Commercial Glucoamylase Research Articles

Modified simultaneous saccharification and fermentation for the production of bioethanol from highly concentrated raw corn starch

Bioethanol is one of the main bio-based molecules produced mainly from sugar cane, molasses and corn. Its environmental advantages allow it to be considered as safe and the cleanest fuel alternative. Starch is a widespread renewable carbohydrate conventionally used for bioethanol production via energy demanding liquefaction and saccharification processes. Raw starch hydrolysis using enzymes capable of degrading it below the gelatinization temperature significantly simplifies the process and reduces the cost of starch processing. In this study, an innovative modified simultaneous saccharification and fermentation process is proposed for the production of bioethanol from highly concentrated raw corn starch (30 % w/v). A two-step synergistic hydrolysis and fermentation was carried out in a single bioreactor vessel. To ensure high process efficiency, factors influencing the hydrolysis of concentrated raw corn starch by raw starch degrading α-amylase from Bacillus paralicheniformis ATCC 9945a (BliAmy) and commercial glucoamylase were investigated. Box–Behnken experimental design was used to predict the effects of different ratios of added enzymes, glucoamylase addition time, incubation time, and pH on hydrolysis yield. Optimal conditions for the highest yield of hydrolysis of raw corn starch (90 %) were obtained after 8 h using 5.0 IU BliAmy per mg of starch and 0.5 % (v/v) glucoamylase at pH 4.5 and 60 °C. Obtained glucose was further fermented with Saccharomyces cerevisiae at 30 °C in the same vessel for bioethanol production. Bioethanol concentration at 129.2 g/L, with productivity of 2.94 g/L/h and ethanol yield (YP/S) at 0.50 g EtOH/g total sugar, equivalent to 87.8 % theoretical yield, was obtained by modified simultaneous saccharification and fermentation. This work enriches the information of bioethanol production and offers a novel strategy for raw starch hydrolysis under industrial conditions.

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

BackgroundStarch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase.ResultsIn this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%.ConclusionsOur study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications

BackgroundGlucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry.ResultsIn this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification.ConclusionsWe provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.

Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate.

The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

Glucoamylase Immobilization in Corncob Powder: Assessment of Enzymatic Hydrolysis of Starch in the Production of Glucose

Amylases are environmentally attractive enzymes and are employed in food processing technology; for example, the starch hydrolysis process is used for glucose syrup production. The use of biocatalysts attached to a support promotes greater stabilization of the enzyme and allows its reuse, this being interesting for industrial applications. The choice of the support for enzyme immobilization must be made according to the process costs, therefore, the employment of agro-industrial byproducts is an alternative. We evaluated the immobilization of commercial glucoamylase in corncob powder (CCP) and the application of the immobilized enzyme (CCP-glucoamylase) in response to starch hydrolysis process The yield of glucoamylase immobilization in CCP was 95%. Soluble glucoamylase and CCP-glucoamylase presented maximum activity temperatures at 50 °C and 60 °C, respectively. Both biocatalysts showed a maximum activity at pH 5. Soluble glucoamylase and CCP-glucoamylase exhibited a good thermal stability at 50 °C, resulting in half-lives of 33 h and 46 h, respectively. Soluble glucoamylase and CCP-glucoamylase reached starch conversion into glucose of 75% and 16%, respectively, after 12 h of reaction, with an enzymatic load of 30 U per gram of starch. However, after an increase in the enzymatic load of CCP-glucoamylase, starch conversion was equivalent CCP-glucoamylase has the potential to hydrolyze starch into glucose and can be reused, thus becoming an enhanced value-added bioproduct and an alternative for industrial applications. A strategy for future industrial application would be the hydrolysis of starch using CCP-glucoamylase in a fixed bed reactor operated continuously.

Very high Gravity (VHG) Bioethanol Production Using Modified Simultaneous Saccharification and Fermentation of Raw Cassava Chips with Molasses by Kluyveromyces marxianus DMKU-KS07

Purpose: The aim of this study was to investigate the enhancement of very high gravity (VHG) bioethanol production using co-substrates of cassava chip hydrolysates and molasses by Kluyveromyces marxianus DMKU-KS07. Methods: The factors effecting cassava chip hydrolysis by raw starch degrading enzyme (RSDE) from the thermophilic bacterium Laceyella sacchari LP175 and commercial glucoamylase were investigated. The obtained raw cassava chip hydrolysate was co-fermented with molasses by the thermotolerant K. marxianus DMKU-KS07 at 42°C under nonsterile system. Results: Factors affecting sugar syrup production from raw cassava chips by synergistic hydrolysis and enzyme saccharification were optimized at temperature, substrate concentration and agitation rate of 50 °C, 250 g/L and 200 rpm, respectively. A yield of 98.6 g/L was obtained at 6 h of incubation, equivalent to 45.9% saccharification and 51.4% hydrolysis of starch, respectively. High bioethanol concentration at 118 g/L, with highest productivity of 2.19 g/L/h and ethanol yield (YP/S) at 0.44 g EtOH/g total sugar, equivalent to 86.3% theoretical yield, was obtained by modified simultaneous saccharification and fermentation (Modified SSF) with co-fermentation of substrates from the enzymatic hydrolysates of raw cassava chips at 42 °C for 12 h. Subsequent addition of molasses increased the final concentration to 100 g total sugar/L at 36 h. Conclusions: Co-fermentation of raw cassava chip hydrolysates with molasses enhanced production of bioethanol at VHG condition, and showed potential for application in ethanol production by enhancing the fermentation process and reducing energy consumption.

Single and Combined Enzymatic Saccharification and Biohydrogen Production from Chlorella sp. Biomass

The saccharification of Chlorella sp. biomass by single and combined commercial carbohydrase enzymes, Termamyl SC, Dextrozyme GA, and Cellic CTec2, was investigated. A substrate concentration of 100-g volatile solids (VS)/L was optimal for all the enzymes used. The optimal enzyme dosages for Termamyl SC, Dextrozyme GA, and Cellic CTec2 were 160-units/g VS, 320-units/g VS, and 40–filter paper units/g VS, respectively. The maximum reducing sugar yield, 130-mg/g VS, was obtained with a Dextrozyme GA treatment, which is equivalent to 38.37% hydrolysis efficiency based on carbohydrate content. A combined-enzyme treatment with Cellic CTec2, Termamyl SC, and Dextrozyme GA raised the reducing sugar yield and hydrolysis efficiency to 186.69-mg/g VS and 54.85%, respectively. Scanning and transmission electron microscopic images indicated morphological alterations in Chlorella sp. cells as an effect of enzyme treatment. Enzyme treatment improved the hydrogen production, hydrogen production rate, and hydrogen yield from Chlorella sp. biomass. The maximum hydrogen yield, 42.24-mL/g VSadded, was obtained from a Dextrozyme GA–hydrolyzed microalgae biomass. This was 82.46% higher than that obtained from untreated biomass (23.15-mL H2/g VSadded). Fourier-transform infrared analysis revealed that the functional groups of Chlorella sp. biomass, especially carbohydrates, were modified by both enzymatic treatment and the hydrogen fermentation process. This work demonstrated the efficient saccharification of Chlorella sp. biomass using combined cell wall– and starch-degrading enzymes. The results also elucidate that the pretreatment of Chlorella sp. biomass by commercial glucoamylase is a promising approach to solubilize internal starch granules and promote biohydrogen production without the addition of any cell wall–degrading enzyme.

A novel combination of enzymatic hydrolysis and microbial fuel cell for electricity production from bakery waste

This work developed a novel two-stage bioprocess for electricity generation from bakery waste (BW). In the first stage, commercial glucoamylase was utilized to hydrolyze the BW to produce soluble BW hydrolysate. It was found that 100 g BW could be converted to 32 g hydrolysis solid and 760 mL BW hydrolysate. The highest glucose production of 21.9 g/L could be achieved within 5 h. In the second stage, the soluble BW hydrolysate was utilized as feedstock for electricity generation in microbial fuel cell (MFC). The maximum voltage of 0.386 V was obtained. The power density reached a peak value of 29.96 mW/m2 when the external resistance was 1090 Ω. It could be potentially utilized to transform high-starch containing raw materials into biofuels production which could reduce the cost of biofuels production effectively for industrial application.

The Development of a Sorghum Bran-Based Biorefining Process to Convert Sorghum Bran into Value Added Products.

Sorghum bran, a starch rich food processing waste, was investigated for the production of glucoamylase in submerged fungal fermentation using Aspergillus awamori. The fermentation parameters, such as cultivation time, substrate concentration, pH, temperature, nitrogen source, mineral source and the medium loading ratio were investigated. The glucoamylase activity was improved from 1.90 U/mL in an initial test, to 19.3 U/mL at 10% (w/v) substrate concentration, pH 6.0, medium loading ratio of 200 mL in 500 mL shaking flask, with the addition of 2.5 g/L yeast extract and essential minerals. Fermentation using 2 L bioreactors under the optimum conditions resulted in a glucoamylase activity of 23.5 U/mL at 72 h, while further increase in sorghum bran concentration to 12.5% (w/v) gave an improved gluco-amylase activity of 37.6 U/mL at 115 h. The crude glucoamylase solution was used for the enzymatic hydrolysis of the sorghum bran. A sorghum bran hydrolysis carried out at 200 rpm, 55 °C for 48 h at a substrate loading ratio of 80 g/L resulted in 11.7 g/L glucose, similar to the results obtained using commercial glucoamylase. Large-scale sorghum bran hydrolysis in 2 L bioreactors using crude glucoamylase solution resulted in a glucose concentration of 38.7 g/L from 200 g/L sorghum bran, corresponding to 94.1% of the theoretical hydrolysis yield.

AmyZ1: a novel \u03b1-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

BackgroundStarch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch.ResultsA novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries.ConclusionsAs a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.

Consolidated bioprocessing of raw starch with Saccharomyces cerevisiae strains expressing fungal alpha-amylase and glucoamylase combinations.

Cost-effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing α-amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S-2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw-starch α-amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular α-amylase activities (2.17 U mL-1 and 2.98 U mL-1, respectively). Both the ApuA and AteA α-amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30°C. When ateA was co-expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA-GlaA] strain produced 45.77 g L-1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 μL STARGEN 002 (54.54 g L-1 ethanol and 70.44% carbon conversion) or 20 μL commercial glucoamylase from Sigma-Aldrich (73.80 g L-1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch-based biofuel production.

Ethanol biosynthesis by fast hydrolysis of cassava bagasse using fungal amylases produced in optimized conditions

The search for a renewable platform to produce high-value biochemicals and energy that are environmentally correct has been a current concern. A fast and inexpensive bioprocess for amylase production, able to hydrolyze complex residues in fermentable sugars to be used for ethanol production was developed. High titer amylase from Rhizopus oligosporus in solid state fermentation (SSF) was obtained by optimizing the medium supplementation using agro-industrial waste as substrate. Statistical experimental design and partial least square (PLS) regression were used to establish a relation between added chemical compounds and enzyme production, showing that urea was the most important nutrient. Crude amylase extract had competitive performance features giving higher productivities in starch hydrolysis than a commercial glucoamylase. The amylase produced was applied in the proportion of 15U/g dry cassava bagasse to obtain cassava bagasse hydrolysate (CBH). More than 42% conversion in reducing sugars was achieved with an efficient 10h single-step hydrolysis at 55°C in a bioreactor. The concentrated CBH was subsequently used in fed batch process producing 89.2% ethanol yield. Furthermore, comparing just the cost of the raw materials sugarcane and CHB, the latter demonstrated to be a lower-cost feedstock for ethanol fermentation.

High-efficient production of citric acid by Aspergillus niger from high concentration of substrate based on the staged-addition glucoamylase strategy

Citric acid (CA), an important platform-compound, has attracted much attention because of its broad applications and huge market demand. To solve high residual sugar at the fermentation end, we put forwarded strategy of pre-saccharification and then fermentation. Results showed that the residual total sugar decreased by 10.4% and the productivity increased by 4.0% and initially high glucose inhibited cell growth. Furthermore, commercial glucoamylase with high low-pH stability was proposed to staged-add in the fermentation process, which timely compensated enzyme loss, ensuring the glucose supply rate. The fermentation productivity was evidently enhanced by 13.3% with residual total sugar decreasing by 31.3%, simplifying the subsequent product separation and extraction process. Our results confirmed that staged-addition glucoamylase strategy was feasible to effective production of CA.

Biohydrogen production from enzymatic hydrolysis of food waste in batch and continuous systems.

In this study, the feasibility of biohydrogen production from enzymatic hydrolysis of food waste was investigated. Food waste (solid-to-liquid ratio of 10%, w/v) was first hydrolyzed by commercial glucoamylase to release glucose (24.35 g/L) in the food waste hydrolysate. Then, the obtained food waste hydrolysate was used as substrate for biohydrogen production in the batch and continuous (continuous stirred tank reactor, CSTR) systems. It was observed that the maximum cumulative hydrogen production of 5850 mL was achieved with a yield of 245.7 mL hydrogen/g glucose (1.97 mol hydrogen/mol glucose) in the batch system. In the continuous system, the effect of hydraulic retention time (HRT) on biohydrogen production from food waste hydrolysate was investigated. The optimal HRT obtained from this study was 6 h with the highest hydrogen production rate of 8.02 mmol/(h·L). Ethanol and acetate were the major soluble microbial products with low propionate production at all HRTs. Enzymatic hydrolysis of food waste could effectively accelerate hydrolysis speed, improve substrate utilization rate and increase hydrogen yield.

Production of raw starch degrading enzyme by the thermophilic filamentous bacterium Laceyella sacchari LP175 and its application for ethanol production from dried cassava chips

Physical factors that enhanced the production of raw starch degrading enzyme by a thermophilic filamentous bacterium, Laceyella sacchari LP175 in a 3 L airlift fermenter, were optimized at a pH of 6.5, a temperature of 45°C, and an aeration rate of 0.5 vvm using a central composite design. This yielded 278 U/mL, at 36 h cultivation, an increase in enzyme production and productivity of 1.53‐ and 2.04‐fold, respectively, as compared with a shaking flask cultivation. Raw starch degrading enzyme produced by L. sacchari LP175 with commercial glucoamylase synergistically enhanced the hydrolysis of 300 g/L dried cassava chips (71% starch content) to 157 g/L reducing sugars, equivalent to 66.3% hydrolysis at pH 6.5 and 50°C for 12 h. The comparative study on bioethanol production in a non‐sterile system from dried cassava chip by three different processes; the maximum production, 90.9 g/L (88% theoretical yield), was obtained by modified simultaneous saccharification and fermentation at 50°C for 6 h and subsequent fermentation by the thermotolerant Kluyveromyces marxianus DMKU‐KS07 at 42°C within 18 h with the highest productivity of 3.79 g/L/h. The other processes tested – separate hydrolysis and fermentation and simultaneous saccharification and fermentation – yielded 83 and 51.6 g/L of ethanol, respectively.

효소 종류에 따른 참죽 추출물의 산화방지 효과

This study was conducted to analyze the functional component contents and antioxidant activities of Cedrela sinensis extracts hydrolyzed using four different enzymes. The yield of Viscozyme extract was the highest among the samples, and all enzymatic extracts, except for the commercial glucoamylase (AMG) extract, had significantly higher total polyphenol and flavonoids contents compared to the non-enzymatic extract (p＜0.05). Viscozyme extract showed the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and Fe 2+ chelating ability among the samples. All enzymatic extracts showed high＞90% 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity, and there was no significant difference between the enzymatic and non-enzymatic extracts. The reducing power of the extracts using Shearzyme or Viscozyme was significantly higher than that of the other samples (p＜0.05). Therefore, the results indicated that all enzymatic extracts (especially Viscozyme extract), except for the AMG extract, showed high antioxidant activity compared to the nonenzymatic extract. These result suggested that the enzymatic extracts of C. sinensis can be used in functional foods.

Production and characterization of raw starch degrading enzyme from a newly isolated thermophilic filamentous bacterium, Laceyella sacchari LP175

Medium optimization for production of raw starch degrading enzyme (RSDE) from a newly isolated thermophilic filamentous bacterium, Laceyella sacchari LP175, is described. Yeast extract and raw cassava starch were found to be the best nitrogen and carbon sources, respectively, for enzyme production. Response surface methodology with Plackett–Burman design and central composite design showed that an optimized concentration of 4.93 g/L raw cassava starch and 2.8 g/L yeast extract in basal medium yielded 181.1 U/mL RSDE activity in a shaking flask at 50°C and pH 7.0 after 48 h. Maximum RDSE activity in crude enzyme was achieved at pH 6.5 and 50°C. The major end product of raw cassava starch hydrolysis was maltose (88%). The Km and Ki values of raw cassava starch and maltose, respectively, were found to be 2.85 mg/mL and 1.04 mg/mL. Raw cassava starch at 100 g/L was hydrolyzed with 200 U/mL RSDE for 12 h, yielding 36.8% hydrolysis. The addition of commercial glucoamylase (1.6 U/mL) synergistically enhanced the hydrolysis reaction, yielding 70% hydrolysis (equivalent to 78.0 g/L glucose). The high efficiency of RSDE produced from L. sacchari LP175 could be useful for replacing conventional enzymes currently used in various raw starch processing industries.

A thermostable glucoamylase from Bispora sp. MEY-1 with stability over a broad pH range and significant starch hydrolysis capacity.

BackgroundGlucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.Methodology/Principal FindingsA glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.Conclusion/SignificanceGLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.

Fermentative polyhydroxybutyrate production from a novel feedstock derived from bakery waste.

In this study, Halomonas boliviensis was cultivated on bakery waste hydrolysate and seawater in batch and fed-batch cultures for polyhydroxybutyrate (PHB) production. Results demonstrated that bakery waste hydrolysate and seawater could be efficiently utilized by Halomonas boliviensis while PHB contents between 10 and 30% (w/w) were obtained. Furthermore, three methods for bakery waste hydrolysis were investigated for feedstock preparation. These include: (1) use of crude enzyme extracts from Aspergillus awamori, (2) Aspergillus awamori solid mashes, and (3) commercial glucoamylase. In the first method, the resultant free amino nitrogen (FAN) concentration in hydrolysates was 150 and 250 mg L−1 after 20 hours at enzyme-to-solid ratios of 6.9 and 13.1 U g−1, respectively. In both cases, the final glucose concentration was around 130–150 g L−1. In the second method, the resultant FAN and glucose concentrations were 250 mg L−1 and 150 g L−1, respectively. In the third method, highest glucose and lowest FAN concentrations of 170–200 g L−1 and 100 mg L−1, respectively, were obtained in hydrolysates after only 5 hours. The present work has generated promising information contributing to the sustainable production of bioplastic using bakery waste hydrolysate.

Molecular cloning and characterization of a thermostable α-amylase exhibiting an unusually high activity

An α-amylase gene was cloned from the thermophilic bacterium Bacillus subtilis isolated from Indonesian oil palm shell waste. The gene expressed an extracellular enzyme. Optimal hydrolysis conditions for the enzyme were 70 o C and pH 6.0. The specific activity of the enzyme was 16.0 kU per mg of protein, which was higher than for other thermostable amylases. Hydrolytic products of the enzyme using starch and glycogen were mainly maltohexaose and maltopentaose. The enzyme had a Km value of 0.099 mg/mL for amylopectin, more than 10 times lower than for amylose. The catalytic efficiency of the enzyme using amylopectin was 39,200 mL/mg·s and was 3,270 mL/mg·s using amylose. The enzyme liquefied corn starch at pH 5.0, which was successfully converted to glucose using commercial glucoamylase and pullulanase without pH adjustment. The enzyme has advantages for industrial applications.