Gene Cloning And Characterization Of α-Galactosidases From Thermophilic Microorganisms

α-Galactosidase has both hydrolysis ability to catalyze the breakage of α-D-galactoside bond andtransglycosylation activity, and is widely used in the feed, medicine, food and chemical industries. Toalleviate the anti-nutritional effect of galactooligosaccharides in soybean meal and promote animalhealth, α-galactosidase is generally supplemented in the feed stuff. Due to the high cost, low yield, andthermoliability under high-temperature feed processing conditions, application of α-galactosidase ismuch limited. The objective of this study is to clone novel α-galactosidase genes from thermophilicmicroorganisms, to construct high-yield secretory engineering strains, and to provide cost-effectiveα-galactosidase candidates with excellent properties and high productivity for potential industrialapplication.Thermophilic Neosartorya fischeri P1, Talaromyces leycettanus JCM12802and Alicyclobacillussp. A4of our laboratory stock showed significant ability to produce high-temeprature-activeα-galactosidases and were used as the donor strains. By using degenerate PCR and genome sequenceanalysis, three α-galactosidase genes of glycoside hydrolase family27(gal27A and gal27B from N.fischeri and agaA from T. leycettanus) and two of family36(gal36P1from N. fischeri P1and gal36A4from Alicyclobacillus sp. A4) were obtained. BLAST analysis indicated that deduced amino acidsequences of these genes shared50–96%identities with that of functionally characterizedα-galactosidases. Homology modeling suggested that these proteins had a typical structure of (β/α)8barrel. The five gene products were successfully purified directly or purified from the cultures ofheterlogous hosts, and were subject to biochemical characterization individually.An acid, thermophilic α-galactosidase Gal27A was purified from N. fischeri P1, which internalpeptide sequences were similar to that of deduced gal27A. Native Gal27A and recombinant rGal27A,rGal27B and rAgaA produced in Pichia pastoris had a pH optimum of4.0–4.5and retained stable at pH3.0–11.0(>80%activity). The temperature optimum of rGal27B was75°C, higher than all knownfamily27counterparts and Gal27A (70°C), rAgaA (70°C) and rGal27A (60°C). When usinggalactomannan as the substrate, these recombinant enzymes exhibited degrading ability of differentdegrees. rGal27A showed the highest yield up to3.2g/L, and had a synergistic action with themannanase Man5P1to degrade galactomannan. rGal27B had the highest specific activity (477U/mg)and distinct ability to degrade natural substrates (raffinose and stachyose). rAgaA was highlythermostable, retaining>80%activity after60-min incubation at65°C.Both rGal36A4and rGal36P1were produced in Escherichia coli and purified to electrophoretichomogeneity. rGal36A4had the pH and temperature optima at6.0and60°C, and retained stable at pH5.011.0and up to60°C. The enzyme was specific for α-1,6-glycosidic bond and displayed wideacceptor specificity for transglycosylation. rGal36P1exhibited optimal activities at pH6.5and45°C,retained stable at pH5.0–11.0and up to45°C, and had great ability to degrade melibiose, raffinose andstachyose. In comparison with α-galactosidases of family27that release α-galactose from polymericgalactomannan, those of family36only degrade oligosaccharides (melibiose, raffinose, and stachyose). The reason might be that the large galactomannan molecules limit their access to the active site offamily36α-galactosidases.In summary, three α-galactosidase genes of family27and two of family36were cloned from threethermophilic strains. The native protein was purified, and the gene products were expressed at highlevels. Their enzyme properties were characterized, and the mechanism of substrate specificity ispreliminary studied. This study not only enriches the genetic resource of α-galactosidase, but alsoprovides good materials for the basic study and extensive applications.