Joo Shun Tan

Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications

The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase.

Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry

BackgroundLactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications.ResultsLAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase-negative, able to produce β-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics.ConclusionTraditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods.

Primary recovery of a bacteriocin-like inhibitory substance derived from Pediococcus acidilactici Kp10 by an aqueous two-phase system

A polymer-salt aqueous two-phase system (ATPS) consisting of polyethylene-glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth.

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

BackgroundCell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.ResultsThe proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.ConclusionsThe model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

Extractive bioconversion of cyclodextrins by Bacillus cereus cyclodextrin glycosyltransferase in aqueous two-phase system

An extractive bioconversion with Bacillus cereus cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) in aqueous two-phase system (ATPS) was investigated for the synthesis and recovery of cyclodextrins (CDs). Optimum condition for the extractive bioconversion of CDs was achieved in ATPS consisted of 7.7% (w/w) polyethylene glycol (PEG) 20,000 and 10.3% (w/w) dextran T500 with volume ratio (VR) of 4.0. Enzymatic conversion of starch occurred mainly in dextran-rich bottom phase whereas the product, CDs was transferred to top phase and a higher partition coefficient of CDs was achieved. Repetitive batch of CDs synthesis was employed by replenishment of the top phase components and addition of starch every 8h. An average total CDs concentration of 13.7 mg/mL, (4.77 mg/mLα-CD, 5.02 mg/mLβ-CD and 3.91 mg/mLγ-CD) was recovered in the top phase of PEG 20,000/dextran T500 ATPS. This study showed the effectiveness of ATPS application in extractive bioconversion of CDs synthesis with B. cereus CGTase.

Comparative Analyses of Response Surface Methodology and Artificial Neural Network on Medium Optimization for Tetraselmis sp. FTC209 Grown under Mixotrophic Condition

Mixotrophic metabolism was evaluated as an option to augment the growth and lipid production of marine microalga Tetraselmis sp. FTC 209. In this study, a five-level three-factor central composite design (CCD) was implemented in order to enrich the W-30 algal growth medium. Response surface methodology (RSM) was employed to model the effect of three medium variables, that is, glucose (organic C source), NaNO3 (primary N source), and yeast extract (supplementary N, amino acids, and vitamins) on biomass concentration, X max, and lipid yield, P max/X max. RSM capability was also weighed against an artificial neural network (ANN) approach for predicting a composition that would result in maximum lipid productivity, Prlipid. A quadratic regression from RSM and a Levenberg-Marquardt trained ANN network composed of 10 hidden neurons eventually produced comparable results, albeit ANN formulation was observed to yield higher values of response outputs. Finalized glucose (24.05 g/L), NaNO3 (4.70 g/L), and yeast extract (0.93 g/L) concentration, affected an increase of X max to 12.38 g/L and lipid a accumulation of 195.77 mg/g dcw. This contributed to a lipid productivity of 173.11 mg/L per day in the course of two-week cultivation.

Optimization of an induction strategy for improving interferon-α2b production in the periplasm of Escherichia coli using response surface methodology

Induction strategies for the periplasmic production of recombinant human IFN‐α2b (interferon‐α2b) by recombinant Escherichia coli Rosetta‐gami 2(DE3) were optimized in shake‐flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl β‐D‐thiogalactoside) concentration and induction temperature. Second‐order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN‐α2b formation and percentage of soluble IFN‐α2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 °C. The optimized induction strategy reduced inclusion‐body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN‐α2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non‐optimized condition, soluble periplasmic production and the percentage of soluble IFN‐α2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20‐fold and 1.4‐fold respectively.

Fermentation factors influencing the production of bacteriocins by lactic acid bacteria: a review

Lactic acid bacteria (LAB) are of major interest in the food industry primarily by virtue of their biopreservative properties. LAB have ability to produce various types of antimicrobial compounds, the most important being bacteriocins. Bacteriocins and bacteriocin-producing cultures have the potential to increase the shelf-life of foods and contribute towards decreasing the incidence of food-borne diseases. In this respect, food preservation through in situ production of bacteriocins by LAB introduced into the food system would be the most logical approach. However, there is a need to understand the relationship between bacterial growth and bacteriocin production in various types of food system. Bacteriocin production by LAB is dependent on a number of factors such as the types of carbon and nitrogen sources and their concentrations in the media formulation. Other factors which need to be considered are the culture conditions which include pH, temperature and aeration which greatly influence the cultivation performance of bacteriocins producing LAB. Economic aspects pertaining to the optimization of fermentation process for the enhancement of bacteriocin production should also be given due considerations. Failure to acknowledge or recognize this hidden economic element would be a substantial financial loss to the industry especially from the point of view that the product is costly and highly sought after. Thus, the fermentation factors which influence the production of bacteriocins by LAB and the approaches to improve the production not only in term of yield and productivity but also in term of economic and regulation are reviewed in this paper.

Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10

An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.

Recovery of Microquantities of Human Epidermal Growth Factor from Escherichia coli Homogenate and Pichia pastoris Culture Medium using Expanded Bed Adsorption

A rational design of recovery of microquantities of human Epidermal Growth Factor (hEGF) from different complex feedstocks using STREAMLINE Direct HST in expanded bed adsorption (EBA) was approached. The highest adsorption yields were achieved at pH 4.5, which was close to the isoelectric point of protein by utilizing mixed interaction that was offered by the adsorbent. Escherichia coli treated with osmotic shock and Pichia pastoris culture medium spiked with hEGF were applied as feedstocks to evaluate bed stability in the presence of cells. A recovery of 90% was achieved for both cells at pH 4.5. Effects of pH on the P. pastoris culture medium and E. coli homogenate were similar, indicating that both cells have negatively charged surfaces at pH 4.5. The cell transmission index (T) showed that there was no tendency for E. coli homogenate and yeast cells to bind to the matrix at pH 4.5. Because the electrostatic properties of cells and protein are pH dependent, the method presented for screening conditions for biomass and adsorbent is convenient for designing robust and reliable EBA purification processes. Lower ionic strength improved purification of hEGF from E. coli homogenate.