In vitro synthesis of a microfibrillar (1→3)‐β‐glucan by a ryegrass ( Lolium multiflorum ) endosperm (1→3)‐β‐glucan synthase enriched by product entrapment

Abstract

A simple, rapid procedure has been developed to purify (1→3)‐β‐glucan synthase (UDP‐Glc:(1→3)‐β‐glucan 3‐β‐ d ‐glucosyl transferase (EC 2.4.1.34)) over 400‐fold from membrane preparations of Italian ryegrass ( Lolium multiflorum ) and 60‐fold from CHAPS‐extracted membranes. When a CHAPS‐extract of the membranes is treated with 8 mM CaCl 2 , proteinaceous material is precipitated. Although less than 10% of CHAPS‐solubilized protein is removed in this step, the total activity recovered in the supernatant increases fourfold. Thus, CaCl 2 precipitation appears to be important in removing inhibitors of the (1→3)‐β‐glucan synthase. In the presence of 1 mM UDP‐glucose, the supernatant after CaCl 2 treatment produces a high molecular weight, insoluble product that entraps a (1→3)‐β‐glucan synthase of high specific activity. The product‐entrapped enzyme preparation contains six major polypeptides, and comparison of the SDS—PAGE pattern of this fraction with the polypeptide profile of an immunoprecipitated (1→3)‐β‐glucan synthase preparation suggests that polypeptides at 30–31 and 55–58 kDa are the most likely candidates for participation in (1→3)‐β‐glucan synthesis. When the reaction is performed on a larger scale, milligram quantities of product can be seen precipitating from the reaction mixture within 1 h of substrate addition. This product has been characterized by methylation analysis, 1 H‐ and 13 C‐nmr spectroscopy, X‐ray diffraction, electron microscopy, size exclusion chromatography, UV‐induced fluorescence in the presence of the (1→3)‐β‐glucan‐specific fluorochrome from aniline blue, and enzymic hydrolysis with a specific (1→3)‐β‐glucanase. These physical, chemical and enzymic analyses clearly demonstrate that the product is a microfibrillar (1→3)‐β‐glucan with a degree of polymerization of about 1500.