Screening Of Highly Selenium-tolerant Ganoderma Lucidum,extraction Of Selenoproteins And Research Of Special Enzymes For Cell Wall Disruption

Selenium is an indispensable trace element and an important component of various selenoproteins in the human body.For humans,inorganic selenium is toxic and difficult to absorb.Using Ganoderma lucidum as an intermediate carrier for selenium enrichment,through biological transformation,inorganic selenium can be transformed into organic selenium,which is beneficial to humans and less toxic.Selenoprotein is the main form of organic selenium present in fungi.Selenoproteins and selenium-containing peptide molecules are a selenium supplement with special physiological functions,such as anti-tumour effects and immunomodulation.Researchers have paid a lot of attention on how to effectively use selenoprotein-rich resources in the food industry.Most of the large fungal protein extraction processes are based on ultrasound-assisted alkali methods,which have the advantages of high extraction efficiency and easy operation,but the long ultrasound time or high alkali concentration will damage the protein structure,making it difficult to improve the final protein extraction rate.In the enzymatic extraction process,the reaction conditons are mild and high protein yield can be attained,however,it has the disadvantage of high cost.The development of a highly efficient selenoprotein extraction process is of great practical significance for utilization and further processing of selenoprotein.In this paper,a highly selenium-tolerant Ganoderma lucidum strain was obtained by screening and mutagenesis breeding.For obtaining selenoprotein,β-1,3-glucanase and pectinase were heterologously expressed to hydrolyze Ganoderma lucidum cells.The extraction rate of selenoprotein from Ganoderma lucidum was substantially increased by enzymatic hydrolysis assisted by short time ultrasonic wall breaking and alkaline lysis.The main results were as follows:（1）A mycelium strain ST308 of selenium-tolerant Ganoderma lucidum was obtained from a natural environment with high selenium in China,and a highly selenium-tolerant Ganoderma lucidum mutant strain ML2613 was further obtained by ⁶⁰Co-γ-ray mutagenesis breeding.Comparison of the fermentation showed that ST308 and ML2613 grew at approximately the same rate in the fermentation medium.The growth of the starting bacterium ST308 was significantly inhibited in the highly selenium liquid fermentation medium,while the growth rate of ML2613 was significantly higher than that of the starting bacterium,with a dry weight of 17.3 g·L^-1at day 5.（2）The Na₂Se O₃·5H₂O concentration of ML2613 in a 15 L fermentor was 180 mg·L^-1.After 72 h of fermentation,the dry weight of the bacteria was 16.4 g·L^-1,and the selenium content of the selenium-enriched Ganoderma lucidum mycelium was 821.4 mg·L^-1.The crude protein was extracted by ultrasonic-assisted alkaline method,the protein extraction yield was37.2%,the protein isoelectric point p H was 3.0,and the lyophilized Ganoderma lucidum protein was further purified with saturated ammonium sulfate,and the total protein selenium was 623.0mg·kg^-1.The speciation of selenoamino acids in Ganoderma lucidum selenoproteins was analyzed by HPLC-ICP-MS,and the quantitative analysis of three selenoamino acids,selenocysteine,selenomethylcysteine and selenomethionine in the selenoprotein of Ganoderma lucidum were quantified using HPLC-ICP-MS coupled analysis technique at 457.2 mg·kg^-1,6.5mg·kg^-1and 147.2 mg·kg^-1.In Ganoderma lucidum selenoproteins,selenoamino acids are mainly selenocysteine and selenomethionine.The selenium content of selenoamino acids in selenoproteins was calculated to be 276.2 mg·kg^-1,and it was concluded that the selenium content of selenoamino acids accounted for 44.3%of the total selenium in selenoproteins.（3）Heterologous expression of theβ-1,3-glucanase gene from Cellulosimicrobium CICIM6906.The recombinant enzyme BGL6906 was obtained by using E.coli BL21（DE3）as the host bacterium to construct recombinant BL21/6906 and expressβ-1,3-glucanase.the pure enzyme specific activity of recombinant enzyme BGL6906 was 567.8 U·mg^-1 at an optimum p H of 5.5 and an optimum temperature of 50°C when Poria polysaccharide was used as the substrate.The recombinant bacteria were fermented in a 15 L fermenter for 72 h.Most of the enzyme activity was secreted extracellularly and the extracellular enzyme activity reached 67.0 U·m L^-1.The mycelium of Ganoderma lucidum was alternately hydrolysed with BGL6906 and commercially available pectinase,and the morphology of the mycelium changed significantly with the appearance of a small amount of protoplasts.The lysis process of Ganoderma lucidum cells was thus established:the mycelium was subjected to two enzymatic digestions and two filtrations to extract Ganoderma protein by ultrasound-assisted alkaline method,and the protein extraction rate was as high as 76%at 5 min of ultrasound.（4）The protease in commercially available pectinases is highly degrading to BGL6906,therefore pectinases with low protease activity is needed.For this purpose,the expression of pectinase in different hosts of Pichia pastoris and the methods of hydrolysing mycelium of Ganoderma lucidum together with BGL6906 were investigated.The recombinant bacterium GS115/pg605 and GS115/pmj5a were constructed to express polygalacturonase and pectin methylesterase,respectively,using the yeast GS115 as the host bacterium.The enzyme activities of the recombinant pectin hydrolase and esterase were 114.0 U·m L^-1 and 5.6 U·m L^-1,respectively.GS115/pg605 and GS115/pmj5a were fermented in shaking flasks and concentrated,and the concentrated enzyme solution was made into an enzyme mixture with an enzyme activity ratio of 5:1,named as pectinase PGMJ5A.The recombinant bacterium SMD1168/pg605 and SMD1168/pmj5a were constructed to express polygalacturonase and pectin methylesterase,respectively,using the protease-deficient host bacterium Bilobacterium SMD1168 as the host bacterium.The maximum enzyme activity was 194.5 U·m L^-1 for SMD1168/pg605 and 16.2 U·m L^-1 for SMD1168/pmj5a at 156 h.The fermentation broth was collected and concentrated separately,and the concentrated enzyme broth was made into a mixed enzyme broth in the ratio of 5:1 enzyme activity,named as pectinase SPGMJ5A.The mixture of BGL6906 and PGMJ5A hydrolyzed the mycelium of Ganoderma lucidum,and the morphology of the mycelium did not change significantly.It was found that the pectinase expressed by GS115 still had high protease activity,whch caused that BGL6906 was degraded and unable to hydrolyze the cell wall of Ganoderma lucidum synergistically.Although a large amount of protoplasts could not appear,the morphology of the mycelium was obviously changed and its cell wall strength showed weakened to some extent.The protein extraction rate of Ganoderma lucidum was 54%at 10 min of sonication,while the protein extraction rate of the control group was only 27%in the same time.