Highest Enzyme Yield Research Articles

Production Process Optimization of Recombinant Erwinia carotovoral-Asparaginase II in Escherichia coli Fed-Batch Cultures and Analysis of Antileukemic Potential.

The aims of this work were to optimize the production of Erwinia carotovoral-asparaginase II enzyme in Escherichia coli by different fed-batch cultivation strategies using a benchtop bioreactor and to evaluate the therapeutic potential of the recombinant enzyme against different acute lymphoblastic leukemia cell lines. The highest enzyme activities (∼98,000 U/L) were obtained in cultures using the DO-stat feeding strategy with induction in 18 h of culture. Under these experimental conditions, the maximum values for recombinant l-asparaginase II (rASNase) yield per substrate, rASNase yield per biomass, and productivity were approximately 1204 U/gglucose, 3660 U/gcells, and 3260 U/(L·h), respectively. This condition was efficient for achieving high yields of the recombinant enzyme, which was purified and used in in vitro antileukemic potential tests. Of all the leukemic cell lines tested, RS4;11 showed the highest sensitivity to rASNase, with an IC50 value of approximately 0.0006 U/mL and more than 70% apoptotic cells. The study demonstrated that the cultivation strategies used were efficient for obtaining high yield and productivity of rASNase with therapeutic potential inasmuch as cytotoxic activity and induction of apoptosis were demonstrated for this protein.

Production and characterization of protease enzyme from Acinetobacter pittii using peanut meal as substrate

ABSTRACT A significantly high protease enzyme yield of 617 U/ml was achieved with Acinetobacter pittii as the microorganism and peanut oil meal as the substrate. Peanut oil meal, which consists of proteins (40-60%) and carbohydrates (22-30%), serves as a sufficient source of nitrogen and carbon necessary for microbial growth and production of enzymes. Moreover, peanut meal offers the advantages of being affordable and available in large quantities, making the meal suitable for cost-effective enzyme production. In the present study, two bacterial strains and one fungal strain were selected to produce proteases utilizing peanut oil meal as the substrate. The experimental conditions during the enzyme production, including pH and temperature, were optimized. In addition, the substrate was enriched with various carbon and nitrogen sources. The microbial strains were streaked on nutritional agar (for bacteria) and potato dextrose agar (for fungus). Following an incubation period, the plates were stored at 4°C for further studies. The molecular weight of partially purified proteases of Acinetobacter pittii was found to be ≅ 95.5 kDa. Potassium nitrate was the most ideal nitrogen source (up to 411% increase in activity) and fructose was the best carbon source (425% increase). These enzymes exhibited excellent temperature tolerance and were capable of functioning over a wide pH range. Furthermore, the obtained proteases demonstrated ability to coagulate milk effectively, indicating their potential for various food-related applications.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as "secretion stress" on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields. IMPORTANCE In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

Bioethanol production from immobilized amylase produced by marine Aspergillus flavus AUMC10636

Aspergillus flavus, an amylase-producing fungus, was isolated from the Mediterranean Sea, Alexandria, Egypt. It was selected according to the formation of a clear zone using the Dox-medium containing 1% starch for enzyme production. The maximum amount obtained of amylase production was 22.68 U/mL after 7 days of incubation under static condition. Partially purified amylase with 70% ethanol provoked a single protein band with 54 kDa molecular weight using SDS-PAGE giving a specific activity of 307.35 U/mg. The purified α-amylase exhibited optimal activity of 65.17 and 71.5 U/mL at pH = 6 and 50 °C, respectively. The calculated enzyme kinetics were Km = 5 mg/mL and Vmax = 100 U/mL. Residual α-amylase retained optimum stability at 50 °C by 67.74% and at pH = 6 by 100% after 60 min of incubation. The highest enzyme tolerance occurred at 1.5 mM NaCl concentration. The highest enzyme yield (165.24 U/mL) was provided by the immobilized amylase with 1% of covalent bonding agents solution, providing double the sugars’ byproduct than from the free amylase form, and double the bioethanol fermentation yield with 0.12 g/g sugar/l. Highly specific activity of such enzyme in the immobilized form could offer a highly effective approach for bioethanol production on the industrial scale than the free and other reported forms.

Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System.

Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL−1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.

Sequential optimization of xylanase production using Sapindus mukorossi seed waste in Lechevalieria aerocolonigenes

The production of xylanase from Lechevalieria aerocolonigenes using reetha seed waste as substrate was studied using sequential optimization of fermentation parameters by response surface methodology. Five different lignocellulosic agricultural wastes as a substrate were studied to replace commercially available xylan, amongst which reetha seed waste was found to be the most suitable substrate for xylanase production. A sequential two-stage optimization strategy was used for the fermentation parameter optimization. The Plackett–Burman design was first employed for screening the 6 different physicochemical parameters affecting xylanase production (inoculum concentration, substrate concentration, temperature, pH, media volume, and agitation). The significant factors affecting the xylanase yield were further optimized by Box–Behnken Design in order to obtain the values contributing the highest enzyme yield. Three parameters, namely, temperature, inoculum concentration, and substrate concentration, can be interpreted as the most significant parameters based on the results of Plackett–Burman design. The optimum values by Box-Behnken Design (BBD) are 35 °C temperature, 3 g/L substrate concentration, and inoculum concentration of 4% (v/v) that resulted in maximum xylanase productivity of 5.75 IU/mL at 24 h of the incubation period. Sequential optimization strategy enhanced the xylanase yield by 4.8 fold to that of an unoptimized process.

Evaluation of a Trametes pubescens laccase concentrated extract on allura red AC decolorization without the addition of synthetic mediators

Synthetic dye bioremediation is a topic of great importance since these pollutants possess toxic effects, and huge quantities of them are being discharged into water bodies. Ligninolytic enzyme treatment stands out for being a cost-effective methodology, capable of obtaining high decolorization levels. In this work, a laccase enzyme treatment was evaluated to effectively perform a cycle of dye bioremediation. Furthermore, a dye decolorization improvement was also assessed through laccase immobilization. Particularly, a Trametes pubescens enzyme extract was concentrated, immobilized onto calcium alginate beads, and characterized to assess its dye decolorization potential. Ammonium sulfate precipitation and vacuum evaporation were evaluated to concentrate the crude extract and to decolorize allura red AC. Both treatments reached a high enzyme yield recovery (>90%), but only the vacuum-evaporated extract achieved a high allura red AC decolorization level after 16 h of contact time. This suggested that essential compounds for allura red AC decolorization were present in the crude extract, implying that neither a complete laccase purification process nor an addition of synthetic mediators are necessary. Under optimized immobilization conditions, 94.6% immobilization efficiency and 49.8% activity recovery were obtained with 0:1 alginate:enzyme (v/v), 100 mM CaCl2, and 5.0% w/v sodium alginate. Furthermore, by immobilizing the laccase concentrated extract, both the pH and temperature stabilities were improved. The decolorization of allura red AC by free and immobilized laccase was 68.4% and 4.6%, respectively, showing that although the enzyme stability was improved, dye decolorization was negatively affected. Thus, an efficient allura red AC decolorization was obtained with concentrated-free laccase by a feasible and low-cost methodology.

β-Mannanase Production Using Coffee Industry Waste for Application in Soluble Coffee Processing.

Soluble coffee offers the combined benefits of high added value and practicality for its consumers. The hydrolysis of coffee polysaccharides by the biochemical route, using enzymes, is an eco-friendly and sustainable way to improve the quality of this product, while contributing to the implementation of industrial processes that have lower energy requirements and can reduce environmental impacts. This work describes the production of hydrolytic enzymes by solid-state fermentation (SSF), cultivating filamentous fungi on waste from the coffee industry, followed by their application in the hydrolysis of waste coffee polysaccharides from soluble coffee processing. Different substrate compositions were studied, an ideal microorganism was selected, and the fermentation conditions were optimized. Cultivations for enzymes production were carried out in flasks and in a packed-bed bioreactor. Higher enzyme yield was achieved in the bioreactor, due to better aeration of the substrate. The best β-mannanase production results were found for a substrate composed of a mixture of coffee waste and wheat bran (1:1 w/w), using Aspergillus niger F12. The enzymatic extract proved to be very stable for 24 h, at 50 °C, and was able to hydrolyze a considerable amount of the carbohydrates in the coffee. The addition of a commercial cellulase cocktail to the crude extract increased the hydrolysis yield by 56%. The production of β-mannanase by SSF and its application in the hydrolysis of coffee polysaccharides showed promise for improving soluble coffee processing, offering an attractive way to assist in closing the loops in the coffee industry and creating a circular economy.

Optimization and molecular characterization of novel Aspergillus spp. producing invertase enzyme

Background and objective Invertase, one of the most important enzymes used in industrial and pharmaceutical applications, has been produced from different Aspergillus spp. Statistical and mathematical tools [response surface methodology (RSM)] have been carried out to optimize culture conditions to facilitate maximum enzyme production. This investigation aims to screen the ability of some Aspergillus spp. to produce invertase enzyme, to optimize the culture parameters, and to detect the genetic diversity between them. Materials and methods A total of five fungal strains were tested for the production of invertase. The most promising strain was selected for optimization. Optimization experiments were carried out to obtain maximum enzyme production, and a molecular marker (RAPD-PCR) was used to differentiate between these strains. Results and conclusion Some Aspergillus spp. were examined to produced invertase enzyme after 2 days of cultivation. Aspergillus niger recorded the highest enzyme production (6.8 U/ml). Ten RAPD-PCR primers were used to differentiate between Aspergillus spp. A total of 131 bands were amplified in the five strains of Aspergillus spp. Based on RAPD-PCR analysis, the highest similarities obtained between A. parasiticus and A. niger showed 75%. Moreover, the RAPD-PCR technique is a good tool for differentiation between the Aspergillus spp. To optimize culture condition, RSM was used to increase invertase production by A. niger. Temperature, pH, and incubation time dramatically affect invertase yield. A combination of these factors (pH 5, 35°C, and incubation for 72 h) was optimum for maximum production of invertase (8.2 U/ml). Our results employed RSM to elucidate the combination of effective factors such as temperature, pH, and incubation time to obtain a high yield of an important enzyme, invertase, from A. niger, used as a promising candidate for industrial applications.

Optimization of high endoglucanase yields production from polypore fungus, Microporus xanthopus strain KA038 under solid-state fermentation using green tea waste.

ABSTRACTPolypores are diverse macrofungi that have been extensively studied for their lignocellulolytic enzyme production capabilities. Currently, these enzymes are being used for many industrial purposes. However, the high cost associated with their production is the main barrier to their broader application. This work aimed to study the optimal medium and conditions for endoglucanase production using solid state fermentation. Seven polypore strains were used for endoglucanase activity screening. The fermentation experiments were carried out in 250 ml Erlenmeyer flasks with green tea waste as a substrate. Notably, Microporus xanthopus strain KA038 showed the best level of activity (38.62 IU/gds). Various parameters such as moisture content, nitrogen source, initial pH value, inoculum size and incubation time were considered to determine the optimal conditions for endoglucanase production. The optimal medium consisted of green tea leaves as a carbon source, beef extract as an organic nitrogen source, NH4H2PO4 as an inorganic nitrogen source, pH 7.0 and an incubation temperature at 30°C for 4 days resulted in a high enzyme yield with M. xanthopus strain KA038 (81.8 IU/gds).This article has an associated First Person interview with the first author of the paper.

Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC–MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.

ISOLATION, OPTIMIZATION, AND ANTITUMOR ACTIVITY OF L-ASPARAGINASE EXTRACTED FROM PECTOBACTERIUM CAROTOVORUM AND SERRATIA MARCESCENS ON HUMAN BREAST ADENOCARCINOMA AND HUMAN HEPATOCELLULAR CARCINOMA CANCER CELL LINES

Objectives: The objective of this research was to obtain isolates capable of producing a high yield of L-asparaginase enzyme and to evaluate the antitumor activity of the purified enzyme against different cancer and normal cell lines.
 Methods: Isolation of bacteria was performed by the serial dilution technique of soil samples collected from Cairo, Egypt, using modified M9 agar plates. Culture filtrates of selected isolates were quantitatively screened for L-asparaginase production using well-diffusion and direct nesslerization techniques. Factors influencing L-asparaginase activity were optimized by studying the effect of physical and nutritional conditions on the enzyme activity. The purification of L-asparaginase extracted from both the isolates was achieved using chilled acetone (−20°C), followed by gel filtration on Sephadex G-100. The anticancer activity of the purified enzyme against human breast adenocarcinoma (MCF-7), human hepatocellular carcinoma (HepGII) and homo sapiens human (WISH) cell line was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay.
 Results: Two L-asparaginase producers were identified by Biolog identification system as Pectobacterium carotovorum and Serratia marcescens. Optimization increased the production of L-asparaginase to 4.835 and 5.221 U/ml for P. carotovorum and S. marcescens, respectively. L-asparaginase was extracted, purified, and tested in vitro for cytotoxic activity using 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT assay) against MCF-7, HepGII, and WISH cell line. L-asparaginase from P. carotovorum and S. marcescens was neutral to normal epithelial WISH cells. On the other hand, L-asparaginase from both isolates was cytotoxic to MCF-7 and HepGII cancer cell lines with an half maximal inhibitory concentration of 15 μg/ml and 26 μg/ml and 26 μg/ml and 25 μg/ml, respectively.
 Conclusion: L-asparaginase extracted from P. carotovorum and S. marcescens showed remarkable anticancer activity. Further studies on hypersensitivity action need to be carried out to recommend the use of L-asparaginase as an alternative to commercially available preparations.

A Novel Method of Affinity Tag Cleavage in the Purification of a Recombinant Thermostable Lipase from Aneurinibacillus thermoaerophilus Strain HZ

The development of an efficient and economical purification method is required to obtain a pure and mature recombinant protein in a simple process with high efficiency. Hence, a new technique was invented to cleave the tags from the N-terminal region of recombinant fusion HZ lipase in the absence of protease treatment. The recombinant pET32b/rHZ lipase was overexpressed into E. coli BL21 (DE3). Affinity chromatography was performed as the first step of purification. The stability of the protein was then tested in different temperatures. The fused Trx-His-S-tags to the rHZ lipase was cleaved by treatment of the fusion protein at 20 °C in 100 mM Tris-HCl buffer, pH 8.0. The precipitated tag was removed, and the mature recombinant enzyme was further characterized to specify its properties. A purification yield of 78.9% with 1.3-fold and 21.8 mg total purified mature protein was obtained from 50 mL starting a bacterial culture. N-terminal sequencing of purified recombinant HZ lipase confirmed the elimination of the 17.4 kDa tag from one amino acid before the native start codon (Methionine) of the protein. The mature rHZ lipase was highly active at 65 °C and a pH of 7.0, with a half-life of 2 h 15 min at 55 °C and 45 min at 60 °C. The rHZ lipase showed a preference for the hydrolysis of natural oil with a long carbon chain (C18) and medium-size acyl chain p-nitrophenyl esters (C10). The enzyme remained stable in the presence of nonpolar organic solvents, and its activity was increased by polar organic solvents. This study thus demonstrates a simple and convenient purification method which resulted in the high yield of mature enzyme along with unique and detailed biochemical characterization of rHZ lipase, making the enzyme favorable in various industrial applications.

Production of lipolytic enzymes by bacteria isolated from biological effluent treatment systems.

This work aimed to evaluate the production of lipolytic complexes, produced by microorganisms isolated from a biological treatment system of effluents from a hotel. To select the best lipolytic microorganism for use in biotechnological processes, we tested 45 bacterial isolates recovered from the raw effluent of the hotel's restaurant waste tank. Lipase production was assayed in culture medium supplemented with olive oil and rhodamine B, incubated at 25 °C and 30 °C for 24 h - 48 h. Results showed 22 isolates lipase producers. All isolates were inoculated on medium without yeast extract to select the ones with highest enzyme yields. Out of these, nine isolates showed high lipase activity. The strain with the larger halo was assayed in submerged culture using an orbital shaker and a bioreactor, with three different substrates (olive oil, grape seed oil, and canola oil). Isolate G40 identified as Acinetobacter baylyi was selected to run the production assays because it showed the best result in the solid medium. In the bioreactor, maximum lipase production was obtained after 12 h of culture with the three substrates evaluated: 0,358 U/mL.min-1 in olive oil, 0,352 U/mL.min-1 with grapeseed oil, and 0,348 U/mL.min-1 with canola oil.

Improvement of cellulose degradation by cloning of endo-β-1, 3-1, 4 glucanase (bgls) gene from Bacillus subtilis BTN7A strain.

The aim of this study is to construct a new recombinant strain able to degrade cellulose efficiently. The endo-β-1, 3-1, 4 glucanase (bgls) gene was cloned from Bacillus subtilis BTN7A strain by using PCR technique. The specific primers of bgls gene were deduced. Optimization of PCR mixture and program were identified. The nucleotide sequence of bgls was placed in the public domain (GenBank accession number KM009051.1). The obtained bgls DNA was cloned with pGEM®-T Easy Vector. The recombinant plasmid designated as Bgls-NRC-1 was transformed into E. coli DH5α. The successful cloning of the bgls gene was tested either by PCR or by evaluating its expression in its new bacterial host. The bgls gene was expressed efficiently in E. coli and the enzyme activity of the transformant was compared to the enzyme activity of the donor bacterial strain. The new constructs produce much higher enzyme yields than the donor bacterial strain, they produce about 29% and about 57% higher cellulase specific activity at 37 °C and 55 °C respectively. Optimization of cellulolytic activity of the new recombinant strain were described. The effect of minimal medium supplemented with CMC or cellulose, or complete medium (LB) on bgls expression were tested, the order of cellulase activity production was CMC27.2 > cellulose 21.9 > LB 19.8 U/mg protein, respectively at 24 h. CMC was proved to be the best medium for cellulase production. Results also showed that double the initial inoculum resulted in more cellulase activities in all media.

Statistical Optimization of the production of protease from Bacillus sp. MSK-01 in submerged fermentation

Protease has been in increasing demand in industries due to its hydrolytic nature. In industries, high yield of enzyme is required to meet the industrial need at a relatively cheaper cost. In the present study, the protease from Bacillus sp. MSK-01 was produced in large quantity by submerged fermentation. Statistical techniques including Plackett-Burman and Response surface methodology are useful tools for optimizing many parameters at a time and are used for increasing the protease production from Bacillus sp. MSK-01. 19 different parameters were chosen, out of which 15 factors had positive effect on protease yield. Four maximum influencing factors were peptone, magnesium sulphate, skim milk powder and casein were chosen to further increase the protease yield. 397.3 IU ml-1 of enzyme yield was obtained under optimized conditions which lead to 198 fold increase in the yield of protease from unoptimized condition.

OPTIMIZATION OF CELLULASE ENZYME PRODUCTION FROM Aspergillus oryzae FOR INDUSTRIAL APPLICATIONS

Cellulases are the hydrolytic group of enzymes, responsible for release of sugars in the bioconversion of the cellulosic biomass into a variety of value added industrial products. Fungal isolated cellulases are well studied and playing a significant role in various industrial processes. Enzymatic depolymerisation of cellulosic material has been done by the various fungal isolated enzymes. In the present study, the cultivation conditions for cellulase production from Aspergillus species were optimized. Optimization of scarification conditions such as time course, inoculum size, carbon source and concentration, nitrogen source, various pH levels were performed for the production of extracellular carboxymethyl cellulase and endoglucanase enzyme. The result exhibited, 15 % inoculums size, corncobs 2 % concentration, Urea and medium pH 7 at 30oC supported high yield of carboxymethyl cellulase (38.80 U/ml/min) and exoglucanase enzyme (10.94 U/ml/min) through a submerged fermentation (SmF). In future biotechnological applications in cellulase enzyme production attain a vital role to obtain high degradable yield.

Systematic assessment of Pichia pastoris system for optimized β -galactosidase production

β- Galactosidase (commonly known as β-lactase; EC 3.2.1.23) is a multifunctional enzyme that can catalyze the hydrolysis of terminal non-reducing β-d-galactose residues in β-d-galactosides or transfer the galactosyl residue to saccharide acceptors to yield galacto-oligosaccharides (GOS). β-Galactosidase has a variety of applications in food and medical industries such as hydrolysis of lactose in milk, manufacture of galactooligosaccharides (GOS) and treatment of lactose malabsorption [1]. Although β-galactosidase is an ubiquitous enzyme existing in plants, animals and microorganisms, only a few β-galactosidases from Kluveromyces lactis, Aspergillus niger and Aspergillus oryzae are regarded as safe for food related industry applications. To achieve commercial scale production of β-galactosidase, heterologous expression systems were applied including Saccharomyces cerevisiae and Pichia pastoris [1]. P. pastoris is a methylotrophic yeast with great protein expression potential, and has been used as host for expression of many proteins both experimentally and industrially. P. pastoris has also been used for the extracellular expression of β-galactosidase from Paecilomyces aerugineus [2], Lactobacillus crispatus [3] and strains belonging to Aspergillus spp [4]. Despite its advantage in expression of proteins, P. pastoris system usually needs to be optimized to achieve maximum possible production level for a given protein. In achieving this, potential expression bottlenecks are analyzed and alleviated through perturbing and engineering of P. pastoris at different levels. And this process is often performed in a protein-specific manner, depending on the inherent nature and applications of target protein as well as its interaction with P. pastoris host. Take β-galactosidase for example, while some previous works have reported its successful expression in P. pastoris with reasonably high level, there are still several concerns needing to be addressed before further optimization, for instance: 1) Which kind of promoters are suitable to express β-galactosidase, the inducible or constitutive promoters? The strong AOX1 (alcohol oxidase I) promoter has been the most frequently used one. Nevertheless, the adoption of constitutive promoters has been appreciated in recent years because it does not need methanol to induce the expression, and therefore is safer (especially for food-grade β-galactosidase production) and eases the process control during the fermentation. 2) Unexpected N-glycosylation of foreign proteins are very commonly observed in P. pastoris system and its effects on the activity of expressed proteins remain unpredictable. In some cases, glycosylation is essential for maintaining the activities of expressed enzymes [5], [6], [7], while in other cases, glycosylation can negatively affect the enzyme activity [8], [9], [10]. Although β-galactosidase possesses multiple potential N-glycosylation sites, the effects of N-glycosylation on β-galactosidase activity were rarely investigated. 3) Of thousands of proteins that have been expressed using P. pastoris, the protein expression levels can range from tens of milligrams to tens of grams per liter. β-galactosidase can easily reach several grams per liter of production, which is obviously near the high end of this range. This raises the concern that the high enzyme yield may saturate the protein secretion ability of P. pastoris and thus limit the further improvement of its production level. In order to address the above concerns and systematically assess P. pastoris system for optimized β-galactosidase, we expressed β-galactosidase from K. lactis and A. oryzae in P. pastoris, evaluated different constitutive promoters in addition to AOX1 promoter and examined co-expression of different chaperones in hope of enhancing the secretion of β-galactosidase in P. pastoris. We equally assess the effect of glycosylation on β-galactosidase activity using OCH1 disrupted strain. Combining these strategies, the production level of β-galactosidase from A. Oryzae reached 1434.75 U/mL in 1 L fermentor, which therefore provided a basis for further optimization and industrial scale production of β-galactosidase in future works.

Statistical optimization of L-asparaginase production by using Fusarium solani

Background and objective The enzyme l-asparaginase is important as a therapeutic agent in the treatment of acute lymphocytic leukemia. It has been observed that microorganisms such as yeast and filamentous fungi such as Aspergillus spp., Penicillium spp., and Fusarium spp. are commonly produced asparaginases with fewer adverse effects. Fusarium solani was selected to be the most potent microbial isolate for l-asparaginase production. The factors controlling l-asparaginase production, such as containing media, were implemented to increase the yield of l-asparaginase. Statistical experimental design such as the Plackett–Burman enables finding out the most effective factors that increase the yield of l-asparaginase. Materials and methods Aspergillus rubber, Aspergillus terreus, Epico niger, Penicillium cyclopium, and F. solani were chosen for screening the l-asparaginase activity using phenol red – a pH indicator. l-Asparagine and the modified Czapek Dox media were tested for the production of l-asparaginase from F. solani under different incubation times. To optimize l-asparaginase production by using F. solani, the combined effect of seven variables (l-asparagine, sucrose, NaNo3, K2HPO4, MgSO4, KCl, and time) were assessed using the central composite design. Results and conclusion All the four fungal strains screened for l-asparaginase activity showed positive results in the rapid plate assay method. The medium employed contained asparagine with phenol red, and after incubation, pink zones around the colonies were observed. F. solani was a potential source for a high yield of l-asparaginase enzyme and high substrate specificity. Modified Czapek medium II was the most suitable for l-asparaginase, showing an activity of 121 U/ml. The result on l-asparaginase activity according to the screening Plackett–Burman experiments gives a medium composed of the following (g/l): sucrose (1); l-asparagine (0.5); KH2PO4. (0.75), MgSO4 (0.7); H2O (0.72); KCl (0.72); (NH4)2SO4 (11.7) at an incubation time of 3 days. For further investigation, x1 K2HPO4, x2 sucrose, and x3 time were found to be the most significant variables affecting l-asparaginase activity. The second optimization step was to identify optimal values of the three factors that bring about maximum l-asparaginase activity, using the central composite designed experiment. The results showed that K2HPO4 (2.5 g/l), sucrose (4 g/l), and the time (8 days) were critical in the production of l-asparaginase.

DEVELOPMENT OF CELLULOSE NANOFIBRE (CNF) DERIVED FROM KENAF BAST FIBRE AND IT’S POTENTIAL IN ENZYME IMMOBILIZATION SUPPORT

This research mainly focuses on developing a natural cellulose nanofibre (CNF) from kenaf bast fibre and its potential for enzyme immobilization support. CNF was isolated by using a combination between chemical and mechanical treatments such as alkaline process and high-intensity ultrasonication process to increase the efficiency of hemicellulose and lignin removal, and to reduce its size into nano-order. The morphological study was carried out by using scanning electron microscope (SEM), indicating most of CNF diameter in range of 50-90 nm was obtained. The result of chemical analysis shows that cellulose content of raw bast fibre, bleached pulp fibre and CNF are 66.4 %, 83.7 % and 90.0 %, respectively. By decreasing the size of cellulose fibre, it increases the number of (O–H) group on the surface that plays as important role in enzyme immobilization. Covalent immobilization of cyclodextrin glucanotransferase (CGTase) onto CNF support resulted in about 95.0 % of protein loading with 69.48 % of enzyme activity, indicating high immobilization yield of enzyme. The enzymatic reaction of immobilized CGTase was able to produce more than 40 % yield of α-CD. Reusability profile of immobilized CGTase resulted in more than 60 % of retained activity up to 7 cycles. Therefore, the CNF is highly potential to be applied as enzyme immobilization support.