Optimization Of Fermentation Conditions For β-Glucosidase Production By Penicillium Oxalicum And Preliminary Studies Of The Encoding Gene Cloning

β-Glucosidase, also calledβ-D-glucoside glucohydrolases, can hydrolyze non-reducing end group beta-D-glucosidic bond, and give off glucose and corresponding ligands at the same time. It is paid attention as the rate-limiting enzyme in cellulosedegradation. So far it has been widely used in many fields such as improving tea and juice's flavor, producing gentiooligosaccharide, soybean isoflavones active aglucone, natural pigment gardenia blue, natural antioxidant polyphenol compounds, and so on. This experiment mainly concentrate on strain screening, optimization of fermentation conditions, research of enzymology properties and gene clone. The results are as below:β-Glucosidase can hydrolyze geniposide to genipin, and genipin can be cross-linked to gardenia blue in the presence ofα-amino acid. Based on this principle, a rapid screening medium forβ-glucosidase-producing strains was designed. Through selecting from strains kept in the bioengineering laboratory with this method, Penicillium oxalicum is confirmed the highlyβ-glucosidase producing strain.First, the single factor test of temperature, pH, liquid medium volume, carbon sources, nitrogen sources, inorganic salts, surfactants and irritants during Penicillium oxalicum fermentation were carried out. Then the uniform experiment about proportion of the carbon, nitrogen, inorganic salts and surfactants was designed and performed. Through analyzing the uniform design datum by DPS (data processing system), the optimum fermentation conditions was determined: wheat bran 4%, beef extract 0.2%, NaCl 0.1%, CuSO₄ 0.08%, EDTA 0.2%, KH₂PO₄ 0.2%, optimized initial pH 7.0, 20 mL medium in 250 mL flask, and temperature 30℃. The final enzyme activity ofβ-glucosidase reached 52.18 U/mL, 3.1 times as it in the initial condition.The enzymology properties ofβ-Glucosidase were studied. The enzyme reaction optimum pH value and temperature were 5.0 and 70℃respectively. Different metal ions showed different effects on theβ-Glucosidase activity. Ag+ caused obvious inhibition of its activity, while Mn²⁺ and Al³⁺ effectively promoted it. Fe²⁺, Fe³⁺, Cu²⁺, Ca²⁺, Zn²⁺, Co²⁺, Ni²⁺, Mg²⁺ and K⁺ had no obviously effect. The research about organic solvent showed that 10% ethanol, 10% methanol, 10%-40% butarol, 10%-20% ethyl acetate and 10% acetone all could more or less enhance the enzyme activity, and yet they showed obvious inhibition with higher concentration. Theβ-Glucosidase could keep stability up to 20h under 70℃and 12h in pH 2.2-8.0.In the study ofβ-Glucosidase gene of Penicillium oxalicum, the middle fragment was obtained through RNA extraction and RT-PCR. The 5'sequence was obtained through Genome Walking. Unfortunately, the full length of the gene had not been obtained.