Grapes Of Beta-glucosidase Producing Strains And Optimization Of Culture Conditions And Beta-glucosidase Application

β-glucosidase is a key enzyme of cellulose system,which plays an important role in the cellulose degradation,moreover,they can be applied for flavor enhancement in food and beverage industries.Their activities directly affect the efficiency of cellulose degradation.However,theβ-glucosidase activities of currently known cellulase-producing strains are generally low.Improvement for these strains by genetic engineering is time-consuming and arduous.Screening of theβ-glucosidase producing strains directly from nature is an effective way.The main results of this paper are as follows:(1) A new method named "glycosides aescinate plate" in the screening ofβ-glucosidase producing strains was developed inspired by the bacteria "bile-aescinate glycosides" experiment,which was applied in the rapid screening ofβ-glucosidase producing strains.After enrichment cultivation of soil samples with decomposed straw,thirteenβ-glucosidase producing strains were isolated on the glycosides aescinate plate.Three strains showed higher activities.One strain named Peni-1 with the highestβ-glucosidase activity was used as starting strain for further study.By observing the morphology characteristics and analyzing the 18S rDNA sequence of strain Peni-1,the strain was identified as Penicillium decumbens.(2) In order to enhance the enzyme activity of the strain Penicillium decumbens Peni-1,the fermentation medium and fermentation conditions were optimized., The optimum medium was obtained by single factor and orthogonal experiments, that is,wheat bran 3%,meal 3%,microcrystalline cellulose 0.6%,(NH₄)₂SO₄ 0.4%,KH₂PO₄ 0.1%,MgSO₄ 0.05%,CaCl₂ 0.05%,trace examples of liquid 1ml/ L medium;the optimized cultivation conditions were as follows:50 ml medium in 300 ml cone-shaped bottle,200 r/min,30℃.β-glucosidase enzyme activity of the strain Peni-1 increased 3 times under optimized cultivation condition after 6 days' fermentation.The value of the enzyme activity reached 15.3 IU/ml,which is higher than previously reported strains of Penicillium,Aspergillus,Trichoderma genus. (3)β-glucosidase enzymatic properties of Penicillium decumbens Peni-1 were also studied.The optimum temperature for the enzyme is 70℃,β-glucosidase activity reached 44.3 IU/ml,at 70℃,which increased by 3 times compared with that at 50℃.β-glucosidase of the strain Peni-1 is stable at a temperature range of 0～70℃.The optimum pH for the enzyme is pH 4.5,the enzyme is stable at a pH range of pH 3.0～10.0.(4)β-glucosidase gene of Peni-1 was also cloned.Peni-1β-glucosidase gene sequences showed a a similarity of 97%with that of the strain Penicillium decumbens JU-A10β-glucosidase,but some differences in their amino acid residues were still found.This work provides a reference for the next research.(5) Except as a key component in the cellulose degradation enzyme complex,β-glucosidase is also widely used as flavor enhancer in food and beverage industries.In this paper,β-glucosidase was used to hydrolyze polydatin in Chinese herb Polygonum cuspidatum for the production of resveratrol.This is a new application ofβ-glucosidase.Polydatin in Polygonum cuspidatum was beta-glycoside as the most glycosides in natural plants.Therefore,polydatin can be hydrolyzed to resveratrol byβ-glucosidase quickly and efficiently.The conversion rate of polydatin is beyond 90%.The transformation of polydatin to resveratrol byβ-glucosidase in large scale demands for the high activity and stability ofβ-glucosidase.In this paper,Peni-1β-glucosidase was immobilized and used to hydrolyze polydatin.The reuse results indicated that the conversion rate maintained at more than 85%after the same batch of immobilizedβ-glucosidase was used to hydrolyze polydatin continuously for 6 times.The amount of the resveratrol extracted from Polygonum Cuspidate after transformation byβ-glucosidase reached 5.8 mg/g.This makes the strain be a good candidate in the future industrial production.