α-Amylase, glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16

Highlights

• The α-amylase gene, the glucoamylase gene and the isopullulanase gene in A. melanogenum P16 were cloned and characterized.

• The triple mutant DT15 could yield 58.14 g/L of pullulan with Mw of 3.02 × 10⁶ Da.

• After the genes were complemented, pullulan production and Mw of pullulan were restored.

Abstract

A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 10⁶ Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ± 3.5 g/L of pullulan with a Mw of 0.35 × 10⁶ Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.

Introduction

Pullulan is a linear and un-branched exo-polysaccharide which mainly consists of maltotriose units attached by α (1 → 6) glycosidic linkage. The main producers are different strains of Aureobasdium spp. [1]. Pullulan is a water soluble, biodegradable, non-toxic, non-immunogenic, non-carcinogenic and non-mutagenic biopolymer, can form thin films which are oil resistant, transparent and oxygen impermeable and has adhesive property during drying so that it can be widely used in food, pharmaceutical and cosmetic industries [2]. Each repeating unit of pullulan consists of nine hydroxyl groups for the chemical substitution reactions. After chemical modification, pullulan can be used in tissue engineering and grafting, a carrier for drug delivery and gene delivery, medical imaging and so on [2]. One of the most important applications of pullulan should be as a major material of the drug capsules and cosmetics. To date, most of the drug capsules have been made of gelatin which has many drawbacks such as contamination of the animal pathogens since it is obtained from several animal body parts. It has been reported that the drug capsules made of pullulan have many advantages and useful properties and are warmly welcome by the vegetarians and Muslims, diabetics, and patients with restricted diet [3]. If pullulan is used as the material for making the drug capsule and as a component of the cosmetic, it is required that the molecular weight (Mw) of the pullulan should be more than 2.0 × 10⁶ Da due to its high viscosity, a very strong strength and high stability [4]. However, so far, the application of pullulan in various fields was actually limited due to both low production yield and low Mw of the pullulan. For example, the commercial pullulans produced by Hayashibara Company, the principle producer of commercial pullulan in the world include food grade (designated as PF) and deionized (PI) products with mean Mw of 1.0 × 10⁵ or 2.0 × 10⁵ Da [5].

Therefore, it is very important how to yield the pullulan with high Mw. In our previous study [4], changes in a culture medium can increase Mw of the produced pullulan by Aureobasidium melanogenum P16, a high pullulan producer. Yu et al. [6] and Wang et al. [7] also have the similar findings.

During cultivation and fermentation, it has been well documented that the Mw of pullulan greatly decreases in the late stationary growth phase due to the presence of isopullulanase, α-amylase and glucoamylase secreted into the medium (Fig. 1) [8, 9]. In our previous study [4], it was found that after the compositional change of a pullulan production medium, a Mw of the pullulan produced by A. melanogenum P16 increased, but pullulan titer decreased. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase. It has been reported that pullulan also contains as a minor structural feature, a low percentage of α-1,6-linked maltotetraose subunits which are substrates for α-amylase [9]. This was also proven by Prasongsuk et al. [10] who added the α-amylase inhibitor acarbose to the culture medium, showing that pullulan of slightly higher Mw was obtained from late cultures. It appeared that the enzyme responsible for pullulan degradation during the cultivation was also likely glucoamylase B [11]. Furthermore, A. pullulans has been reported to produce other enzymes that might attack pullulan, including glucoamylase [10] and pullulanase [12]. However, so far, there have been no genetic evidences to show that the pullulanase, α-amylase and glucoamylase are implicated in high Mw pullulan biosynthesis. Therefore, further study of the relationship between pullulanase, α-amylase and glucoamylase activities and the Mw of pullulan is needed, including an analysis of α-amylase gene, glucoamylase gene and pullulanase gene. Furthermore, a more practical approach for yielding a higher Mw pullulan might be to use α-amylase, glucoamylase and pullulanase negative mutants (Fig. 1).

The aims of the study were to clone, characterize, delete and complement α-amylase, glucoamylase and isopullulanase genes in A. melanogenum P16 in order to confirm the hypothesis that the Mw of the produced pullulan could be determined by the three enzymes.