Purification,Characterization And Applications Of A Novel β-galactosidase From Lotus Seeds In Lactose Hydrolysis

Lactose intolerance is a global problem,and in particular,the incidence rate in China is as high as 85%.If not effectively treated,it can lead to abdominal bloating,gas,stomach cramps and diarrhea.β-galactosidase is an essential glycoside hydrolase for the degradation of lactose and the solution of lactose intolerance.Microbialβ-galactosidase is the most widely studied,but there are few studies on plantβ-galactosidase with high safety and low price.Lotus seeds are among the first to be included in the dual use list of medicine and food in China,with abundant resources,high protein content,and high enzyme safety.Research has found that the crude enzyme solution of lotus seeds containsβ-galactosidase activity,andβ-galactosidase from this source has not been reported.It is safe and inexpensive to develop newβ-galactosidase for providing resources.Thus,in this paper,lotus seeds were used to produceβ-galactosidase and the following studies were carried out on the enzyme:isolation and purification,enzymatic characterization,application research of lactose hydrolysis function and immobilization research.Below are key research findings.（1）A method was developed for extraction ofβ-galactosidase enzyme from lotus seeds.Firstly,the lotus seeds powder was defatted,and the crude enzyme was extracted at pH5 which gave enzyme activity of 2.8 U/m L.Purification ofβ-galactosidase from crude extract was performed using various steps.Purification steps included ammonium sulfate precipitation and then using the Phenyl-Sepharose 6FF hydrophobic interaction chromatography and SP-Sepharose Fast Flow cation exchange chromatography.The purification yield of the target enzyme reached 7.4 fold with a specific activity of 1.3 U/mg.The purified enzyme was analyzed by SDS-PAGE which showed a single band having relative molecular weight of about 50 k Da which was confirmed to be aβ-galactosidase by zymography.（2）The biochemical characteristics of the purifiedβ-galactosidase from lotus seeds were studied.The optimal pH and temperature of the target enzyme was 4.0 and50℃,respectively.The enzyme had good thermal stability in the range of temperature20-50℃,and remained stable in the range of pH 4.0-10.0 with high enzyme activity.Compared with the control group,β-galactosidase activity was significantly activated by various metal ions such as K⁺（113%）、Na⁺（115%）、Mg²⁺（113%）、Fe²⁺（117%）、Ca²⁺（113%）、Ba²⁺（115%）、Mn²⁺（128%）.Zn²⁺（109%）and Co²⁺（103%）had little effect on the enzyme activity.The enzyme activity was slightly inhibited by Cu²⁺（96%）,and significantly inhibited by Ag⁺（6%）and Hg²⁺（9%）.The metal chelator ethylenediamine tetraacetic acid（EDTA）had no inhibitory effect on the target enzyme,and sulfhydryl reagentsβ-mercaptoethanol（92%）had no significant inhibitory effect on the target enzyme,while the denaturant sodium dodecyl sulfate（SDS）completely inhibited the activity of the target enzyme.Galactose（106%）,fructose（105%）and sucrose（108%）with 0.2 mol/L concentration could activate the target enzyme to some extent,while lactose（96%）and glucose（88%）could inhibit the target enzyme to some extent.High concentration（0.5 mol/L）of glucose（77%）inhibited the activity of the target enzyme,but in high concentration of galactose（0.5 mol/L）,the target enzyme still showed full activity（100%）.To sum up,lotus seedsβ-galactosidase does not need divalent metal ions to maintain its catalytic activity.Cysteine may not participate in the catalytic site of the enzyme.Galactose,the product of lactose hydrolysis at high concentration,has no inhibitory effect on it.（3）Both crude enzyme and purified enzyme could hydrolyze lactose.In different concentration of lactose hydrolysis experiments,the target enzyme not only degraded lactose,but also generated prebiotic galactooligosaccharides（GOS）.Lotus seeds powder had dual functions of hydrolyzing lactose and generating GOS in milk and whey.Simulate of the tolerance ofβ-galactosidase from lotus seeds to the gastric juice environment in vitro was performed at 37℃for 4 hours.The target enzyme was still active in the gastric environment.These results indicate that lotus seedsβ-galactosidase from plants has good activity and hydrolytic ability,and hence it has the potential to develop and apply to the food industry.（4）Theβ-galactosidase from lotus seeds was immobilised on chitosan microsphere.Compared with free enzyme,immobilized enzyme showed better enzyme activity at higher temperature and had certain thermal stability in the temperature range of 20-70℃.The optimal pH range was expanded,with a wider pH stability in the range of 3.0-10.5.Galactose and glucose had different degrees of inhibition on the immobilized enzyme.It is worth noting that free enzyme（100%）was not inhibited by galactose but immobilisedβ-galactosidase（52%）was significantly inhibited by galactose at high concentration（0.5mol/L）.However,the inhibition of immobilized enzyme（90%）by glucose was significantly reduced compared with free enzyme（77%）at high concentration（0.5mol/L）.To sum up,the immobilizedβ-galactosidase has obviously stronger stability and higher enzyme activity in terms of temperature and pH,and interesting properties and hence its structure and function need to be further explored to provide more potential for development and application.