Production Of Α-galactosidase Research Articles

Statistical optimization and sequential scale-up of α-galactosidase production by Actinoplanes utahensis B1 from shake flask to pilot scale

α-Galactosidase (α-GAL) is a class of hydrolase that releases galactose from galacto-oligosaccharides and synthetic substrates such as pNPG. In this study, the production of α-GAL by Actinoplanes utahensis B1 in submerged fermentation was enhanced by using statistical methods. The effects of temperature, pH, and inoculum percentage on enzyme secretion were optimized using BBD of RSM. The optimized process was scaled up from the shake flask to the laboratory scale (5 L) and to pilot scale (30 L) using KLa based scale-up strategy. By using BBD, a maximum yield of 62.5 U/mL was obtained at a temperature of 28 °C, a pH of 6.9, and an inoculum of 6.4%. Scale-up was performed successfully and achieved a yield of 74.4 U/mL and 76.8 U/mL in laboratory scale and pilot scale fermenters. The TOST was performed to validate the scale-up strategy and the results showed a confidence level of 95% for both scales indicating the perfect execution of scale-up procedure. Through the implementation of BBD and scale-up strategy, the overall enzyme yield has been significantly increased to 76%. This is the first article to explore the scale-up of α-GAL from the A. utahensis B1 strain and provide valuable insights for industrial applications.

Enhancement of α-galactosidase production using novel Actinoplanes utahensis B1 strain: sequential optimization and purification of enzyme.

α-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzymes, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing α-1,6 linked terminal galacto-oligosaccharide residues such as melibiose, raffinose, and branched polysaccharides such as galacto-glucomannans and galactomannans. In this study,Actinoplanes utahensis B1 was explored for α-galactosidase production, yield improvement, and activity enhancement by purification. Initially, nine media components were screened usingthe Plackett-Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as the best-supporting nutrients for the highest enzyme secretion by A. Utahensis B1. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in α-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified α-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the isolation of Actinoplanes utahensis B1 and optimization of the process for the α-galactosidase production as well as single-step purification.

Advanced strategies for production of soy-processing enzyme.

Enzyme production is critical and often costly for biorefinery. It is challenging to produce enzymes with not only high titers but also proper combinations of all required activities in a single fermentation. This work aimed at improving productivity and composition of the multiple enzyme activities required for hydrolysis of complex soybean carbohydrate in a single fermentation. A previously selected Aspergillus niger strain was used for its high carbohydrases and low protease production. Strategies of fed-batch substrate addition and programmed pH-decrease rates were evaluated. Cheap soybean hull (SH) was confirmed to induce production of all necessary carbohydrases. Surprisingly, fed-batch SH addition, originally thought to sustain substrate-inducer availability and reduce feedback repression by sugars, did not increase pectinase and cellulase production significantly and even lowered the α-galactosidase production, when compared with batch fermentation having the same total SH amount (all added initially). On the other hand, the pH-decrease rate could be effectively optimized for production of complex enzyme mixtures. The best fermentation was programmed to lower pH from 7 to 4 in 84h, at a drop rate of .0357 per h. It produced the highest pectinase (19.1 ± .04U/mL), α-galactosidase (15.7 ± .4U/mL), and cellulase (.88 ± .06 FPU/mL). Producing these high enzyme activities in a single fermentation significantly improves the effectiveness and economics of enzymatic soy processing, which, e.g., can hydrolyze the 30%-35% carbohydrate in soybean meal to sugars, with minimal protein degradation, to generate high-value protein-rich products and a hydrolysate as fermentation feedstock.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and V max of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

Molecular advances in microbial α-galactosidases: challenges and prospects.

α-Galactosidase (α-D-galactosidase galactohydrolase; EC 3.2.1.22), is an industrially important enzyme that hydrolyzes the galactose residues in galactooligosaccharides and polysaccharides. The industrial production of α-galactosidase is currently insufficient owing to the high production cost, low production efficiency and low enzyme activity. Recent years have witnessed an increase in the worldwide research on molecular techniques to improve the production efficiency of microbial α-galactosidases. Cloning and overexpression of the gene sequences coding for α-galactosidases can not only increase the enzyme yield but can confer industrially beneficial characteristics to the enzyme protein. This review focuses on the molecular advances in the overexpression of α-galactosidases in bacterial and yeast/fungal expression systems. Recombinant α-galactosidases have improved biochemical and hydrolytic properties compared to their native counterparts. Metabolic engineering of microorganisms to produce high yields of α-galactosidase can also assist in the production of value-added products. Developing new variants of α-galactosidases through directed evolution can yield enzymes with increased catalytic activity and altered regioselectivity. The bottlenecks in the recombinant production of α-galactosidases are also discussed. The knowledge about the hurdles in the overexpression of recombinant proteins illuminates the emerging possibilities of developing a successful microbial cell factory and widens the opportunities for the production of industrially beneficial α-galactosidases.

A statistical strategy for optimizing the production of α-galactosidase by a newly isolated Aspergillus niger NRC114 and assessing its efficacy in improving soymilk properties

Backgroundα-Galactosidase is widely distributed in plants, microorganisms, and animals, and it is produced by different fungal sources. Many studies have confirmed the valuable applications of α-galactosidase enzymes for various biotechnological purposes, like the processing of soymilk. ResultsAspergillus niger NRC114 was exploited to produce the extracellular α-galactosidase. One factor per time (OFT) and central composite design (CCD) approaches were applied to determine the optimum parameters and enhance the enzyme production. The CCD model choices of pH 4.73, 1.25% mannose, 0.959% meat extract, and 6-day incubation period have succeeded in obtaining 25.22 U/mL of enzyme compared to the 6.4 U/mL produced using OFT studies. Treatment of soymilk by α-galactosidase caused an increase in total phenols and flavonoids by 27.3% and 19.9%, respectively. Antioxidant measurements revealed a significant increase in the enzyme-treated soymilk. Through HPLC analysis, the appearance of sucrose, fructose, and glucose in the enzyme-treated soymilk was detected due to the degradation of stachyose and raffinose. The main volatile compounds in raw soymilk were acids (45.04%) and aldehydes (34.25%), which showed a remarkable decrease of 7.82% and 20.03% after treatment by α-galactosidase. ConclusionsTo increase α-galactosidase production, the OFT and CCD approaches were used, and CCD was found to be four times more effective than OFT. The produced enzyme proved potent enough to improve the properties of soymilk, avoiding flatulence and undesirable tastes and odors. Graphical Abstract▪

The Transcription Factor Gene tclB2 Regulates Mannanolytic Enzyme Production in the Fungus Talaromyces cellulolyticus.

The filamentous fungus Talaromyces cellulolyticus is a well-characterized cellulolytic and hemicellulolytic enzyme producer. In this study, the function of the tclB2 gene, which is a homolog of the manR/clrB/clr-2 gene in other filamentous fungi, in mannanolytic enzyme production by T. cellulolyticus was investigated. When a tclB2-disrupted strain (YDTclB) was grown in the presence of glucomannan, the production of β-mannanase, β-mannosidase, and α-galactosidase was decreased at the protein and transcriptional levels when compared to the control strain. In addition, a tclB2-overexpressing strain (YHTclB) showed higher β-mannanase and β-mannosidase production. When cellulose was used as a carbon source, the expression of genes encoding mannanolytic enzymes also decreased in YDTclB. These results suggested that TclB2 contributes to mannanolytic enzyme production in T. cellulolyticus. This work is the first study to identify a transcriptional regulator of mannanolytic enzyme genes in T. cellulolyticus.

Cloning and expression of a novel α-galactosidase from Lactobacillus amylolyticus L6 with hydrolytic and transgalactosyl properties.

Lactobacillus amylolyticus L6, a gram-positive amylolytic bacterium isolated from naturally fermented tofu whey (NFTW), was able to hydrolyze raffinose and stachyose for the production of α-galactosidase. The cell-free extract of L. amylolyticus L6 was found to exhibit glycosyltransferase activity to synthesize α-galacto-oligosaccharides (GOS) with melibiose as substrate. The coding genes of α-galactosidase were identified in the genome of L. amylolyticus L6. The α-galactosidase (AglB) was placed into GH36 family by amino acid sequence alignments with other α-galactosidases from lactobacilli. The optimal reaction conditions of pH and temperature for AglB were pH 6.0 and 37°C, respectively. Besides, potassium ion was found to improve the activity of AglB while divalent mercury ion, copper ion and zinc ion displayed different degrees of inhibition effect. Under the optimum reaction condition, AglB could catalyze the synthesis of GOS with degree of polymerization (DP) ≥5 by using 300 mM melibiose concentration as substrate. The maximum yield of GOS with (DP) ≥3 could reach 31.56% (w/w). Transgalactosyl properties made AglB a potential candidate for application in the production of GOS.

Особливості продукції α-галактозидази Penicillium restrictum

Особливості продукції α-галактозидази Penicillium restrictum

Streptococcus downii sp. nov., isolated from the oral cavity of a teenager with Down syndrome

A new α-haemolytic streptococcal strain has been isolated from the dental plaque of a teenager with Down syndrome. Genetic and taxonomic analyses place this Streptococcus within the oralis group. It is a Gram-stain-positive, non-motile, non-spore-forming spherical alpha-haemolytic coccus arranged in chains, and it ferments a large number of monosaccharides and disaccharides, as well as polymeric carbohydrates. It differs biochemically from closely related species of Streptococcus due to its production of α-galactosidase, β-galactosidase and N-acetyl-β-d-glucosaminidase and by the absence of arginine dihydrolase deiminase and IgA1-protease. It grows in a temperature range of 25 to 40 °C (optimal growth temperature at 37 °C) and in a pH range of 4.5 to 8 (optimal pH at 7.0). A phylogenetic analysis based on its 16S and 23S rRNA gene sequences placed it close to Streptococcus dentisani CECT 7747T. The ANIb and ANIm values were 93.19 and 93.61 %, respectively, both below the accepted threshold to designate it as a new species of bacteria. A phylogenetic tree based on its core genome placed it close to Streptococcus oralis subsp. dentisani strain CECT 7747T with a distance in the expanded core phylogeny of 0.1298. The in silico DNA-DNA hybridization value was 52.2 % with respect to the closest species, S. oralis subsp. dentisani CECT 7747T. Based on these data, a new species of bacteria within the genus Streptococcus, family Streptococcaceae and order Lactobacillales is described, for which the name of Streptococcus downii sp. nov. is proposed (type strain CECT 9732T=CCUG 73139T).

Aspergillus niger production of pectinase and α-galactosidase for enzymatic soy processing

Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers.

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.

Improving extracellular protein production in Escherichia coli by overexpressing D,D-carboxypeptidase to perturb peptidoglycan network synthesis and structure.

Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli, for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB, and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB, respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA, and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB. Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli. This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli.

Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.

The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger.

α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production. Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35°C. The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

Response surface methodology-based optimization of production media and purification of α-galactosidase in solid-state fermentation by Fusarium moniliforme NCIM 1099

Response surface methodology was used to enhance the production of α-galactosidase from Fusarium moniliforme NCIM 1099 in solid-state fermentation. Plackett–Burman design was employed for selection of critical media constituents which were optimized by central composite rotatable design. Wheat bran, peptone and FeSO4·7H2O were identified as significant medium components using PB design. Further CCRD optimized medium components as wheat bran; 4.62 μg, peptone; 315.42 μg, FeSO4·7H2O; 9.04 μg. RSM methodological optimization increased the enzyme production from 13.17 to 207.33 U/g showing 15.74-fold enhancement. The α-galactosidase was purified by 70% fractionation followed by DEAE anion exchange column chromatography which yields 23.33% with 28.68-fold purification. The molecular weight of α-galactosidase was 57 kDa which was determined by SDS-PAGE analysis. Purified enzyme has optimum pH of 4.0 and was found to be stable in wide pH range of 3.0–9.0. Its optimum temperature was 50 °C, whereas its activity remains above 50% up to 2 h at 75 °C. Hg2+ was found to be a potent inhibitor and Mg2+ acted as an activator of enzyme. No significant change was observed in enzyme activity for galactose concentration, ranging from 1 to 100 mM. The K m values of enzyme for substrates p-nitrophenyl-α-d-galactopyranoside, melibiose and raffinose were 0.20, 1.36, and 3.66 mM, respectively. Low K m and stability to various physiological conditions of enzyme represents its potential which can be exploited in various industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0575-7) contains supplementary material, which is available to authorized users.

Statistical media optimization for enhanced production of α-galactosidase by a novel Rhizopus oryzae strain SUK

Abstract A novel fungus producing extracellular α-galactosidase was isolated from soybean cultivated soil of Solapur, India. It was identified as Rhizopus oryzae strain SUK using 28S rDNA sequencing. Response surface methodology was used for the optimization of medium components to achieve enhanced production of α-galactosidase by newly isolated Rhizopus oryzae strain SUK in solid state fermentation (SSF). Plackett Burman (PB) design was employed for selection of critical media constituents which were identified as wheat bran, soybean meal and molasses. Further optimization was carried out using Central composite rotatable design (CCRD) which gave optimized media components for production of α-galactosidase with wheat bran; 4 g, soybean meal; 0.5 g, molasses; 0.5 g. α-galactosidase production obtained experimentally has close agreement with the model prediction value and the model was proven to be adequate. Initial enzyme activity was 20.17 U/g which was increased up to 218.54 U/g by CCRD with 17.74fold enhancement proving the efficacy of experimental design for process optimization. TLC analysis confirmed the ability of produced α-galactosidase to hydrolyze the non-digestible oligosaccharides in soymilk, which finally increments nutritional value of soymilk. Stereochemistry of hydrolysis of glycosidic linkage was determined by NMR spectroscopy.

Selection of lactic acid bacteria strain for simultaneous production of α- and β-galactosidases

Galactosidases are group of enzymes gaining increasing attention in recent times due to the fact they catalyse hydrolysis of galatose-containing oligosaccharide, which reduce digestibility of various food and feed products. Microbial production of galactosidases is the most efficient and widely used. The aim of this study was to selects strain of lactic acid bacteria which can be used as producer of both, α- and β-galactosidases, since bacteria with such properties can be used for improving digestibility of products containing soybean and milk derivatives. In preliminary selection only intracellular activity of α- and β-galactosidases was detected and Lactobacillus reuteri was selected as the most adequate producer of both enzymes. In subsequent experiments, fermentation was optimized with respect to produced intracellular activity of both enzymes using spectrophotometric enzyme assays. Enzymes were characterized with respect to their temperature and pH optima. It was revealed that the most effective factor for production of enzymes is the selection of inducer. The addition of lactose induced production of β-galactosidase, while raffinose was the most efficient inducer of α-galactosidase production. Further improvement of enzyme production was achieved by optimization of inoculum concentration.

Deinococcus radioresistens sp. nov., a UV and gamma radiation-resistant bacterium isolated from mountain soil.

Two Gram-negative, non-motile, short rod-shaped bacterial strains, designated as 8A(T) and 28A, were isolated from Mount Deogyusan, Jeonbuk Province, South Korea. The isolates were analyzed by a polyphasic approach, revealing variations in their phenotypic characters but high DNA-DNA hybridisation values reciprocally, confirming that they belong to the same species. Both the isolates also showed a high resistance to UV compared with Deinococcus radiodurans, and a gamma-radiation resistance similar to other members of the genus Deinococcus. Phylogenetic analysis with the 16S rRNA gene sequences of closely related species indicated their similarities were below 97 %. Chemotaxonomic data showed the most abundant fatty acids to be C16:1ω7c and C16:0. The strains can be distinguished from closely related species by the production of esterase (C4) and α-galactosidase, and by their ability to assimilate L-alanine, L-histidine and N-acetyl-D-glucosamine. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus radioresistens sp. nov. is proposed. The type strain is 8A(T) (KEMB 9004-109(T) = JCM 19777(T)), and a second strain is 28A (KEMB 9004-113 = JCM 19778).

Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

BackgroundTrichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling.ResultsConstructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a method was developed for gravity-based density separation and protein body solubilisation. The secreted protein had similar specific activity to that of the commercially available mammalian form. The intracellular version had 5-10-fold lower activity due to the enzymes incompatibility with alkaline pH. The secretion strain achieved 10% lower total biomass than either the parental or the intracellular strain. The patterns of gene induction for intracellular and parental strains were similar, whereas the secretion strain had a broader spectrum of gene expression level changes. Identification of the genes involved indicated strong secretion stress in the secretion strain and to a lesser extent also in intracellular production. Genes involved in the unfolded protein response (UPR) and ER-associated degradation were induced by GLA production, including; hac1, pdi1, prp1, cnx1, der1, and bap31.ConclusionsActive human α-galactosidase could most effectively be produced intracellularly in Trichoderma reesei at >0.5 g/l by avoidance of the extracellular environment, although purification was challenging due to specific activity losses. Strain analysis revealed that in addition to the issues with secreted proteases, the processes of secretion stress including UPR and ER degradation remain as bottlenecks for heterologous protein production. Genetic engineering to eliminate these bottlenecks is the logical path towards establishing a strain capable of producing sensitive heterologous proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0091-y) contains supplementary material, which is available to authorized users.