Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup.
An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.
Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala-147 002, Punjab, India. rssingh11@lycos.com
Ram Sarup
RS
Singh
Rajesh
R
Dhaliwal
Munish
M
Puri
D027761:Asparagus Plant / Q000737:chemistry; D002413:Cations, Divalent / Q000494:pharmacology; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D003911:Dextrans; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000302:isolation & purification / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology*; D019668:Liliaceae; D008670:Metals / Q000494:pharmacology; D029595:Orchidaceae; D010936:Plant Extracts / Q000378:metabolism; D011887:Raffinose / Q000378:metabolism; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D002413:Cations, Divalent; D003911:Dextrans; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D010936:Plant Extracts; D005632:Fructose; D013395:Sucrose; D007444:Inulin; C025614:sephadex; D006026:Glycoside Hydrolases; C023086:inulinase; D011887:Raffinose