Invertase of Saccharomyces cerevisiae SAA-612: Production, characterization and application in synthesis of fructo-oligosaccharides

Highlights

• Among nine yeast strains screened, Saccharomyces cerevisiae SAA-612 showed highest invertase activity.

• Response Surface Methodology led to 24 fold enhancement in production of invertase from Saccharomyces cerevisiae SAA-612.

• The invertase from S. cerevisiae SAA-612 exhibited good potential for the synthesis of fructo-oligosaccharides (FOS).

Abstract

Production and characterization of invertase from Saccharomyces cerevisiae SAA-612 were carried out and transfructosylation activity was also studied for the synthesis of fructo-oligosaccharides (FOS). Optimization of invertase production following Response Surface Methodology resulted in 24 fold increase in the production of invertase activity of S. cerevisiae SAA-612. Optimum temperature and pH for invertase activity were observed to be 40 °C and 6.0, respectively. The enzyme was found to be stable at 30 °C and 40 °C. K_m and V_max of invertase of S. cerevisiae SAA-612 were calculated to be 11 mM and 434.7 U/mg protein, respectively. The maximum FOS synthesis was observed with 250 mg sucrose and 2.5 U invertase in one ml reaction, 5.5 pH, 40 °C and 4–8 h of incubation. This investigation can be further extended to explore the prebiotic potential of synthesized FOS.

Introduction

Invertases or β-fructofuranosidases (EC.3.2.1.26) are members of the GH32 family of glycoside hydrolases that catalyse the hydrolysis of sucrose to give an equimolar mixture of monosaccharide d-glucose and d-fructose called invert sugar. Invertases are widely distributed in the biosphere and mainly isolated from plants (Alberto et al., 2004, Hussain et al., 2009) like Japanese pear fruit (Pyrus pyrifolia), pea (Pisum sativum), oat (Avena sativa), sugarcane (Saccharum officinarum) and microorganisms e.g. Saccharomyces cerevisiae (Kulshrestha, Tyagi, Sindhi, & Yadavilli, 2013;; Ansari, Yadav, & Lal, 2013), Aspergillus sp. (Nyugen et al., 2005, Guimaraes et al., 2009), Candida utilis (Belcarz, Ginalska, Lobarzewski, & Penel, 2002), Neurospora crassa, Fusarium oxysporium, Phytophthora meganosperma, Schizosaccharomyces pombe and Schwanniomyces occidentalis (Silveira, Oliveira, Carvajal, & Bon, 2000).

Invertase is extensively used in confectioneries, food industries and in the production of high fructose sugar syrup from sucrose (Kumar et al., 2001, Uma et al., 2010). It is used to produce artificial honey and able to catalyse transfructosylation to produce fructo-oligosaccharides (FOS) such as kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). However, inulinase has also been used to synthesize fructo-oligosaccharides (FOS) from sucrose (Santos & Maugeri, 2007) or by the hydrolysis of inulin (Silva et al., 2013). Fructo-oligosaccharides are well known as neo-sugars and have numerous beneficial properties (Gill et al., 2006, Yun, 1996). It has a wide range of applications in food, bread, and beverage products (Kurakake et al., 2010, Park et al., 2003). FOS can be obtained synthetically from agave fructans by acid-catalyzed hydrolysis, from sucrose via microbial action of β-fructosyltransferases or by β-fructofuranosidases (Avila-Fernandez et al., 2011, Balken et al., 1991, Ghazi et al., 2005, Chiang et al., 1997).

The formation of FOS via enzymatic methods is preferred due to high substrate specificity and selectivity of the enzymes. FOS from sucrose are considered as new alternative sweeteners with functional properties, also called soluble fibres, having a number of desirable characteristics such as low calories, no cariogenicity and safety for diabetics. Fructo-oligosaccharides are also known as prebiotics, since they stimulate the growth of probiotic organisms. The ingestion of fructo-oligosaccharides (FOS) has been shown to stimulate bifidobacteria in the lower gut. The gut microflora performs three major tasks: colonization resistance to pathogens, modulation of gastrointestinal and systemic immune responses, and nutritional support (Crittenden and Playne, 2006, Farthing, 2004). These prebiotics along with probiotics have many health benefits such as promotion of normal colon transit time, production of short-chain fatty acids (Swennen, Courtin, & Delcour, 2006), enhancement of mineral absorption (Beynen, Baas, & Hoekemeijer, 2002), favourable modulation of lipid levels (Fiordaliso, Kok, & Desager, 1995), improved gut mucosal barrier and immune function (Gibson et al., 2005, Murasaki et al., 1999), influences on glucose and insulin levels (Swennen et al., 2006) and reduction in colon cancer risk (Pool-Zobel, 2005). Saccharomyces cerevisiae is the organism of choice for invertase production because of its capability to ferment sucrose. Present study is focused on characterization of invertase from Saccharomyces cerevisiae SAA-612 isolated from the traditional alcoholic beverage (chhang) of Himachal Pradesh and its use in the synthesis of fructo-oligosaccharides (FOS).