligosaccharides (RFOs) such as raffinose and stachyose are main natural substrates of α-galactosidase. They are ubiquitously found in various legume seeds and vegetables, and negatively affect the quality or yield of bio-industrial products. For instance, RFOs in soybean have been considered as anti-nutritional factors because they cannot be digested in human and monogastric animal gut, resulting in flatulence and abdominal distress [4]. Raffinose in sugar beet prevented the crystallization of beet sugar, decreasing sucrose yield [2]. Due to the increased industrial demand for enzyme suitable for the removal of RFOs, many α-galactosidases have been studied [5]. However, most of them seemed to be unsuitable in practical application mainly because of their low hydrolysis ability toward RFOs [6,7]. Only a few α-galactosidases such as those from Rhizomucor miehei [8] and Bacillus megaterium [9] had been reported to have relatively high activity toward RFOs (several dozens of U mg−1), but they also required long hydrolysis time of more than 1 h to remove RFOs from soymilk. Thus, there is a great demand for α-galactosidase highly active on RFOs. Besides their hydrolysis ability toward RFOs, the stability of enzymes is also a critical parameter in practical application. The processing of soybean products includes blanching at >80 °C, and beet sugar production is usually performed under the condition of 65–70 °C [10]. Therefore, thermostable α-galactosidases can be well applied in these processes without cooling or after slight cooling. Furthermore, high reaction temperature can decrease the risk of contamination by mesophilic microbes and increase the hydrolysis rate of RFOs. On the other hand, α-galactosidases are often used as food and feed additives. In this case, the ingested enzymes are inactivated in the low gastric pH condition and degraded by digestive proteases [11]. Moreover, α-galactosidases are also used in combination with proteases during the processing of food and feed [12]. Due to the advantage of stable α-galactosidases in removing RFOs, several enzymes had been characterized with good thermostability, pH stability or protease resistance [8,10,[13], [14], [15], [16]]. However, there had been very few reports on a single α-galactosidase with good thermostability, pH stability and protease-resistance altogether.