Biochemical characterization of the ß-glucosidase Glu1B from Coptotermes formosanus produced in Pichia pastoris.

ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.

Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity.

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1-1/MPG1 genes rather than increased heterologous gene transcription.

Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity.

Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain A. niger GH1 tannase, which was also produced in P. pastoris. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate's access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in P. pastoris, the tannase described could be used in food and beverage processing at low and medium temperatures.

Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters.

Glargine is a long-acting insulin analog with less hypoglycemia risk. Like human insulin, glargine is a globular protein composed of two polypeptide chains linked by two disulfide bonds. Pichia pastoris KM71 Muts strains were engineered to produce and secrete insulin glargine through the cleavage of two Kex2 sites. Nevertheless, the recombinant product was the single-chain insulin glargine (glargine precursor) instead of the expected double-chain glargine. Molecular model analysis of the dimeric and hexameric forms of the single-chain glargine showed buried Kex2 sites that prevent intracellular glargine precursor processing. The effect of the methanol-feeding strategy (methanol limited fed-batch vs. methanol non-limited fed-batch) and the induction temperature (28 °C vs. 24 °C) on the cell growth and production parameters in bioreactor cultures was also evaluated. Exponential growth at a constant specific growth rate was observed in all the cultures. The volumetric productivities and specific substrate consumption rates were directly proportional to the specific growth rate. The lower temperature led to increased metabolic activity of the yeast cells, which increased the specific growth rate. The methanol non-limited fed-batch culture at 24 °C showed the highest values for the process parameters. After 75 h of induction, 0.122 g/L of glargine precursor was obtained from the culture medium.

Biochemical characterization of recombinant Penaeus vannamei trypsinogen.

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.4), which are digestive serine proteases. Despite knowing the properties of trypsins from Pacific white shrimp, Penaeus vannamei, the biochemical properties of shrimp trypsinogens including activation mechanisms and kinetics are unknown, due to difficulties isolating them from natural sources. In the present work, we describe the purification and biochemical characterization of four trypsinogen-like isoforms from recombinant P. vannamei trypsinogen, with a special emphasis on understanding its activation kinetics. The major trypsinogen-like isoform had an apparent molecular mass of 29 kDa. The other three forms of recombinant trypsinogen were: an N-glycosylated form of 32 kDa, a possibly O-glycosylated form of 41 kDa, and a likely double-chain form with a subunit of 23 kDa. The autoactivation profile of three-recombinant trypsinogen-like isoforms showed increased trypsin activity at a rate that was higher than that of bovine trypsinogen. This confirms the hypothesis proposed in the literature of a rapid trypsinogen autoactivation in the absence of aspartates in the activation peptide as it is for P. vannamei trypsinogen.

Tannase sequence from a xerophilic Aspergillus niger Strain and production of the enzyme in Pichia pastoris.

Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an α/ß-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.

Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris.

ß-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry.